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cavernomas are vascular lesions composed of thin - walled , dilated capillary spaces with no intervening brain tissue . the prevalence of cavernomas in the general population is 0.50.6% [ 2 , 3 ] . they become symptomatic either through epileptic seizures or focal neurological symptoms attributable to rupture and subsequent bleeding . cavernomas in the frontal lobe or in arterial border zones have a high risk . de novo cavernomas are known to develop in response to cranial irradiation for the treatment of primary brain tumours , prophylactic cns radiotherapy for acute lymphoblastic lymphoma and total body irradiation ( tbi ) as conditioning therapy for haematopoietic stem cell transplantation ( hsct ) [ 6 , 7 ] . the pathophysiological mechanisms of radiation - induced cavernomas ( rics ) remain elusive . as children treated with cranial radiotherapy or tbi are at higher risk , hypotheses assume that the immature brain of paediatric patients is more vulnerable to actinic harm [ 1 , 7 ] . radiotherapy induces vessel wall necrosis leading to endothelial swelling , dilation of the vessel lumen , hyalinization and fibroses which predisposes to cavernoma formation [ 1 , 7 ] . others speculate that tiny , invisible cavernomas are already present before radiotherapy and growth is subsequently triggered . finally , expression of vascular endothelial growth factor ( vegf ) is increased in response to irradiation [ 1 , 7 , 9 ] . analysed 189 cerebral cavernomas , including a subgroup of patients treated with prior cerebral radiotherapy . results indicated a correlation between cranial irradiation and cavernoma formation , especially in younger children at the time of radiotherapy . the majority of adult de novo cavernomas occurred after radiation doses > 30 gy . the median age at the time of cavernoma detection varies between 8 and 11.7 years in unselected patient cohorts [ 2 , 7 ] . in children , the interval between cranial radiotherapy and the detection of de novo cavernomas is approximately 8 years [ 2 , 7 ] . a 45-year - old man was admitted to our hospital with a series of focal and generalized epileptic seizures . ecg and thoracic x - ray were also unremarkable . computed tomography of the brain showed a haemorrhagic partially calcified lesion in the right parietal lobe . subsequent mri studies revealed a haemorrhagic transformed and space - occupying lesion diagnosed as a cavernoma ( fig . 1 , fig . 2 ) . according to the clinical symptoms and the anatomical correlation , the cavernoma was the underlying lesion responsible for the new onset of focal epileptic seizures . the patient 's previous medical history included intensive treatment with various chemotherapeutic agents including cytarabine / idarubicin ( first cycle ) , cytarabine / amsacrine ( second cycle ) and cytarabine / etoposide ( third cycle ) for the treatment of acute myeloid leukaemia ( aml ) , subtype m2 , in 1999 . after early bone marrow recurrence in march 2000 , he received reinduction chemotherapy ( s - ham ) and an allogenic hsct in july 2000 . as part of the therapy , he was treated with tbi with 12 gy in 6 fractions . after diverse infections ( sinusitis , invasive pulmonary aspergillosis and mucositis ) he finally achieved complete remission . later on , he suffered from treatment - related secondary arterial hypertension most probably due to cyclosporine - associated renal impairment . he frequently complained about headaches which led to a first cerebral mri in 2006 to exclude secondary causes . 3 ) . until 2011 , neither a neurological evaluation nor a further radiological evaluation was performed . according to the well - documented history including former cerebral mri studies here , we report a 45-year - old patient with a ric after intensive and multimodal treatment for aml . the development of a cerebral ric is underlined by an entirely normal high - resolution cerebral mri 5 years before the diagnosis . patients irradiated for the treatment of medulloblastomas , gliomas and acute lymphoblastic lymphoma are predominantly at risk for the development of rics [ 6 , 7 ] . however , a growing body of evidence indicates that not only primary brain tumours but also haematological diseases treated with any cerebral radiotherapy are prone to the risk of cerebral cavernoma development . investigated a population of children ( n = 56 ) between 3 and 14 years of age who had received radiotherapy for leukaemia . patients either received 1824 gy for cranial irradiation or 9.912 gy for tbi before hsct . cerebral cavernoma was the most frequent finding in 57% of the patients 11 years after therapy . another study investigated 68 patients with various haematological malignancies treated with radiotherapy in the context of hsct . the median age for the entire study cohort was 7 years ( range 024 ) and the median age at follow - up was 28 years ( range 1443 ) . all of these aml patients were subdivided in a heterogeneous patient group irradiated with 612 gy as a conditioning therapy for hsct ( n = 39 ) . therefore , we present here the first adult aml patient undergoing hsct in combination with tbi with a documented history of a ric . furthermore , our patient is much older compared to most other reports , as rics mainly develop in the second decade [ 7 , 11 ] . tbi with a cumulative radiation dose of 12 gy as a trigger for a ric is considerably low . an analysis of 15 patients older than 10 years of age indicated that the vast majority of the patients received radiation doses 30 gy . however , in the same report , the authors demonstrated a possible ric in a region with radiation doses < 18 gy . investigated a cohort of juvenile and adolescent long - term survivors of hsct ( n = 19 ) who did or did not receive radiotherapy as part of conditioning treatment . they demonstrated that 46.2% ( n = 36 ) of the patients irradiated with 612 gy developed cerebral rics . this clearly discloses cerebral irradiation itself , and higher doses in particular , as a risk factor in the context of hsct [ 2 , 3 ] . the probability of developing a ric 25 years after hsct was 61.5% in 28 out of 68 children . radiation dosages of 612 gy might be considered as the threshold level for cavernoma formation . moreover , faraci et al . reported that a radiation dose < 1 gy might be sufficient to trigger vegf release and induce neoangiogenesis leading to cavernomas . these data fit well with the low radiation dosage applied to our patient reported herein . controversially , a retrospective analysis of 552 patients ( < 18 years ) did not confirm a correlation between radiation dose and cerebral cavernoma formation within the radiation field . these authors identified various neoplastic lesions within a certain distance of the maximal irradiation area . it was speculated that cells with proliferative potential are killed in the high radiation areas compared to sublethal dna damage of cells in low - dose areas . the median time from irradiation to cerebral cavernoma formation is reported to be 8.9 years . one could speculate that the lower radiation dose may have accounted for the longer time span . this is supported by the fact that higher radiation doses lead to earlier formation of cerebral cavernomas in children < 10 years of age . an analysis of 40 patients with rics revealed that half are detected in the age group between 10 and 19 years . additionally , a literature review of 84 cases with rics reported a median age of 8 years at the time of radiation and a median interval to cavernoma formation of 8 years . interestingly , patients over 15 years of age at irradiation had a marginally higher risk than younger patients . cns irradiation leads to several vascular effects such as endothelial swelling and adventitial vessel proliferation , causing focal ischemia . hypoxia stimulates expression of hypoxia - inducible factor-1 and vegf ; the latter being the strongest proangiogenic factor . higher expression of these biological factors has been demonstrated in younger patients , which partially explains susceptibility of vascular malformations in response to radiotherapy in children . taken together , higher radiation dose and younger patient age are negative risk factors for the development of rics . since our patient received tbi , one would probably expect multiple cavernomas in the body or at least in the brain . however , most patients develop only a single cavernoma , whereas multiple cavernomas are extraordinary rare . this observation supports the hypothesis of a preexisting vascular lesion not detectable on mri scans as the predisposing factor for a radiotherapy - induced proliferation trigger for cavernomas . . demonstrated in a cohort of incidental cavernomas that the annual rate of bleeding was 6.19% in patients initially presenting with haemorrhage , 2.18% in patients with symptoms not related to haemorrhage and 0.33% in patients with an incidental finding . it was also shown that infratentorial cavernomas are more likely to be associated with symptomatic bleeding compared to supratentorial cavernomas . this can easily be explained due to the anatomical characteristics of the posterior fossa . of note , de novo cavernomas have an increased risk for bleeding compared to congenital cavernomas but are mainly located supratentorially . in conclusion , cerebral cavernomas should be considered in the differential diagnosis of focal neurological symptoms in any patient who has received previous cranial radiotherapy . the case reported here accumulates evidence that a cumulative radiation dosage > 30 gy has to be taken into account as a risk factor for cerebral cavernoma formation also in adults [ 3 , 6 ] .

cerebral cavernomas are thin - walled vascular lesions composed of dilated capillary spaces . de novo formation of cavernomas after cerebral radiotherapy has been suspected since 1994 . they are mostly seen in children after irradiation of brain tumours . radiation dose and the developing juvenile brain are predisposing factors causing cavernomas . however , the underlying mechanisms are still far from being understood . in adults , radiation - induced cavernomas ( rics ) usually occur 10 years after a high cumulative radiation dosage of > 30 gy . here , we report a 45-year - old man with new - onset focal epileptic seizures caused by a haemorrhagic lesion noted on cerebral computed tomography scan . brain mri showed the typical appearance of a ruptured cavernoma . of note , a cerebral mri scan 5 years earlier showed no corresponding lesion . the patient had been treated with haematopoietic stem cell transplantation for acute myeloid leukaemia ( aml ) 16 years before . as part of this procedure , total body irradiation ( tbi ) consisting of 12 gy was administered . according to the data from the literature , the typical delay from irradiation and a former normal brain mri scan , we assume that our patient suffers from a ric . to our knowledge , this is the first documented adult aml patient with a ric treated with tbi . we aim to increase awareness among neurologists for the association of cranial irradiation or tbi and de novo cavernomas in patients suffering from malignant diseases .

past theoretical mechanisms have failed to explain the observed , postoperative hypothermia in laparoscopic patients . mismatch of tissue and inflation gas thermal capacities rule out any substantial global tissue temperature reductions during laparoscopic procedures . overlooked , however , is the possibility that severe hypothermia is due to local super - cooling of tissue caused by evaporation from tissue surfaces of peritoneal fluid water into the dry jet of insufflation gas . this mechanism is examined analytically and experimentally and is found to be real and significant . patient hypothermia during laparoscopic surgery is widely reported in recent literature . while this clinical condition and its undesirable consequences to the patient are well understood , the exact cause of the additional hypothermia due to laparoscopy is not clear . it is reported that the heat capacity effects of the co2 insufflation gas are not sufficient to cause physiologically significant , bulk tissue cooling . the question remains , therefore , what is the cause of a patient 's additional hypothermic response observed during laparoscopic surgery ? at least one possible causative factor could be severe cooling of local tissue surfaces through the evaporation of peritoneal fluid by the jet of dry insufflation gas ( usually co2 ) impinging on the epithelial surface while the gas is being introduced to the peritoneal cavity through a trocar or other device , such as a veress needle . this study was designed to explore the possibility that such a mechanism could result in substantial cooling of surface tissue during the initial abdominal insufflation and/or subsequent insufflation during the laparoscopic procedure . a theoretical analysis was completed of the evaporative cooling effects caused by the simultaneous tissue - conductive , and gas - convective , jet heat and mass transport that reasonably could be expected to occur during typical insufflation . laboratory measurements of the temperature transient occurring when a dry co2 gas jet impinged on simulated and animal tissue material was obtained to verify the results of these theoretical calculations . the experimental studies included measurement of the effects of procedure variables : insufflation gas flow rates ; height 's of the gas jet ; tissue cooling - area size ; type of gas entry port ; tissue type ; and , most importantly , the thermal / humidity condition of the insufflation gas stream . table 1 summarizes the range of these variables for which theoretical and experimental results were obtained in this work . evaporation jet cooling of the epithelial tissue occurs when the end of a veress needle or trocar is positioned close to a tissue surface , as shown in figure 1 . the insufflation gas exits from the end of the veress needle or trocar ( the nozzle of figure 1 ) in a free vertical jet . when the gas jet reaches the epithelial surface ( impingement surface of figure 1 ) , the gas flow is redirected horizontally and flows radially away from the centerline of the entry port on the impingement surface ( the stagnation point ) . because of a very rapid flow transition on the epithelial surface , high heat and mass transfer rates are generated in the area of the stagnation point with the maximum effect occurring at the stagnation point . when the insufflation gas is dry , as it is in all laparoscopic procedures , a large evaporation - driving force is generated in the resulting gas phase boundary layer , with velocity profile transitions , in very high evaporation rates around the stagnation point . the energy needed to evaporate fluid from peritoneal tissue surfaces is from jet evaporation and cools the surface at a rapid rate . how fast , how long , and how much tissue will be cooled and to what temperature is a complex function of the gas flow rate ( f 1/m ) , the height of the gas injection device above the tissue ( h mm ) , the dimension of the gas injection device ( d , the diameter of the circular entrance port , or w , the width of the annular jet from a trocar ) , the size of the cooled tissue area ( measured by r , the distance from the stagnation point for the circular jet , or x , the distance from the center of slot width w of an annular jet ) , the magnitude of the tissue 's thermal parameters of thermal conductivity and diffusivity and , most important , the relative humidity of the insufflation gas . the theoretical analysis and . experimental verification of the theoretical results were obtained through in vitro measurements of jet cooling of synthetic and animal tissue surfaces at various flow and geometry conditions known to influence the cooling rates . the rapid tissue cooling rates were measured with single and four thermocouple ( t / c ) configurations that detected tissue surface temperature at the stagnation point and with the four t / c configuration , at progressively larger distances ( r ) from the stagnation point ( figure 1 ) . temperatures were collected by computer data acquisition at rates of 5 to 15 points per second and stored for analysis and display . great care was taken during the experimental measurements with the apparatus seen in figure 1 to assure that the surrounding conditions of temperature ( t ) and humidity simulated conditions that are experienced in the human abdomen . much theoretical and experimental work has been done on heat and mass transfer into and from circular and slot gas jet streams . the mass transfer elements of these studies , however , have been limited to sublimation of a solid into a gas jet ; and gas jet evaporative mass transfer of a liquid surface has been studied very little . for this study , we have used the well known and reliable heat - mass transfer analog for convective flow , making it relatively simple to circumvent this lack of direct experimental correlations . referring to figure 1 , the impingement surface of our model is wet with peritoneal fluid and is cooled by latent evaporative heat flux into the jet of dry co2 insufflation gas . the heat transfer flux due to this evaporative mass transfer is characterized by the equation , where he is the evaporative heat transfer coefficient , ps , the vapor pressure of the surface fluid ( a function of surface temperature ) , and is the jet gas 's relative humidity ( assumed zero for this analysis ) . as has been discussed , the heat and mass transfer coefficients needed in equation 1 vary considerably from a maximum at the stagnation point to decreasingly lower values at positions r ( or x for a slot jet ) away from the circular jet center line . the integral mean values for the transfer coefficients in these regions in terms of r and x ; the diameter , d , of the circular jet ( or w , the width of the slot jet ) ; and the height of the nozzle , h , from the impingement surface have previously been determined and is called the martin 's nusselt number . from the martin 's nusselt number correlations we obtained mean ( with r or x ) heat transfer coefficients , he . the classic heat - mass transfer analog is in the form where sh is the sherwood number , nu = nusselt number , sc = schmidt number , and pr is the prandtl number . equation 2 , with sc = 0.49 , pr = 0.75 for diffusion of water through co2 , permitted the calculation of the mass transfer coefficients hd . the evaporative heat transfer coefficient , he of equation 1 , was determined by the simple conversion of the mass transfer coefficient , hd , from concentration units to partial pressure units , with assumptions of ideal gases and the boundary layer mean temperatures and the known values of water 's heat of vaporization . this led to the following simple relationship : thus , with martin 's correlations for nusselt number as a function of entry port type ( gas jet stream ) , size , orientation , flow conditions , and height , h , above the impingement surface , and with equations 13 described above , it is possible to determine the mean ( between the stagnation point and the position r or x ) heat flux due to evaporation for any set of flow and entry port configuration conditions . these values then become the critical boundary conditions for the unsteady state , tissue - conduction problem . the speed that peritoneal tissue will be cooled by evaporative jet cooling is governed by the jet 's evaporative surface heat flux , jh , e , the size of the jet , and the conduction characteristics of the tissue . to simplify the analysis , it was assumed that the very high heat fluxes caused by the evaporative gas jet would completely dominate tissue internal heating due to metabolism and perfusion near the tissue surface . additionally , it was assumed that the tissue dimensions would be large in comparison to that of the gas jet , for example , very large conduction pathways . under these conditions , the general transient heat conduction differential equation reduces to where , for our model , the reduced temperature , theta , is defined as and the boundary conditions for evaporative surface cooling , with c = je / k where k is the surface tissue 's thermal conductivity , are as follows : this classic transient - conduction problem has been solved by many . the solution for the general case , as well as the special situation of x = 0 , or the impingement surface temperature of our model can be expressed as where , for our jet evaporation system , c = jh , e / k and solutions to equation 5 are complicated by the fact that the value for c = jh , e / k is itself a nonlinear function of surface temperature , t , through jh , e and its dependence on the vapor pressure term ps which , in turn , is a nonlinear function of surface temperature . this results in an equation 5 that is transcendental in t and must be solved numerically by iterative , non - constant interval , finite - difference techniques . figure 2 shows the results of an equation 5 calculation for the surface tissue temperature transient , caused by evaporative gas jet cooling of a circular area of 0.79 sq . the data of this figure are calculated for a veress needle height over the tissue of 5 mm and for insufflation gas flows of 1 to 11 liters per minute . a typical set of surface temperature cooling curves for evaporative jet cooling via a trocar gas injection device is shown in figure 3 . as with the veress needle theoretical results , the effect of insufflation gas flow rate is indicated parametrically for co2 flows from 1 to 11 liters per minute . experimental measurements have been made to verify the results of the theoretical analysis of laparoscopic evaporative gas jet cooling . the first ( phase 1 ) was a series of tests using a single thermocouple ( t / c ) measurement , at the stagnation point = t1 , of the temperature transient detected in 3 mm thick sections of synthetic materials ; cellular polyurethane ( cp ) and woven rayonpolyester ( wr ) . data were collected at a rate of five points a second for phase 1 measurements . a second series of tests ( phase 2 ) consisted of measurements using cellular polyurethane and woven rayon , and 3 mm sections of animal tissue consisting of turkey or ham . a four thermocouple array , with the first t / c ( t1 ) , was located at the stagnation point , and the remaining t / cs ( t2 , t3 , & t4 ) were spaced on a radial line 1 , 2 and 4 mm from the first thermocouple , respectively . phase 2 temperature data were collected simultaneously from each t / c at a rate of approximately 15 points per second for each thermocouple . the t / cs for this series , as were those of phase 1 , were small ( 0.2 mm diameter ) sensors , with fast response times ( time constant 0.6 sec ) , and positioned from below , at or just slightly below ( 0.2 mm ) the impingement surface . a 1 mm , inside diameter , veress needle and a standard 10 mm trocar were used as the insufflation gas injection ports . these were positioned 2 to 5 mm above the tissue surfaces , and gas flows were varied between two and eight liters per minute . the experimental jet / impingement surface configuration of figure 1 was totally enclosed in a transparent container to provide an isolated environment that reproduced the gas space , temperature , and humidity conditions that the gas injection device and impingement surface experience during a laparoscopic procedure , ( co2 , 37c and = 100% ) . during all tests the tissue surface was irrigated with a temperature controlled sterile saline drip at 37c . figure 4 shows the results of phase 1 experimental tests of evaporative jet cooling at the stagnation point on a wet cellular polyurethane impingement surface at 37c . experimental temperature transient - cp material . the results of a typical phase 2 experimental test are seen in figure 5 , which shows the surface temperature cooling transient for ham tissue when evaporatively cooled by a 1 mm circular jet at approximately 6 liters per minute flow rate . these data are typical of the measurements obtained at the flow rates and jet height tests of this study . figure 6 shows typical impingement surface cooling transients resulting from an approximately 6 liters per minute co2 flow rate from a 10 mm trocar . experimental temperature transients - thick ham . experimental temperature transients - thick ham . the effects of changes in insufflation gas flow rate and gas port height above the impingement surface on tissue temperature decrease with the initiation of evaporative surface cooling . the average of replicate runs of temperature measurements of thermocouple tc1 ( the impingement point temperature ) , measured five seconds after initiation of the insufflation gas flow , is plotted against various gas flows . the parameters of figure 7 represent lines of constant height , h , of the tip of the injector above the tissue surface gas flow effects on tissue temperature after five seconds . to confirm that the rapid temperature drops demonstrated in the tissue tests of this study were caused primarily by evaporation of tissue - surface liquid , a series of experiments were carried out in which the insufflation gas was both heated ( to 37c ) and humidified ( to > 95 relative humidity ) prior to its introduction to the insufflation gas injection port . a typical result of these tests is shown in figure 8 , where thermocouples tc1 through tc4 measured the tissue surface temperatures at various distances from the impingement point for approximately eight seconds after initiation of the gas flow rate of approximately 6 liters per minute . experimental temperature transients - thick ham . the results of the analytical modeling of evaporative jet cooling of tissue surfaces gives dramatic indications of rapid and significant cooling of these surfaces , as seen in figures 2 and 3 . these results predicted that , for circular jets , tissue surface temperatures could drop to under 20c in less than three seconds after initiation of insufflation gas flow and cool a tissue area of more than 0.75 square centimeter . our modeling of this cooling effect showed serious tissue temperature drops for a wide range of delivery port types , which resulted from jet streaming of gas and , importantly , for jet heights ( h ) up to 1 cm . comparison of figures 2 and 3 demonstrates that the decrease of surface temperature is relatively independent of type of gas injection device , although the veress needle injector caused a somewhat faster tissue - cooling rate ( 1.2c / s ) than the 10 mm trocar . results , however , from the trocar analysis , predicted that considerably more tissue surface area would experience the cooling effect ( 1.9 cm ) . phase 1 experimental testing ( figure 4 ) showed very large cooling rates of 30c / s in the first second of gas flow ; larger , in fact , than cooling rates predicted by the analytical model . the temperature for these tests tended to reach an equilibrium level within two to five seconds of the initiation of the evaporative gas jet . this effect most certainly is due to the jet dispersing and evaporating the fluid in the region of the t / c . under these conditions , the slower evaporation rate could be offset by internal heat conduction to the tissue surface . phase 2 results ( figures 57 ) confirm the theoretical prediction of the degree of evaporative cooling on wet tissue both qualitatively and quantitatively . at insufflation flow rates above 51/m , significant areas of tissue ( > 2 cm ) were cooled to temperatures less than 18c within six seconds of initiating gas flow . these cooling effects , as can be seen in figure 7 , were relatively independent of the height , h , of the gas port site above the tissue surface . that these cooling effects are caused by evaporation of surface fluid into jets of dry co2 is demonstrated by the results of figure 8 , where pre - heated and humidified insufflation gas jet streams with flow rates over 5 1/m caused less than 1c of local surface cooling . in some cases , local tissue was heated approximately 1c . these small temperature rises or drops experienced with the introduction of heated / humidified insufflation gas were caused by small temperature differences ( positive or negative 1c ) between the temperature of the incoming gas and the temperature of the injection device . the degree of tissue cooling , with either the veress needle or the trocar gas injector were found , in phase 2 experiments , to be independent of the types of tissue tested . the differences in maximum cooling rate and/or the minimum temperature reached by the tissue in the first five to seven seconds of evaporative jet cooling were not statistically significant for the different types of tissue tested . the crossing of tc1 and tc2 curves after two seconds of evaporative cooling as shown in figure 7 , most likely is due to drying around the stagnation point . tissue heat conduction from the sub - surface regions toward the stagnation point ( sp ) would heat this region after most of the resident liquid is evaporated and its cooling effect decreased . the analytical modeling and experimental testing of this study show that substantial evaporative cooling occurs to extensive local regions of wet surface tissue when dry insufflation gas flows against its surface . cooling rates of 20c / s and local tissue temperature reductions of 20c were measured . these findings suggest that local neurological reaction and tissue damage are possible from such hypothermic events and that this can be overcome . clinical observations and improvement of peri- and postoperative patient hypothermia by heating and humidifying the gas during laparoscopy has been previously demonstrated . this study shows further that the effects of insufflation gas jet evaporative cooling during laparoscopy are completely eliminated by humidifying the insufflation gas stream .

background and objectives : explanations for laparoscopic - induced hypothermia fail to explain clinical observations . it is possible that water evaporation occurs from the jet stream of gas inflation resulting in tissue surface super - cooling leading to tissue damage and drying.methods:theoretical calculations based on thermal conductivity , mass transfer effects and heat flux considerations correlated closely with synthetic and tissue experiments . thermocouple measurements at a rate of 15 data points per second were performed.results:cooling rates of 10 to 25 degrees centigrade per second for high flow rates were found based on gas flow rate and effective size of gas delivery site . these rapid temperature drops extended beyond a 2 cm2 diameter.conclusions:evaporative cooling accounts for significant hypothermia . the cooling is dependent on the lack of water vapor in the gases currently used during laparoscopy . cooling rates are independent of height from tissue and geometry of delivery port . heating and hydrating the gas to a physiologic condition eliminates hypothermia and tissue dessication .

due to its tragic and unexpected nature , sudden cardiac death ( scd ) in athletes has been brought to public attention in recent decades . the incidence of scd has been reported to be approximately 1 - 2 per 100,000 person - years , with a 2.5 times higher risk in athletes when compared with nonathletes . different studies have reported various cardiac diseases as the most common cause . in studies done in the united states , hypertrophic cardiomyopathy ( hcm ) was the most common cause , followed by congenital coronary artery anomalies ( cca ) , myocarditis , and arrhythmogenic right ventricular cardiomyopathy ( arvc ) ; ion channelopathies such as long qt and brugada syndrome were also identified . scd can also be induced by a traumatic blow to the chest ( commotio cordis ) . also , the incidence of scd is known to be greater in males than females [ 14 ] . soccer is the most popular sport in the world , played by men , women , and children of all ages . unfortunately , soccer has been plagued in recent years by deaths at the professional level during internationally televised games . this paper aims to review the physiology of soccer , the relationship between scd in soccer and other sports , the commonest causes of scd in young athletes , and the current screening guidelines in place by the fdration internationale de football association ( fifa ) for soccer . searching primarily the pubmed database , a search of american and international articles available in english was performed . there was no definite time period set due to the limited studies on scd and sport , but keywords were used to find peer - reviewed articles and case studies on the current reporting of soccer and scd . keywords used in the search were soccer , soccer cardiac , soccer and sudden cardiac death , and athlete sudden cardiac death . authors in the selected studies defined scd as cardiac arrest during or within 112 hours of competition [ 14 , 617 ] . a young competitive athlete was defined as an individual < 35 years old who participated in 2 h a week of physical training or participated in organized team or individual sports that required regular competition against others as a central component , placed a high premium on excellence and achievement , and required systematic and , in most instances , vigorous training [ 2 , 18 ] . while some studies included subjects over the age of 35 , the results focused on subjects from those studies under the age of 35 . this paper aimed to highlight the reported causes of scd in young competitive soccer players , as well as the most common causes of scd in young competitive athletes overall . the game of soccer relies on aspects of psychological , physiological , and biomechanical / technical skill . players at the highest levels of competition master all of these components , but players of all levels excel at certain skills over others . however , in recent years there has been a trend towards selecting players with more favorable anthropometric profiles to compete at the highest level . studies conducted by filming soccer players found that distances covered at the top level during a 90-minute game are about 1012 km for players on the field and 4 km for the goalkeeper . during a soccer match , sprinting occurs approximately every 90 seconds and lasts 24 seconds [ 20 , 21 ] . the game is dynamic considering that in addition to running , there is also twisting of the torso to change direction , heading , tackling , and holding the ball against defensive pressure . energy production while playing soccer is mainly dependent on aerobic metabolism ; however , the work intensity can approach the anaerobic threshold , which is defined as the highest exercise intensity where the production and removal of lactate are equal ( usually at 8090% of maximum heart rate ) . table 1 lists the cardiovascular diseases that have been reported in scd while playing soccer and other sports . the listed results focused on athletes under the age of 35 , since it is known that athletes over 35 experienced scd most commonly due to atherosclerotic cad [ 1 , 2 , 4 ] . several of the studies in table 1 report soccer as the most common sport associated with scd [ 1 , 3 , 4 , 6 ] . however , it is only the study done by corrado in the veneto region of italy that reported that the incidence of scd associated with soccer was not statistically significant when compared with other sports , nor was any sport associated with a specific form of fatal disease . in a study done on scd in spain , the subjects under 30 were not found to have any predominant cause of scd associated with soccer . in another worldwide study where soccer was found to be the most common sport associated with scd , the authors concluded that this was simply due to this sport being played more than others in regions from which the studies were reported and that in fact it was most likely the sports with high cardiovascular demand and isotonic work that put athletes at greatest risk for scd . on june 26 , 2003 , an internationally televised semifinal match was played for the fifa confederations cup between columbia and cameroon at stade de gerland in lyon , france . during the 72nd minute of the match , marc vivien fo , a 28 year - old veteran midfielder of the cameroon national soccer team collapsed on the center circle [ 23 , 24 ] . attempts to revive him with cpr were started on the field , and after 45 minutes of resuscitation efforts , he died shortly after arriving to the stadium 's medical center . on autopsy hcm is known to be the most common cause of scd in young athletes in the united states . it has a prevalence of 1 in 500 in several countries including usa , europe , japan , china , and east africa . the pathophysiology lies in autosomal dominant mutations in 11 or more genes encoding thick and thin contractile myofilament protein components of the sarcomere or the adjacent z - disc . suspicion for this diagnosis is suggested by cardiac symptoms , with the findings of a murmur or abnormal electrocardiogram . abnormal ecg patterns are present in the majority of hcm patients ( 7595% ) ; these findings include markedly increased r- or s - wave voltages , deep and prolonged q - waves , and deeply inverted t - waves . imaging findings show an absolute increase in the left ventricular wall thickness ( to 21 - 22 mm on average ) , which can also be associated with mild right ventricular hypertrophy . athletes who are suspected to have low - risk hcm can participate in leisurely sports with yearly followup ; qualifying criteria includes no scd in relatives , no symptoms , no lvh or ventricular arrhythmias , and normal diastolic filling / relaxation . athletes confirmed to have hcm with any of the aforementioned characteristics are to be restricted from competitive sports as it is believed that high - intensity sports predispose to subendocardial ischemia that leads to ventricular arrhythmias . a study by corrado showed that cca were associated with the highest risk of scd in young competitive athletes , and various studies have reported that cca are the second most common cause of scd in athletes under 35 , associated with 1525% of cases [ 1 , 2 , 4 , 2931 ] . table 1 lists case studies of scd in soccer that have been found to be due to cca on autopsy . studies have shown that the most common malformation reported in scd series both in the young and in the athlete is the origin of a coronary artery from a wrong aortic sinus of valsalva , either the right from the left coronary sinus or the left from the right coronary sinus , with a proximal course between the aorta and the pulmonary trunk . the diagnosis is most commonly confirmed by transthoracic echocardiography in children and is supplemented by mri and ct angiography . timely diagnosis of cca is critical because ( 1 ) athletes must be restricted from competitive activity to prevent scd and ( 2 ) cca are surgically correctable . on august 25 , 2007 , a soccer game between la liga spanish teams sevilla and , 22-year - old sevilla defender antonio puerta crouched next to the penalty box , then collapsed . he was substituted and managed to walk off the field , when he reached the locker room he collapsed again , and was pronounced dead at virgen del roco university hospital ; his autopsy revealed arvc . in the veneto region of italy , arvc is the cardiovascular disease that conveys the second highest risk of sports related sudden death , and in spain it was found to be a predominant pathology associated with scd in athletes < 30 years old [ 1 , 8 ] . it is currently estimated that disease prevalence is between 1 in 2000 and 1 in 5000 . arvc is characterized by structural and functional abnormalities of the right ventricle , ranging from regional wall motion abnormalities and ventricular aneurysms to global ventricular dilation and dysfunction ; it may also involve the left ventricle . symptoms of arvc include palpitations , syncope , cardiac arrest , or scd in adolescents or young individuals . the presence of t - wave inversions in v1-v3 or premature ventricular complexes ( pvcs ) of lbbb morphology on 12 lead ecg are the clues noted during cardiovascular screening . however , less commonly patients may present with what appears to be congestive heart failure ( chf ) due to dcm . patients with arvc should not under any circumstance be allowed to participate in competitive sports in order to prevent scd [ 28 , 36 ] . dundela f.c . is a northern irish intermediate - level professional soccer team . on august 25 , 1995 , the team 's captain michael goddard was struck on the chest by a ball and collapsed ; he was found to be in cardiac arrest and died shortly afterwards . commotio cordis is defined as when blunt trauma to the chest leads to ventricular fibrillation and therefore cardiac arrest ( most commonly during the t - wave upstroke on ecg , causing a pvc , which leads to ventricular fibrillation ) . due to a rise in reported incidents , a study recently done by maron compared the international cases of commotio cordis with those inside the us . the results of this study showed that in both groups commotio cordis occurred among young males and that resuscitation and defibrillation rates did not differ between us and non - us subjects . although a difference was found in the sports involved : in the us the sports most commonly involved were baseball / softball and american football , in non - us subjects the most common sport was soccer , followed by cricket and hockey . a notable finding in the international subjects was that in seven of the cases involving soccer , a traumatic blow caused by a soccer ball to the chest led to commotio cordis . this finding contradicts the previous notion that air - filled projectiles conveyed a lesser risk than those with a solid core ( baseballs , lacrosse balls ) . however , this study is limited by the fact that the data is predicated on incidents reported to the commotio registry and may not represent all cases of commotio cordis worldwide . in the aforementioned studies reviewing scd and sport , there has always been a percentage of the sample in which the cause of scd could not be determined by autopsy . however , the reports have been largely variable , ranging anywhere from 1% to 16.3% [ 1 , 2 , 4 , 7 , 8 ] . a study in the uk reported soccer as the most common sport associated with scd in ages 1135 , but the autopsies of athletes from all sports had a morphologically normal heart in 23% of cases . studies done in the us and uk with the families of an individual who experienced scd found that in 4050% of families studied , relatives had an ion channel disorder such as catecholaminergic polymorphic ventricular tachycardia , long qt syndrome , and brugada syndrome [ 39 , 40 ] . in soccer , like all other sports , doping has become a relevant issue , as evidenced in the 1994 world cup when the argentinian superstar diego maradona was expelled from the tournament after testing positive for several banned substances . however , the studies that have been mentioned either excluded toxicological confounders as a cause for scd , or had such a small percentage of cases that they were not addressed [ 1 , 2 , 68 ] . androgen abuse has been shown to have the direct effects of cardiac hypertrophy and myocardial fibrosis , and indirect effects of hypertension , dyslipidemia , arrhythmia , and myocardial infarction . one study reviewing the autopsy findings of four body builders who experienced scd found cardiac hypertrophy and fibrosis in the myocytes of these subjects . other medications that may play a role in soccer players ' scd include but are not limited to nsaids , antihistamines , and herbal supplements . of note , cox-2-selective nsaids prescribed for musculoskeletal and arthritic complaints have been shown to have an increased risk of adverse cardiac events . furthermore , some second - generation antihistamines such as terfenadine and astemizole have been shown to reach high serum levels through drug and food interactions thereby predisposing to qt prolongation and ventricular arrhythmias . the studies reviewed above show that in italy and much of the rest of the world , the sport in which scd occurs most often is soccer [ 1 , 6 ] . however , in the united states , the sports most commonly associated with scd are basketball and american football . therefore , it is reasonable to conclude that the regional difference in scd and sport are likely due to the most common sport being played and that it is the increased cardiovascular demand that predisposes to scd rather than the sport itself . this study is limited in that most of the studies reviewed do not report whether there is in fact a correlation between soccer and a form of scd . however , the one study that did assess this variable was the study done in the veneto region of italy , which found no statistical significance between the incidence of scd in soccer when compared with other sports , nor did it find a relationship between soccer and an underlying cause of scd . also , while the sample was considerably smaller , the study of scd in spain also did not find a predominant cause of scd associated with soccer . the most common cardiac abnormality implicated in scd in young athletes < 35 years old in the united states is hcm , followed by cca and ion channelopathies , while in italy the most common causes of scd in young athletes were cca , arvc , and premature coronary artery disease . a study of scd in spain showed arvc to be the most common cause ; however , in the uk isolated lvh ( confirmed by microscopy to not have myocyte disarray ) was most common , followed by arvc [ 6 , 8 ] . these results show that there may be a greater prevalence of arvc in european nations compared to the united states . it is worth mentioning that preparticipation screening with ecgs in italy is suspected to be the reason why there is a decreased incidence of scd due to increased identification and management of hcm . reviewed studies around the world and also found cca and hcm to be the most common causes of scd but suggested that these pathologies may be the most common because they are the easiest to identify , and that more occult etiologies such as arrhythmias and arvc may be underrepresented . as noted previously , in each of the studies discussed there was a percentage of the sample in which no cause was found [ 1 , 2 , 4 , 68 ] . moreover , given the high prevalence of ion channel disorders found in family members of individuals who experienced scd , more research is needed on the efficacy of screening the relatives of these individuals and prevention of scd [ 39 , 40 ] . while most studies excluded positive toxicological findings , the cardiac effects of steroids and other medications may predispose to scd [ 4345 ] . since soccer players stereotypically have a slender build for speed and agility , steroid use may seem counterintuitive , but some players have tested positive [ 41 , 46 ] . fifa which is the worldwide governing body of soccer , has taken a staunch stance against doping and in 2001 suspended legendary dutch players edgar davids and frank de boer after testing positive for the anabolic steroid nandrolone . in evaluating these studies , sampling bias is an issue considering that the research on scd is based on autopsy results from reported cases , and there is the possibility that cases may go unreported . there is a need for a mandatory reporting database of these incidents in the united states and other countries . until there is better reporting of these incidents , the true incidence and etiology of scd in soccer and other sports will remain unknown . the current guidelines for preparticipation screening have not reached a consensus ; in 2004 and 2005 the european society of cardiology and international olympic committee published notably similar guidelines , which contrasted the american guidelines [ 4 , 28 ] . the main difference was the addition of a 12-lead ecg to the history and physical examination . this decision had been based on the study done by corrado that showed a significantly decreased incidence of scd due to hcm in the italian population . however , the low specificity of ecg as a screening tool in an athletic population is a major disadvantage for its use [ 4749 ] . in 2005 , fifa took action to prevent scd in soccer . prior to the 2006 world cup in germany , the fifa medical assessment and research centre developed and implemented a comprehensive precompetition medical assessment tailored specifically to this population . the cardiovascular screening included a personal and family history , physical examination , a 12-lead resting ecg , as well as an exercise ecg and an echocardiogram . the results showed that cardiovascular preparticipation screening in international elite soccer teams seemed appropriate and that while ecg and echocardiography with further standardization could be useful , exercise stress testing remained questionable . moreover , it was previously believed that preparticipation screening distressed soccer players due to the fear of being removed from competition . however , a study done with norwegian professional soccer players found that the players felt more confident after screening and would recommend it to other players . future studies on scd in soccer and other sports involves more detailed reporting of scd by cause and associated sport . there was a case by zeller et al . in table 1 of scd in a 26-year - old soccer player whose only suggestive finding was marked early repolarization on ecg . furthermore , a recent study by lengyel reported a statistically significant qt prolongation at rest in professional soccer players when compared to aged matched controls . future studies need to be directed towards identifying further cardiac risk factors that may lead to scd in soccer players . after the death of marc vivien foe in 2003 , fifa reacted by making it mandatory to equip all stadiums with automated external defibrillators ( aeds ) as well as to have available medical and paramedical personnel who are able to manage emergencies such as cardiac arrest . a remarkable result of these initiatives can be attributed to the case the 24-year - old fabrice muamba , who on march 17 , 2012 , collapsed on the field during an internationally televised game between bolton and tottenham of the english premier league . resuscitation began on the field and muamba is reported to have been in cardiac arrest for 78 minutes and to have received a total of 15 defibrillator shocks . while he has now retired from soccer , muamba has made a full recovery with no neurological deficits , a medical miracle likely related to the onsite aed and well trained personel rapidly responding to this emergency . because soccer is the most commonly played sport worldwide , more of those considered at risk experienced an episode of scd while playing the sport . several studies reported that soccer was the most common sport associated with scd , and that the causes of scd were similar for soccer as among sporting activities in general ( hcm , cca , and arvc ) . therefore , we conclude that preparticipation screening in soccer players should focus some effort on screening for those structural and/or electrical abnormalities associated with these conditions . given the popularity of the sport worldwide and recent deaths of soccer players on the field , further research on this topic is encouraged .

sudden cardiac death ( scd ) in young competitive athletes ( < 35 years old ) is a tragic event that has been brought to public attention in the past few decades . the incidence of scd is reported to be 1 - 2/100,000 per year , with athletes at a 2.5 times higher risk . soccer is the most popular sport in the world , played by people of all ages . however , unfortunately it is cardiovascular diseases such as hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy that have subtly missed screening and claimed the lives of soccer stars such as marc vivien foe and antonio puerta during live action on the field and on an internationally televised stage . this paper covers the physiological demands of soccer and the relationship between soccer and scd . it also reviews the most common causes of scd in young athletes , discusses the current guidelines in place by the fdration internationale de football association ( fifa ) for screening among professional soccer players , and the precautions that have been put in place to prevent scd on the field in professional soccer .

this procedure can be performed in 500 l siliconized tubes or 6 ml polystyrene round - bottom facs tubes . the latter is normally used when carrying out viability analysis in conjunction with other procedures . all volumes given are for 6 ml polystyrene round - bottom facs tubes . for 500 l siliconized tubes , the optimum concentration for flow cytometry analysis is 2 - 4 x 10 cells per 200 l volume . cell loss may result from this procedure and thus we recommend that each sample consist of 4 x 10 cells at the start of the procedure . harvest cells - refer to specific procedures for corresponding cell lines or primary cell isolations . resuspend cells in 2 ml 1 x phosphate buffered saline ( pbs ) ( no calcium , no magnesium ) . add annexin v according to the manufacturer 's recommendations [ e.g. 5 l annexin v alexa fluor 488 ( molecular probes , a13201 ) ] . add 4 l of pi ( sigma , cat # p-4864 - 10ml ) that has been diluted 1:10 in 1 x annexin v binding buffer ( i.e. 1 l pi with 9 l 1 x annexin v binding buffer ) . this will yield a final pi concentration of 2 g / ml in each sample . resuspend cells in 500 l 1 x annexin v binding buffer and 500 l 2% formaldehyde to create a 1% formaldehyde ( fixative ) solution . mix tubes by gentle flicking . if the latter method is chosen , make sure to be consistent across all tubes labeled with annexin v / pi ( including compensation controls ) . centrifuge tubes at 425 x g for eight minutes and decant the supernatant . repeat steps 2.10 and 2.11 . add 16 l of 1:100 diluted rnase a ( sigma , r4642 ) to give a final concentration of 50 g / ml . incubate for 15 min at 37c . alternatively , samples can be used for subsequent staining steps if annexin / pi staining is being performed in parallel with other procedures . examples of cell types and cell lines that have varying degrees of false - positive pi staining are shown in figure 1 . to account for the false - positive events , cells were fixed and then treated with rnase a. this results in a significant decrease in the number of false - positive events , compared to cells that did n't undergo rnase treatment ( figure 2b ) . figure 2c shows representative images of cells that underwent rnase treatment , compared to cells that had no rnase treatment . figure 3 shows how the modified annexin v / pi protocol , with fixation and rnase treatment steps , improves upon conventional protocols by limiting the number of false - positive staining events . we have previously evaluated extent of false positive staining across primary cells across a broad range of animal models as well as in a variety of cell lines . representative images show extent of false positive staining in three unique cell populations ( one commonly used cell line - raw macrophages and two primary cells - murine bone marrow macrophages ( bmm ) and goldfish primary kidney macrophages ( pkm ) ) . true positive events display the expected co - localization between pi and draq5 within the nuclear compartment ( yellow areas depict colocalization between pi and draq5 ) . draq5 is highly membrane permeable dye that accurately stains nuclear compartment in both live and dead cells . for easier visual determination of colocalization draq5 stain ( a ) we assessed accuracy of pi nuclear staining based on degree of similarity of a number of well - characterized nuclear stains ( brdu , 7-aad and dapi ) to draq5 . brdu is a synthetic nucleoside that is incorporated into the dna of proliferating cells , and as such will only stain within the nuclear compartment . 7-aad has a high affinity for dna and , similar to pi , can not cross an intact plasma membrane . dapi also has a strong affinity for dna and is the most commonly used nuclear stain in fluorescent microscopy . the degree of similarity was > 99% between brdu , 7-aad , dapi and draq5 , highlighting their ability to accurately stain the cell nucleus in raw 264.7 macrophages . in contrast , cytoplasmic pi staining based on conventional protocols leads to a marked decrease in percent similarity of staining relative to draq5 . ( b ) similar results are observed in two primary cells tested : murine bone marrow macrophages ( bmm ) and goldfish primary kidney macrophages ( pkm ) . incorporation of 50 g / ml rnase a at specific stage within the staining procedure removes non - nuclear pi staining and significantly increases degree of similarity relative to draq5 staining . ( c ) representative images of primary kidney macrophages show the degree of similarity for cells with and without rnase treatment . for easier visual determination of differences between treatments primary goldfish kidney macrophages ( pkm ) were stained with conventional and modified annexin v / pi protocols . the scatterplot on the left shows conventional annexin v / pi staining ( no rnase ) of pkm cells . the scatterplot on the right shows pkm cells stained with the modified annexin v / pi protocol ( with rnase ) . the addition of rnase results in a marked reduction of pi staining due to the removal of false positive stains . pkm cells were then analyzed based on distinct populations within the cultures- early progenitors ( ep ) , monocytes ( mono ) and mature macrophages ( mat mac ) . the reduction in the number of positive pi events , due to removal of false positive events is more pronounced in large mature macrophage cells than in smaller early progenitor cells . conclusions based on conventional protocols would have erroneously suggested a positive correlation in cellular death for more mature macrophage subsets . harvest cells - refer to specific procedures for corresponding cell lines or primary cell isolations . resuspend cells in 2 ml 1 x phosphate buffered saline ( pbs ) ( no calcium , no magnesium ) . add annexin v according to the manufacturer 's recommendations [ e.g. 5 l annexin v alexa fluor 488 ( molecular probes , a13201 ) ] . add 4 l of pi ( sigma , cat # p-4864 - 10ml ) that has been diluted 1:10 in 1 x annexin v binding buffer ( i.e. 1 l pi with 9 l 1 x annexin v binding buffer ) . this will yield a final pi concentration of 2 g / ml in each sample . resuspend cells in 500 l 1 x annexin v binding buffer and 500 l 2% formaldehyde to create a 1% formaldehyde ( fixative ) solution . if the latter method is chosen , make sure to be consistent across all tubes labeled with annexin v / pi ( including compensation controls ) . centrifuge tubes at 425 x g for eight minutes and decant the supernatant . repeat steps 2.10 and 2.11 . add 16 l of 1:100 diluted rnase a ( sigma , r4642 ) to give a final concentration of 50 g / ml . incubate for 15 min at 37c . alternatively , samples can be used for subsequent staining steps if annexin / pi staining is being performed in parallel with other procedures . examples of cell types and cell lines that have varying degrees of false - positive pi staining are shown in figure 1 . to account for the false - positive events , cells were fixed and then treated with rnase a. this results in a significant decrease in the number of false - positive events , compared to cells that did n't undergo rnase treatment ( figure 2b ) . figure 2c shows representative images of cells that underwent rnase treatment , compared to cells that had no rnase treatment . figure 3 shows how the modified annexin v / pi protocol , with fixation and rnase treatment steps , improves upon conventional protocols by limiting the number of false - positive staining events . we have previously evaluated extent of false positive staining across primary cells across a broad range of animal models as well as in a variety of cell lines . representative images show extent of false positive staining in three unique cell populations ( one commonly used cell line - raw macrophages and two primary cells - murine bone marrow macrophages ( bmm ) and goldfish primary kidney macrophages ( pkm ) ) . true positive events display the expected co - localization between pi and draq5 within the nuclear compartment ( yellow areas depict colocalization between pi and draq5 ) . draq5 is highly membrane permeable dye that accurately stains nuclear compartment in both live and dead cells . for easier visual determination of colocalization draq5 stain ( a ) we assessed accuracy of pi nuclear staining based on degree of similarity of a number of well - characterized nuclear stains ( brdu , 7-aad and dapi ) to draq5 . brdu is a synthetic nucleoside that is incorporated into the dna of proliferating cells , and as such will only stain within the nuclear compartment . 7-aad has a high affinity for dna and , similar to pi , can not cross an intact plasma membrane . dapi also has a strong affinity for dna and is the most commonly used nuclear stain in fluorescent microscopy . the degree of similarity was > 99% between brdu , 7-aad , dapi and draq5 , highlighting their ability to accurately stain the cell nucleus in raw 264.7 macrophages . in contrast , cytoplasmic pi staining based on conventional protocols leads to a marked decrease in percent similarity of staining relative to draq5 . ( b ) similar results are observed in two primary cells tested : murine bone marrow macrophages ( bmm ) and goldfish primary kidney macrophages ( pkm ) . incorporation of 50 g / ml rnase a at specific stage within the staining procedure removes non - nuclear pi staining and significantly increases degree of similarity relative to draq5 staining . ( c ) representative images of primary kidney macrophages show the degree of similarity for cells with and without rnase treatment . primary goldfish kidney macrophages ( pkm ) were stained with conventional and modified annexin v / pi protocols . the scatterplot on the left shows conventional annexin v / pi staining ( no rnase ) of pkm cells . the scatterplot on the right shows pkm cells stained with the modified annexin v / pi protocol ( with rnase ) . the addition of rnase results in a marked reduction of pi staining due to the removal of false positive stains . pkm cells were then analyzed based on distinct populations within the cultures- early progenitors ( ep ) , monocytes ( mono ) and mature macrophages ( mat mac ) . the reduction in the number of positive pi events , due to removal of false positive events is more pronounced in large mature macrophage cells than in smaller early progenitor cells . conclusions based on conventional protocols would have erroneously suggested a positive correlation in cellular death for more mature macrophage subsets . the annexin v / pi protocol presented here is a modified version of conventional protocols and takes into account the presence of rna in the cytoplasm which also has high affinity for pi . introduction of rnase a ( 50 g / ml ) following a 1% formaldehyde fixation step late in the staining procedure significantly improves accuracy of nuclear pi staining . no negative effects are observed in nuclear pi staining or plasma membrane annexin v staining . without rnase a treatment , up to 40% of pi stain results can lead to false positive events , which leads to a potentially significant and negative impact on downstream conclusions . our modified annexin v / pi staining protocol is simple and has been used effectively in a broad range of cell types ( primary cells : murine bone marrow macrophages and splenocytes ; swine lung resident cells , lung alveolar macrophages , mesenteric lymph node isolates , peripheral blood leukocytes , splenocytes ; chicken blastoderm cells ; goldfish primary kidney macrophages ; carp peripheral blood leukocytes ; cell lines : raw 264.7 macrophages , jurkat t cells , swine 3d4/31 macrophages , ccl-71 goldfish fin fibroblasts , 3b11 catfish b cells ) . it is important to note that cells are differentially affected by false positive pi staining , with primary cells generally having a greater number of false positive events . the differences false positive pi staining among these cellular populations may be partially attributed to differences in cell size , but may also result from differences in rna content . as such , incorporation of this procedure is particularly relevant for experimental systems that utilize , among others , cells undergoing genotoxic stress , cells treated with cell cycle arrest drugs such as thymidine or hydroxyurea , virally - infected cells , and studies on embryonic cells where developmental progression is characterized by discrete changes in cellular rna synthesis .

studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry - based methods . propidium iodide ( pi ) is widely used in conjunction with annexin v to determine if cells are viable , apoptotic , or necrotic through differences in plasma membrane integrity and permeability1,2 . the annexin v/ pi protocol is a commonly used approach for studying apoptotic cells3 . pi is used more often than other nuclear stains because it is economical , stable and a good indicator of cell viability , based on its capacity to exclude dye in living cells 4,5 . the ability of pi to enter a cell is dependent upon the permeability of the membrane ; pi does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6 . in late apoptotic and necrotic cells , the integrity of the plasma and nuclear membranes decreases7,8 , allowing pi to pass through the membranes , intercalate into nucleic acids , and display red fluorescence 1,2,9 . unfortunately , we find that conventional annexin v/ pi protocols lead to a significant number of false positive events ( up to 40% ) , which are associated with pi staining of rna within the cytoplasmic compartment10 . primary cells and cell lines in a broad range of animal models are affected , with large cells ( nuclear : cytoplasmic ratios < 0.5 ) showing the highest occurrence10 . herein , we demonstrate a modified annexin v/ pi method that provides a significant improvement for assessment of cell death compared to conventional methods . this protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of rnase a into cells following staining . both the timing and concentration of rnase a have been optimized for removal of cytoplasmic rna . the result is a significant improvement over conventional annexin v/ pi protocols ( < 5% events with cytoplasmic pi staining ) .

meibomian gland dysfunction ( mgd ) is considered to be a discrete disease entity without prominent inflammatory alterations of the lid margins and a frequent cause of wetting deficiencies of the ocular surface leading to dry eye disease . obstructive mgd is characterized by hyperkeratinization of the ductal epithelium and increased viscosity of the meibum resulting in obstruction of the meibomian gland duct and orifice [ 1 , 2 ] . obstructive mgd is reported to be much more frequent in the general population and increases with age . age , hormonal disturbances , and environmental influences have been considered in the pathogenesis of both obstructive and seborrheic mgd . the effect of androgens on meibomian gland function has been studied in a group of patients with an average of 70.9 years , and androgen deficiency is considered as a critical factor in the pathogenesis of mgd and dry eye [ 4 , 5 ] . very little information exists concerning the correlation of serum levels of sex hormones , thyroid hormones , thyroglobulin , cortisol , and prolactin with seborrheic mgd between 2030 years of age . the purpose of this study is to compare the serum levels of dehydroepiandrosterone sulphate ( dhea - s ) , testosterone , estrodiol , 17-hydroxyprogesterone ( 17-oh - prog ) , prolactin , follicle - stimulating hormone ( fsh ) , luteinizing hormone ( lh ) , thyroid - stimulating hormone ( tsh ) , bound and unbound thyroid hormones ( t3 , t4 ) , thyroglobulin , and cortisol between patients with seborrheic mgd and controls in a gender - based design between 2030 years of age . this is a retrospective case - control study involving 50 patients , 31 male and 19 female with a mean standard deviation ( sd ) of 23.92 5.73 years , and 50 controls 19 male and 31 female with a mean age of 23.36 4.61 years . the study is approved by the human studies committee of the middle east technical university ( ankara , turkey ) and were conducted in accordance with guidelines established by the declaration of helsinki . ocular symptoms including ocular fatigue , discharge , foreign body sensation , dryness , discomfort , sticky sensation , pain , epiphoria , itching , redness , heavy sensation , glare , excessive blinking , and history of chalazion or hordeolum were scored from 0 to 14 according to the number of symptoms present . lid margin abnormalities including irregular lid margin , vascular engorgement , plugged meibomian gland orifices , and anterior or posterior replacement of mucocutaneous junction , were scored from 0 to 4 depending on the number of abnormalities present . superficial punctate keratopathy ( spk ) in the cornea was scored from 0 to 3 [ 3 , 6 ] . the tear film break - up time ( but ) was measured consecutively three times after instillation of fluorescein 1% ( fluorescite injection ) and the median value was adopted . assessment of meibomian gland function was done by using two techniques : observation of meibomian gland orifices by using biomicroscopy and transillumination observation techniques by using a light probe ( meibography ) [ 7 , 8 ] . meibography was performed by using a transillumination device for vitrectomy with a 20-gauge fiberoptic light probe ( bausch and lomb millennium , rochester , ny ) including tungsten - halogen ad metal halide . meiboscore for upper and lower eyelids was used as previously described : grade 0 ( no loss of meibomian glands ) , grade 1 ( loss of less than one - third of the total area of the meibomian glands ) , grade 2 ( loss of between one - third and two - thirds of the total area ) , and grade 3 ( loss of over two - thirds of the total area ) [ 7 , 8 ] . meibomian gland expression was evaluated by applying moderate digital pressure on the tarsus of the upper eyelid , and the degree of ease with which meibum secretion was induced was evaluated as grade 0 ( clear meibum , easily expressed ) , grade 1 ( cloudy meibum , expressed with mild pressure ) , grade 2 ( cloudy meibum expressed with more than moderate pressure ) , and grade 3 ( no meibum expressed even with hard pressure ) . a diagnosis of seborrheic mgd was made on the basis of an ocular symptom score of 3 or more and a lid margin score of 2 or more [ 3 , 6 ] . patients with meiboscore 1 or more , a schirmer value of 5 mm or less , the tear break - up time of less than 5 seconds , and spk score of 1 or more , were excluded from the study . exclusion criteria for both groups include ocular allergies , contact lens wearing , history of eye surgery , and systemic or ocular diseases that might interfere tear film production or function . patients displayed ocular symptoms for at least 3 months and received no topical or systemic therapy for at least 4 weeks . morning fasting values of testosterone , lh , fsh , dhea - s , 17-oh - prog , tsh , bound and unbound t3 and t4 , prolactin , thyroglobulin , and cortisol were determined in 1 milliliter ( ml ) serum of patients and controls on the same day by using vidas pc ( biomerieux , france ) with macroelisa technique . the mean score of ocular symptoms of patients with seborrheic mgd was 9.48 over 14.0 , and the mean score of lid margin abnormalities was 2.98 over 3.0 . the tear film but was in the normal range , and there were no loss of the meibomian glands in both groups . the patients had the score of meibomian gland expression either grade 0 or grade 1 . table 1 discloses mean and standard deviation ( sd ) of serum levels of tsh , bound and unbound t3 and t4 , fsh , lh , 17-oh - prog , estrodiol , prolactin , testosterone , dhea - s , cortisol , and thyroglobulin of the patients and controls in both genders . the mean ( sd ) of serum testosterone level was 2.27 ( 2.72 ) ng / ml in controls and 4.78 ( 3.67 ) ng / ml in patients ( table 1 ) . the data mean ( sd ) of serum testosterone level in both genders of patients and controls was 3.52 ( 3.45 ) ( table 4 ) . there was statistically significant difference of mean serum values of testosterone in both genders with respect to patients with seborrheic mgd and controls ( p = 0.000 , 95% confidence interval ( ci ) 1.22 , 3.79 ; table 4 ) . the mean ( sd ) of serum dhea - s level was 370.70 ( 146.50 ) g / ml in patients with seborrheic mgd and 238.50 ( 97.72 ) g / ml in controls ( table 1 ) . the data mean ( sd ) of serum dhea - s level in both genders of patients and controls was 304.6 ( 140.6 ) g / ml ( table 4 ) . there was statistically significant difference of mean serum values of dhea - s in both genders with respect to patients with seborrheic mgd and controls ( p = 0.000 , 95% ci 82.78 , 181.63 ; table 4 ) . table 2 discloses the mean ( sd ) of serum hormone levels in female gender with respect to patients with seborrheic mgd and controls . the data mean of tsh in female gender of both patients and controls was 1.95 ( 1.17 ) iu / ml ( table 4 ) . the mean value of tsh in female controls was 1.64 ( 0.89 ) iu / ml , and in female patients with seborrheic mgd it was 2.459 ( 1.40 ) iu / ml . there was statistically significant difference of mean serum values of tsh in female gender with respect to patients with seborrheic mgd and controls ( p = 0.014 , 95% ci 0.17 , 1.48 ; table 4 ) . the data mean of serum prolactin level in female gender of both patients and control was 19.97 ( 9.74 ) ng / ml ( table 4 ) . the mean value of prolactin in female controls was 17.79 ( 7.45 ) ng / ml and in female patients with seborrheic mgd was 23.51 ( 12.0 ) ng / ml ( table 2 ) . there was statistically significant difference of mean serum values of prolactin in female gender with respect to patients with seborrheic mgd and controls ( p = 0.043 , 95% ci 0.19 , 11.24 ; table 4 ) . the data mean of serum testosterone level in female gender of both patients and controls was 0.49 ( 0.29 ) ng / ml ( table 4 ) . the mean value of testosterone in female controls was 0.37 ( 0.16 ) ng / ml and in female patients with seborrheic mgd was 0.69 ( 0.08 ) ng / ml ( table 2 ) . there was statistically significant difference of mean serum values of testosterone in female gender with respect to patients with seborrheic mgd and controls ( p = 0.001 , 95% ci 0.15 , 0.49 ; table 4 ) . the data mean of serum dhea - s level in female gender of both patients and controls was 255.20 ( 122.70 ) g / ml . the mean value of dhea - s in female controls was 212.07 ( 86.32 ) g / ml and in female patients with mgd was 325.57 ( 141.78 ) g / ml ( table 2 ) . there was statistically significant difference of mean serum values of dhea - s in female gender with respect to patients with seborrheic mgd and controls ( p = 0.001 , 95% ci 48.80 , 178.19 ; table 4 ) . table 3 shows the mean ( sd ) of serum hormone levels in male gender with respect to the patients with seborrheic mgd and controls . the data mean of serum testosterone level in male gender of both patients and controls was 6.56 ( 2.28 ) ng / ml ( table 4 ) . the mean value of serum testosterone in male controls was 5.376 ( 1.91 ) ng / ml and in patients with seborrheic mgd was 7.281 ( 2.20 ) ng / ml ( table 3 ) . there was statistically significant difference of mean serum values of testosterone in male gender with respect to patients with seborrheic mgd and controls ( p = 0.003 , 95% ci 0.68 , 3.14 ; table 4 ) . the data mean of serum dhea - s level in male gender of both patients and controls was 354.0 ( 141.10 ) g / ml . the mean value of serum dhea - s level in male patients with seborrheic mgd was 398.38 ( 144.6 ) g / ml , and in controls was 281.63 ( 102.0 ) g / ml . there was statistically significant differences of mean serum levels of dhea - s in male gender with respect to patients with seborrheic mgd and controls ( p = 0.003 , 95% ci 40.42 , 193.07 ; table 4 ) . no statistically significant differences of mean serum levels of the hormones including bound and unbound t3 and t4 , fsh , lh , 17-oh - prog , estrodiol , cortisol , and thyroglobulin in both genders were found between the patients with seborrheic mgd and controls ( p > 0.05 ) . this is the first time a case - control comparative study has been undertaken to assess the endocrine aspects of seborrheic mgd in a gender - based design . it represents the first report of increased serum levels of testosterone and dhea - s in both genders with seborrheic mgd and increased serum levels of tsh and prolactin only in female gender with seborrheic mgd . androgens ( testosterone ) are reported to control meibomian gland function , regulate the quality and/or quantity of lipids produced by this tissue , and promote the formation of the tear film 's lipid layer [ 5 , 11 ] . previous studies related to androgen deficiency revealed significant and striking alterations in the lipid patterns of meibomian gland secretions , and it was considered to be an important etiologic factor in the pathogenesis of evaporative dry eye [ 4 , 5 ] . however , testosterone excess is considered to provoke or aggravate seborrhea at a significance rate of p < 0.01 . the effect of androgens on human skin is reported to increase sebaceous gland growth and differentiation , produce acne and seborrhea . we demonstrated statistically significant increase of serum levels of testosterone in both genders with seborrheic mgd with respect to control genders . the mean score of ocular symptoms of the patients with seborrheic mgd was 9.48 over 14.0 , and the mean score of lid margin abnormalities was 2.98 over 4.0 . the value of serum testosterone was considered to be a significant factor affecting the severity of seborrehic mgd in both genders . adrenal glands secrete large amounts of dhea and dhea - s which are then converted into potent androgens ( testosterone and dehydrotestosterone ) or estrogens by stereogenic enzymes in the peripheral sites that permit target tissues to adjust the formation and metabolism of androgens and estrogens to local requirements . increased serum levels of dhea - s have been reported in patients with seborrheic dermatitis , acne vulgaris , alopecia , and hirsutism [ 1517 ] . the aging process is paralleled by a dramatic decline in the serum concentrations of dhea and dhea - sulphate . in addition , there is a marked decline in the circulating levels of dhea in postmenopausal women . our study includes the patients with seborrheic mgd at young age , mean age of 23 , and age - matched controls . we demonstrate significant increase in serum levels of dhea - s in both genders of patients with respect to control genders . we consider increase in serum values of dhea - s in patients with seborrheic mgd could be used as diagnostic marker , and could be correlated with severity of the disease . estrogens , glucocorticoids , and prolactin are also considered to influence sebaceous gland function by stimulating proliferation of sebocyte [ 19 , 20 ] . our study revealed significant increase in serum levels of prolactin in female gender of patients with seborrheic mgd as compared to control gender . however , there was no significant increase in serum levels of prolactin in male patients with seborrheic mgd as compared to control gender . our study revealed significant increase serum levels of tsh only in female gender of the patients with seborrheic mgd with respect to the control gender . the correlation of serum levels of tsh with seborrheic mgd also remains to be determined in larger cohorts . no significant difference of serum levels of other hormones including bound and unbound t3 and t4 , fsh , lh , progesteron , estrodiol , cortisol and thyroglobulin were found between patients with seborheic mgd and controls in both genders . in conclusion : increased serum levels of testosterone and dhea - s in both genders should be considered as diagnostic markers for seborrheic mgd and may affect severity of the disease . however , the correlations of serum levels of tsh and prolactin with seborrheic mgd need to be further investigated .

purpose . to compare the hormone levels of patients with seborrheic meibomian gland dysfunction with controls . procedures . this is a retrospective case - control study involving 50 patients and 50 controls . blood workup for hormones was studied in both groups by using macroelisa ( enzyme - linked immunosorbent assay ) . statistical evaluation was done by using spss 15.0 independent samples t - test . results . there were statistically significant differences of serum testosterone and dehydroepiandrosterone sulphate levels between patients and controls ( p = 0.000 ) . female gender showed statistically significant differences of serum thyroid - stimulating hormone and prolactin levels between patients and controls ( p = 0.014 and p = 0.043 ) , in addition to serum testosterone and dehydroepiandrosterone sulphate levels ( p = 0.000 and p = 0.001 ) . however , male gender showed statistically significant differences of only serum testosterone and dehydroepiandrosterone sulphate levels between patients and controls . ( p = 0.003 and p = 0.003 resp . ) . conclusions . increased serum levels of testosterone and dehydroepiandrosterone sulphate in both genders should be considered as diagnostic markers for seborrheic meibomian gland dysfunction .

amyand 's hernia is an extremely rare condition in which the inguinal hernia sac contains the appendix . this unusual situation is estimated to occur in approximately one percent of adult inguinal hernia cases . we describe a case of acute appendicitis in the inguinal hernia sac with an incarceration in the external ring ; this led to distal appendicitis even though the appendix had a healthy body and base . our report includes a review of the literature and further evaluation of predisposing factors in this case . an 85-year - old - male arrived at our hospital in 2006 presenting a right groin mass . his history included hypertension , coronary artery disease , and transient infarction accident , of which all were receiving regular medical treatment . additionally , he had recently experienced urinary frequency and nocturia . a right groin mass had been protruding for 1 month prior to hospital admission , which increased in size when standing and before stool passage , but decreased in size after stool passage or lying down . the patient was scheduled for elective surgery . the oblique conventional incision between external and internal rings swelling and induration protruded outside the external ring toward the scrotum , leaving a mark on the vermiform organ ( fig . the body and base of the appendix was healthy and a moderate amount of clear ascites was found in the hernial sac . the distal portion of the appendix was attached to the distal portion of the hernial sac , which lay outside the external ring of the right groin . the mobilized cecum and ascending colon were far away from the paracolic space , apparently sliding until occupying the neck of the hernial sac . the patient 's postoperative condition was uneventful and he was discharged on the next day . he was followed up at our opd one week later and the right groin looked good . pathology revealed an acute suppurative appendicitis with mixed inflammatory cells in the appendiceal wall , presenting many eosinophils and small foci of granulomatous lesion ( fig . this rare condition was named after the first surgeon to perform appendectomy , claudius amyand , an english surgeon of the 18th century who first described a case of acute appendicitis in a hernial sac in an 11-year - old child . the first appendectomy ever to be performed in the usa , more than a century later , was also a case of amyand 's hernia in which an abscess with gangrenous appendicitis was found inside the hernial sac . the incidence of amyand 's hernia is estimated to occur in approximately one percent of adult inguinal hernia repair cases . acute appendicitis occurs much less frequently , and perforated appendix and periappendicular abscess formation within an inguinal hernia sac is an extremely rare clinical entity . ryan presented 11 of 8,692 cases of acute appendicitis occurring in external hernia of all forms . reviewed seven cases of acute appendicitis in a series of external hernia ( three inguinal ) collected in 8 years . a detailed description of the historical background and the management of amyand 's hernia was presented by r. hutchinson in 1993 . other investigators have infrequently presented case reports and literature reviews [ 7 , 8 ] . the incidence of acute appendicitis occurring in a hernial sac varies between the 0.13% reported by ryan et al . in 1937 and the 1% reported by carey in 1967 . more recently , c. d'alia and his colleagues reported their unique case of acute appendicitis within the hernial sac in 1,341 inguinal hernia operations performed over 13 years in messina university hospital in 2003 . most reported cases of amyand 's hernia have occurred in the right inguinal region and only a few reported cases have occurred in the left inguinal region [ 8 , 11 ] . the present case , diagnosed during surgery , is the first case of amyand 's hernia in our hospital , a 40-year - old district hospital with 800 beds . acute appendicitis only occurred over the tip of the appendix located outside the external ring . the blood supply was affected by the narrowed external ring and distal appendicitis developed subsequently . the body and base of the appendix appeared to be healthy ; the wide base of the hernial sac in the internal ring caused the vermiform organ to have a strange appearance . neither turbid ascites nor pus formation was found inside the hernial sac , making an excellent prognosis . we preferred to perform the repair via the traditional procedure ( bassini 's method ) to prevent the possibility of infection by the insertion of a foreign body ( mesh ) . treatment procedures have been well discussed in many recent case reports , including mesh hernioplasty , endoscopic hernioplasty or the traditional method of hernioplasty , although controversy still exists . sharma et al . briefly discussed the management of amyand 's hernia among 18 patients in a 15-year period , including appendectomy followed by bassini 's repair , mesh hernioplasty after reduction of a normal appendix , and bassini 's hernia repair plus a lower midline laparotomy for pelvic washout . discussions in recent reports emphasized the incidence and management of amyand 's hernia , highlighting the rarity and causes of the abnormality . abu - dalu and urca have suggested that the appendix becomes more vulnerable to trauma in amyand 's hernia and is ultimately retained by adhesions . its blood supply may subsequently be cut off or significantly reduced , resulting in inflammation and bacterial overgrowth . contraction of the abdominal muscles and other sudden increases in intra - abdominal pressure may cause compression of the appendix , resulting in further inflammation . apart from the above opinions , we found a mobilized cecum and ascending colon in our case , allowing facile advancement of the appendix into the hernial sac . the tip of the appendix proceeded through the narrowed external ring and incarceration developed , followed by adhesion and non - reducibility of the segment . the cecum was pulled close to the internal ring . the congenital anomaly ( mobilized ascending colon and cecum ) is considered to be a predisposing factor for developing amyand 's hernia . in addition , our pathology revealed many eosinophils in the inflamed appendix . most often , the stained white cell differential in acute appendicitis is neutrophil - predominant . the situation may be the result of an incarceration but no strangulation of the distal appendix for one week ( prolonged reduction of blood supply without complete interruption ) , leading to a subacute inflammation of the organ . computed tomography scans may be useful in diagnosis , but are typically not performed routinely . we did not order abdominal ct scanning for our patient because he did not present as an emergency and strangulation was not considered during the preoperative period . differential diagnosis should include strangulated hernia , strangulated omentocele , richter 's hernia , testicular tumor with hemorrhage , acute hydrocele , inguinal adenitis , and epidydimitis . the treatment for hernial appendicitis is appendectomy through the herniotomy with primary hernia repair using the same incision . only lyass et al . reported a delayed wound closure due to a retroperitoneal abscess secondary to the appendicular inflammation . mesh is not suggested in the contaminated abdominal wall defects because of greater risk of wound infection and appendiceal stump fistula . laparoscopic reduction of amyand 's hernia has been described previously [ 15 , 16 ] . amyand 's hernia is a rare condition that has been frequently diagnosed accidentally during a hernioplasty , and especially an incarcerated inguinal hernia diagnosed in the emergency room . it is closely linked to peritoneal spread of the septic process and may be life - threatening , with an incidence of mortality varying between 14 and 30% [ 9 , 17 , 18 ] ; mortality risk is most likely associated with perforated appendix with or without periappendicular abscess formation , or even peritonitis . consequently , our recommendation is that acute appendicitis should be considered as a potential cause for increasing morbidity in patients with incarcerated hernias . awareness of this clinical entity would be useful in the preoperative evaluation of patients with non - reducible or incarcerated inguinal hernias .

amyand 's hernia is an extremely rare condition in which the appendix is positioned in the inguinal hernia sac . acute appendicitis is much less common in this situation and few reports are found in the literature . we report a case of acute appendicitis with the tip of the appendix incarcerated outside the external ring of the right groin . a mobilized cecum and ascending colon were noticed during surgery . we conducted a review of the literature , emphasizing possible causes and suggesting a predisposing factor for the condition .

a double - blind study was performed to evaluate the efficacy of mepivacaine and lignocaine , among 40 patients in the department of oral and maxillofacial surgery at coorg institute of dental sciences , who required surgical extractions of mesioangular bilaterally impacted third molars . criteria are followed in selection of patients : patient selected were between the age group of 4075 yearsmedically compromised patient was not consideredthe patients with known history of allergy to the local anesthetic agent were excludedthe patient should be willing and co - operativethe patient should be within reach for further follow - up and assessmentno signs of inflammation in the extraction sitesfrom all the patients who are going to participate , a written informed consent during the pretreatment screening periods were taken . patient selected were between the age group of 4075 years medically compromised patient was not considered the patients with known history of allergy to the local anesthetic agent were excluded the patient should be willing and co - operative the patient should be within reach for further follow - up and assessment no signs of inflammation in the extraction sites from all the patients who are going to participate , a written informed consent during the pretreatment screening periods were taken . ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . lignocaine ip - 36 mg adrenaline as bitartarte ip - 0.0125 mg methylparaben - 1.0 mg mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine usp - 36 mg adrenaline usp - 0.0125 mg methylparaben - 1.0 mg disposable synringes 2 ml visual analog scale ( vas ) periosteal elevator / explorer . as the solution was not available readily , the solution was prepared by a chemist in department of chemistry , sri venkateswara university , tirupathi . 1000 concentration epinephrine was diluted to 1:27,500 concentration by adding sterile water . 1 ml of 1:27,500 concentration of epinephrine - containing solution was added to 2 ml of 3% mepivacaine to obtain 2% mepivacaine with 1:80,000 of epinephrine . the investigation was conducted as a randomized , double - blind study . to avoid investigator bias and to strengthen the credibility of the patient subjective evaluation , to accomplish this goal , a special procedure was instituted . this code was readily available to the principle investigator and other faculty members in case a medical emergency were to arise . each patient was assigned a patient number depending on when he entered the study . a master code blood pressure and heart rate were recorded before the administration of the local anesthetic agent and again at 5 min , 15 min , 30 min , 1 h , 2 h , after local anesthetic administration . the standardized local anesthetic technique was used to anesthetize , inferior , alveolar , lingual , and long buccal nerves by administering 1.8 ml coded solution with a disposable syringe and needle by direct technique . onset of anesthetic agent action ( in seconds ) is determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patient 's response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia , total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . if more than 4 ml of the solution were necessary to achieve surgical anesthesia , then the case was considered as anesthetic failure and was eliminated from the study . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . duration of anesthesia was calculated by taking the total time from surgical anesthesia to the time numbness wore off . the duration of the surgical procedure was recorded from the time of incision to the last suture placed . during the procedure , intra - operative pain experience was recorded on a vas and visual - verbal scale ( vvs ) . subject receives a pain assessment form and asked to record his or her intraoperative and postoperative pain experience on vas and vvs at intervals of 5 min , 30 min , 1 h , 2 h , 3 h. all patients are provided with analgesics medications ( diclofenac sodium 50 mg 8 hourly for 3 days ) and were instructed to take medications only if they felt pain . the patient is asked to note the time of the return of normal sensation to lower lip , the time of onset pain and time of first rescue medication taken . all patients were reviewed at 3 and 7 postoperative days where pain assessment forms were collected . the following parameters are assessed , the volume of anesthetic solution used during the surgery ( in ml)onset of anesthetic agent action ( in seconds ) , determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patients response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensationthe intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scalesystolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 hpostoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . the volume of anesthetic solution used during the surgery ( in ml ) onset of anesthetic agent action ( in seconds ) , determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patients response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensationthe intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scalesystolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 hpostoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensation the intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scale systolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 h postoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . the significant comparison was analyzed by using the independent - samples t - test ; anova ; crosstabs procedure . local anesthetic agent lignocaine 2% with 1:80,000 adrenaline lignocaine ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . lignocaine 2% with 1:80,000 adrenaline lignocaine ip - 36 mgadrenaline as bitartarte ip - 0.0125 mgnacl ip - 0.5.mgmethylparaben - 1.0 mgwater -1 ml . lignocaine ip - 36 mg adrenaline as bitartarte ip - 0.0125 mg methylparaben - 1.0 mg mepivacaine 2% with 1:80,000 adrenaline mepivacaine usp - 36 mgadrenaline usp - 0.0125 mgmethylparaben - 1.0 mgwater -1 ml . mepivacaine usp - 36 mg adrenaline usp - 0.0125 mg methylparaben - 1.0 mg disposable synringes 2 ml visual analog scale ( vas ) periosteal elevator / explorer . as the solution was not available readily , the solution was prepared by a chemist in department of chemistry , sri venkateswara university , tirupathi . was diluted to 1:27,500 concentration by adding sterile water . 1 ml of 1:27,500 concentration of epinephrine - containing solution was added to 2 ml of 3% mepivacaine to obtain 2% mepivacaine with 1:80,000 of epinephrine . the investigation was conducted as a randomized , double - blind study . to avoid investigator bias and to strengthen the credibility of the patient subjective evaluation , to accomplish this goal , a special procedure was instituted . this code was readily available to the principle investigator and other faculty members in case a medical emergency were to arise . each patient was assigned a patient number depending on when he entered the study . a master code blood pressure and heart rate were recorded before the administration of the local anesthetic agent and again at 5 min , 15 min , 30 min , 1 h , 2 h , after local anesthetic administration . the standardized local anesthetic technique was used to anesthetize , inferior , alveolar , lingual , and long buccal nerves by administering 1.8 ml coded solution with a disposable syringe and needle by direct technique . onset of anesthetic agent action ( in seconds ) is determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patient 's response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia , total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . if more than 4 ml of the solution were necessary to achieve surgical anesthesia , then the case was considered as anesthetic failure and was eliminated from the study . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . duration of anesthesia was calculated by taking the total time from surgical anesthesia to the time numbness wore off . the duration of the surgical procedure was recorded from the time of incision to the last suture placed . during the procedure , intra - operative pain experience was recorded on a vas and visual - verbal scale ( vvs ) . subject receives a pain assessment form and asked to record his or her intraoperative and postoperative pain experience on vas and vvs at intervals of 5 min , 30 min , 1 h , 2 h , 3 h. all patients are provided with analgesics medications ( diclofenac sodium 50 mg 8 hourly for 3 days ) and were instructed to take medications only if they felt pain . the patient is asked to note the time of the return of normal sensation to lower lip , the time of onset pain and time of first rescue medication taken . all analgesic medication taken all patients were reviewed at 3 and 7 postoperative days where pain assessment forms were collected . the following parameters are assessed , the volume of anesthetic solution used during the surgery ( in ml)onset of anesthetic agent action ( in seconds ) , determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patients response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensationthe intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scalesystolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 hpostoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . the volume of anesthetic solution used during the surgery ( in ml ) onset of anesthetic agent action ( in seconds ) , determined by loss of sensibility of inferior lip , corresponding half of the tongue , and the mucosa , and patients response to a sharp explorer ( pinprick testing ) . a 26 gauge sterile injection needle will be used for sharp objective testing on buccal attached gingival between mandibular canine and first premolar . the onset of anesthesia tested every 30 s to the time when objective test not elect any sensation . if the onset of anesthesia total was not obtained within 10 min , anesthesia would be regarded as unsuccessful . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensationthe intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scalesystolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 hpostoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . postoperatively , patients were evaluated with pinpricks to establish the duration of anesthesia testing being repeated every 30 min after surgery to the time point when patients felt blunt . sensation then continued every 10 min to no sensation the intensity of anesthesia during the surgical procedure and postoperatively will be determined by 2 scores after the completion of the treatment ( 1 ) a 110 vas ( 2 ) six - point verbal rating pain scale systolic blood pressure , diastolic blood pressure , mean arterial pressure , heart rate are recorded before and during the administration of local anesthesia , 5 min , 15 min , 30 min , 1 h , 2 h postoperative pain was assessed by noting the time of intake of 1 painkiller and further intake successive of tablets . the significant comparison was analyzed by using the independent - samples t - test ; anova ; crosstabs procedure . following completion of the clinical study on the patients the measurement and data taken from all the patients were tabulates for statistical studies after decoding the drugs used . the study comprised 26 males ( 57.8% ) and 19 females ( 42.2% ) , found to be approximately significant ( p = 0.601 ) . the study had contained 3 patients ( 6.7% ) in group a solution and 6 patients ( 13.3% ) in group b solution below 45 years age ; 9 patients ( 20% ) in group a solution and 10 patients ( 22.2% ) in group b solution between 46 and 50 years age group ; 13 patients ( 28.9% ) in group a solution and 9 patients ( 20.0% ) in group b solution between 51 - 55age group ; 12 patients ( 26.7% ) in group a solution and 12 patients ( 26.7% ) in group b solution between 56 - 60 age group ; 8 patients ( 17.8% ) in group a solution and 8 patients ( 17.8% ) in group b solution above 60 years age group . the mean age of the patients group a solution was 54.78 and in group b solution was 53.64 . the mean time for onset of anesthesia with mepivacaine was 4.2 min whereas with lidocaine was 4.6 min , and the difference was statistically significant ( p = 0.018 ) . none of the patients complained of pain sensation during incision placement and reflection of the flap , which was started 5 min after onset of anesthesia . during the elevation and removal of teeth 3 patients ( 6.7% ) belonged to group a and had received lidocaine as a supplemental dose of 1.5 ml , and later on they had satisfactory anesthesia . the difference was found to be approximately significant relating to depth of anesthesia ( p = 0.078 ) . the mean duration of action of anesthesia was measured as change of sensation for onset of paresthesia of lower lip to the return of normal sensation , with mepivacaine group had 177.17 min and that of lidocaine was 166.71 min . the differences were found to be significant ( p = 0.085 ) . with regard to the course of pain , according to the patient 's evaluation on vas and venovenous bypass ( vvb ) scale at each determined time point , there was no significant difference between the scores of pain reported by the patients treated with mepivacaine and lidocaine ( p = 0.000 ) . the mean postoperative analgesia required in the first 3 to 4 postoperative hour was same for mepivacaine group ( 4.000 tablets ) and lidocaine group ( 4.170 tablets ) . with respect to hemodynamic changes , there was a sharp increase of pulse rate with respect to both the solutions at 5 min after postinjection of local anesthetics . however , there was no statically significant difference in systolic and diastolic blood pressure before , during the surgery and after the suture placement for both groups ( p = 0.681 ) and ( p = 0.270 ) [ tables 17 and graphs 18 ] onset and duration of anesthesia tables comparison of blood pressure tables comparison of diastolic blood pressure postoperative pain score ( vas scale ) postoperative pain score ( vvb scale ) onset of anesthesia graph duration of anesthesia comparison of systolic blood pressure graph comparison of diastolic blood pressure graph postoperative pain score ( vas scale ) graph . dental profession is one among the branches of healing sciences , where clinicians are more likely to be continuously and directly involved with pain , fear , and anxiety in the ambulatory and otherwise healthy patients . certainly pain in difficult , if not impossible , to quantify and okeson has stated that pain is personal psychological experience and observe can play no legitimate part in the direct measurement . the overall control of pain in minor oral surgical procedures , besides of the tissue handling , consists of the ability to place accurately an anesthetic solution in proper anatomical relationship to the structures to be operated upon . despite this , there is no doubt that the majority of patients who now attend for treatment can be managed without resort to anything other than the conventional method of regional blockage or local analgesia . the present study was done to determine the effectiveness of 2% mepivacaine with 1:80,000 adrenaline in comparison with lidocaine hydrochloride with 1:80,000 adrenaline , as local anesthetics in surgical extraction of bilateral mesioangular impacted third molars . mepivacaine belongs to amide group of local anesthetics , which acts by decreasing the rate of depolarization of action potential in nerve fibers by impending the initial rise in sodium conductance , thus preventing the generation of conduction of nerve impulses . the degree of the protein binding of a local anesthetic directly determines the duration of anesthesia . mepivacaine has got a high affinity for the protein components of nerve and , therefore , is less liable to diffuse from the injection site and be absorbed into systemic circulation . thus , the duration of action of mepivacaine is slightly greater than the duration of lignocaine . mepivacaine was found to provide satisfactory anesthesia in the present study without the occurrence of any complication or side effects . the depth of anesthesia of intra - operative anesthesia with mepivacaine has been found to be superior to that with lidocaine in previous studies . postoperative pain assessments were made using a standard vas and vvs , which are sensitive to and simple means of pain assessment . the result showed there was a significant reduction in pain , experienced by mepivacaine group between 2 and 3 h postoperatively . maximum postoperative pain is usually experienced in the first 68 h. the use of mepivacaine with a longer duration of action reduces the postoperative pain , discomfort , and analgesic intake during the immediate postoperative period . in the present study , the mean analgesic consumption for the first 4 days shows less for mepivacaine group ( 4.000 tablets ) compared to lidocaine group ( 4.170 tablets ) . although the anesthetic agent does not have effect on pain and inflammation directly , longer - acting local anesthetics may allow the patients to rest more easily and thus permit the body to withstand pain more effectively during the 1 postoperative day . the mean systolic and diastolic blood pressure remained relatively constant in the current study , and no statistically significant differences were found between the two local anesthetic agents . this is in agreement with other authors who reported no change in blood pressure with the administration of adrenaline containing a local anesthetic . lidocaine and mepivacaine with the same vasoconstrictor have similar action and both solutions are effective in surgical procedures . there were also no significant differences between them in relation to the intensity of postoperative pain .

objective : the purpose of this prospective , randomized , double - blind study was to compare the anesthetic efficacy of 2% mepivacaine and 2% lidocaine ( both with 1:80,000 epinephrine ) for inferior alveolar nerve block in mesioangular bilaterally impacted third molar extraction.study design : forty patients with mesioangular bilaterally impacted third molars were taken for the study ; either 2% mepivacaine or 2% lidocaine is given in a double - blind manner . surgery started 5 min after solution deposition . success was defined as no or mild discomfort ( visual analog scale [ vas ] recordings ) during the surgical procedure.results:the mean time for onset period 4.2 min and 4.6 min ( p = 0.018 ) . the mean duration anesthesia 177.17 min 166.71 min ( p = 0.085 ) . no significant difference between the scores of pain reported by the patients by vas and venovenous bypass treated with mepivacaine and lidocaine ( p = 0.000 ) . slight increased postoperative analgesics required for mepivacaine group ( 4.000 tablets ) and lidocaine group ( 4.170 tablets ) ( p = 0.335 ) . the sharp increase of pulse rate with respect to both the solutions at 5 min after postinjection of local anesthetics . however , there was no statically significant difference in systolic and diastolic blood ( p = 0.681 ) and ( p = 0.270).conclusion : lidocaine and mepivacaine with the same vasoconstrictor have similar action and both solutions are effective in surgical procedures . there were also no significant differences between them in relation to the intensity of postoperative pain .

the main objective of endodontic treatment is to provide hermetic obturation of the root canal system with an inert , biocompatible , and dimensionally stable filling material . according to a satisfactory obturation of the root canal , the filling material and the endodontic instruments should be limited to the root canal without extending to periapical tissues or other neighbouring structures ( poveda et al . ; 2006 ) . filling material , broken file , and gutta - percha extruted in the periapical area , the connective tissue tends to absorb the foreign body or more frequently surround it with a fibrous capsule . there are numerous examples reported in the literature that cite and document many disabling complications to the alveolar bone , neurovascular anatomy , and maxillary sinus following overextension of root canal filling materials . neural complications , a consequence of endodontic obturation as well as other server outcomes to overextended obturating material , are serious problem . these injuries require a thoughtful strategy for prevention during endodontic procedures as well as a responsible systematic approach to management , should the outcome of endodontic therapy produce an injury . this monograph will focus on measures that can prevent obturation mishaps which occur under the most vulnerable circumstances during the course of endodontic therapy . correction of such mishap may be accomplished in one of several ways depending on the type and extent of procedural accident . endodontic mishaps are either ( a ) access related , ( b ) instrumentation related , ( c ) obturation related , or ( d ) miscellaneous . a 30 years male patient reported to the department of conservative dentistry and endodontic , faculty of dental sciences , csm medical university , lucknow , with a chief complaint of continuous pain and heaviness in the infraorbital region and pus discharge . his medical history was insignificant and dental history revealed that his maxillary lateral incisor and canine of left side got fractured due to trauma 1 year back . he had been treated in a private clinic with root canal treatment , but patient was not totally asymptomatic . after 10 months , he had developed swelling and pain with pus discharge in upper left anterior region . the consultant dentist in that private clinic had extracted both the teeth but pus discharge continued and pain was present , which was refractory to several courses of antibiotics and analgesics . intraoral examination showed sinus in relation to maxillary left lateral incisor and canine region with unhealed sockets . extra oral examination showed slight swelling in the left maxillary lateral incisor and canine region . radiograph revealed the foreign body [ gutta - percha ] in the upper left anterior infraorbital region near the nasal floor [ figure 1 ] . routine blood investigation was done and was planned for surgical intervention to remove foreign body . mucoperiosteal flap was raised and curettage was done to remove the granulation tissues [ figure 2 ] . a foreign body was visible in the bone , which was removed by taking care of nasal floor [ figure 3 ] . the object was identified as no-40 gutta - percha point [ figure 4 ] . the cavity was cleaned and flap was repositioned and sutured . after 7 days , sutures were removed and patient was found to be totally asymptomatic and socket was also in healing stage [ figure 5 ] . radiograph showing foreign body in relation to # 22 and # 23 photograph showing operative procedure photograph showing retrieval of gutta - percha retrieved gutta - percha postoperative radiograph one of the most iatrogenic complications in endodontic is overfilling of the root canal , which has a negative effect on prognosis of endodontically treated teeth ( brkic et al . ; more than a half of the overfilled teeth heal satisfactorily after proper endodontic therapy , but in case of injury of any nerve or presence of obturating material in soft tissues or sinus spaces a surgical approach is necessary ( brkic et al . ; endodontic etiology can affect the maxilllary sinus , which include extension of periapical infections into the sinus , the introduction of endodontic instruments , and materials beyond the apices of posterior teeth in close proximity to the sinus ( hauman et al . , 2002 ) . about 125 cases of odontogenic chronic sinusitis were reviewed retrospectively . in the case described above , the overextention of filling material from the apical foramen of the root canal did show damage of the periapical tissues according to the normal bone density of the area shown from the radiographic examination . overextension filling of left lateral incisor and canine root canal were the causal factors of a chronic inflammation of the corresponding sites . the nerve supply is from the maxillary division of the trigeminal nerve , with branches coming from the posterior , middle , and anterior superior portions . the inflammatory effects of overfilled endodontic materials as well as dental sepsis can affect the differential diagnosis of pain localized to the sinuses . however , as long as the overextension is not in contact with vital structures , such as the inferior alveolar nerve or sinuses , and the apical terminus is well filled in three dimensions , permanent harm is potentially small , unless the obturation materials contain paraformaldehyde . on the other hand , overextension of the root canal filling material risks are serious and possibly permanent consequences should the underlying inferior alveolar nerve be adjacent to the root terminus or initially penetrated with files to create a mishap scenario that includes the possibility for severe injury . in most cases , irritation of the periapical tissues from extrusion of endodontic cement is transitory with subsequent reabsorption of the excess material , leading to complete healing in a few months . this phenomenon may be considered to be an expected complication , and at times , even sought after by many dentists as a sign of a successfully completed intervention . there is notable controversy in the literature , regarding the presence of cement beyond the apex . some authors , among them schilder ( 1967 ) , refute the hypothesis that the presence of cement beyond the apex favors healing of the periapical lesions , maintaining their benign nature . he asserts that extrusion beyond the apex must be avoided solely in the interest of potential discomfort created for the patient during the obturation phase . other authors have reported significant cytotoxicity of both commonly used cements and gutta - percha following research studies carried out in vitro with sem ( scanning electron microscope ) . this cytotoxicity can induce periradicular inflammation or necrosis of the periodontal ligament , and for this reason , overfilling should be avoided as much as possible because it can lead to failure of short - term treatment or a long negative prognosis . in reality , the prognosis for an endodontically treated tooth with overfilling depends on the response of the periradicular tissue to the canal obturation material which is , in its own way , a consequence of the complex , and at times an unpredictable interaction between the materials and the host defences . according to the american dental association , overfilling by more than 2 mm past the radiological apex represents a technical error ascribable to over - instrumentation , inadequate measuring , or a lack of an apical stop . however , the latter was difficult to obtain , as in the presence of resorbed roots caused by inflammatory processes or by particularly wide apices . vertical condensation of warm gutta - percha during the obturation phase offers a higher probability of closure of the lateral and accessory canals . at the same time however , warm vertical compaction techniques also result in a greater risk of the obturation material being extruded into periradicular tissues . the material , usually cement , acts as a lubricant , as it aids in the progression of the principal obturation material ( core ) during the compaction phase . furthermore , it also aids in the filling of the lateral and accessory canals which would otherwise be impossible to fill with a single core of gutta - percha . in addition , it improves the adaptation to discrepancies and irregularities which , even after correct shaping , may persist on the root canal wall . the prognosis for an endodontically treated tooth with overfilling depends on the response of the periradicular tissue to the canal obturation material which is , in its own way , a consequence of the complex , and at times an unpredictable interaction between the materials and the host defences . over instrumentation , in particular , may extrude infected material contained in the canals beyond the apex , interfering , or impeding the healing process of the periapical tissue . gutta - percha cones , which had been extruded past the apices , have demonstrated the presence of a biofilm allows undisturbed growth of the bacteria and renders them particularly resistant to the defences of the host and may be responsible for foreign body reactions . the consequences of overfilling can , therefore , result in infective periapical periodontitis caused by the transport of bacteria beyond the apex and an incomplete cleansing , foreign body reactions , and pain symptoms which are ascribable to irritative stimuli , even in the absence of radiological evidence . some meta - analyses have recognized that , over time , the best results for canal obturations occur when the gutta - percha extrudes 0 - 1 mm from the apex and , on the contrary , when considering measurements of greater than 1 mm ( above or below the apex ) , the results are less favorable . finally , the prognosis for an endodontically treated tooth with overfilling depends on the response of the periradicular tissue to the canal obturation material which is , in its own way , a consequence of the complex and , at times , an unpredictable interaction between the materials and the host defences . knowledge of dento - antral relationships as well as the skill of the operator is important particularly in the prevention of sinusal accidents . the anatomical and clinical significance of the maxillary sinus in relation to conventional and surgical endodontic therapy is considered . clinicians should be aware of the fact that endodontic instruments and filling materials [ solid or liquid ] can be extended in such a degree that can lead to sinus complications , i.e. , sinusitis , due to the proximity of the apices of maxillary posterior teeth to the sinus floor membrane .

complications may occur during and after endodontic treatment , which may be due to negligence of the operator . the surgical treatment of a case presenting pain and persistent pus discharge and swelling due to the extrusion of the root canal filling to the base of the nasal floor between left maxillary lateral and canine teeth is presented in this report . first , carelessness was during root canal treatment that was over obturated and second time during extraction in which overextended gutta - percha remained in the bone , caused the complications like pain , persistent pus discharge , and headache . clinicians should be aware of the fact that endodontic instruments and filling materials ( solid or liquid ) can be extended in such a degree that can lead to neurological or sinus complications .

during a one - year period starting from november 2006 to november 2007 , 52 patients ( 27 male and 25 female ) with crc and 22 cases of non - cancerous lesion as a control group were subjected to the present investigation . all cases were referred to kufa school of medicine teaching hospital from different regions of middle euphrates area of iraq for histopathological evaluation . all cases , whether malignant or non - cancerous lesions , were examined by two histopathologists independently and then subjected to the immunohistochemical method using the abc technique . the total number of malignant cases was 52 ( all were hemicolectomy or segment resection ) . left side cases were 19 , lower rectal tumours were 13 and right side were 20 , while the total number of non - cancerous cases was 22 ( all were endoscopic biopsies ) . the malignant colonic cases were staged according to the tnm ( tumor size , lymph node involvement , distant metastasis ) staging system ( 16 ) . biotin complex ( abc ) method was used for immunohistochemical detection of 0.2 ml ( clone vg1 , code m7273 , lot 00028659 , dako denmark a / s produktionsvej 42 dk-2600 glostrup ) as primary antibody for the detection of vegf protein ( kit k5204 , dako co ) . the antibody sensitivity and a staining kit , code k0673 , from dako co , was also used . the criterion for a positive immune reaction was a dark - brown cytoplasmic precipitate . the intensity scoring of the staining was assessed quantitatively by counting the percentage of positive cells in 100 malignant cells at 40 total magnifications for at least 25 fields and qualitatively as dense and faint staining . a four - scaled scorings system was chosen in this study ; score 0 ( negative ) : no stained malignant cells , score 1 ( weak ) : 510% of malignant cells , score 2 ( moderate ) : < 25% of positive malignant cells , score 3 ( strong ) : 2550% of malignant cells and score 4 ( very strong ) : > 50% of malignant cells ( 16 ) . the results were statistically evaluated with the help of ssps software by using chi - square test and correlation regression test . the positive results for vegf immunohistochemical staining appear as brown cytoplasmic colour . regarding the non - cancerous colonic tissue , 4 ( 18.2% ) out of 22 cases showed positive immunohistochemical stain for vegf , while 27 ( 51.9% ) out of 52 malignant cases showed positive results with a significant difference between these two groups ( p < 0.05 ) ( table 1 , fig . vegf overexpression in relation to grade and stage of colorectal carcinoma vegf overexpression was reported in 52% of adenocarcinoma and 50% of squamous cell carcinoma without any significant difference between the two groups ( p > 0.05 ) ( table 1 , figs . 2 and 3 ) . squamous cell carcinoma of rectum , grade ii exhibiting positive faint , score 1 vegf immunostaining . adenocarcinoma of colon , grade i , stage ii with positive dense vegf immunostaining , score 4 ( 10 ) . i crc , in 11 ( 52.4% ) of grade ii crc , and in seven ( 58.3% ) of grade iii crc . there was good correlation between the grade of tumour and vegf overexpression ( r = 0.96 , p < 0.05 ) ( table 1 ) . vegf overexpression was found to be positive in seven ( 53.8% ) of stage i crc , in 11 ( 52.4% ) of stage ii crc and in nine ( 50% ) of stage iii crc . there was no correlation between the stage of tumour and vegf overexpression ( r = 0.96 , p < 0.05 ) ( table 1 ) . regarding the intensity scorings of positive results of vegf immunohistochemical staining , score 1 was found in 14 ( 51.9% ) cases , score 2 in eight ( 19.65 ) cases , score 3 in four ( 14.8% ) cases and score 4 was found only in one case ( 3.7% ) of crc . there was a strong correlation of vegf immunohistochemical staining scoring with the grade of crc ( table 2 ) . intensity of vascular endothelial growth factors in relation to grade , stage and pattern of immunostaining in colorectal carcinoma in relation to the staining pattern ( dense or faint ) of positive vegf immunohistochemical staining , faint staining was seen in 40.7% ( 11 out of 27 cases ) while the dense mode of immunostaining was reported in 59.3% ( 16 out of 27 ) ( table 2 ) . vegf is considered as the most important , directly acting and potent angiogenic agent that has been shown to be over expressed in crc . in the present investigation , vegf was expressed in 18.2% of non - cancerous colonic tissue indicating that vegf is an effective angiogenic factor in normal physiological conditions in the colon ( 1721 ) . however , a positive vegf immunostaining was found in 51.9% ( 27 out of 52 cases ) of the study group of crc with a significant difference of ( p < 0.05 ) as compared with the non - cancerous control samples . this observation indicates that vegf has a fundamental role in the angiogenesis of crc ( 18 , 2224 ) . a positive correlation between the intensity of vegf immunostaining and histological types of crc has been noted , with a significant difference among these three subtypes in relation to the vegf over expression . there was a positive correlation between the vegf immunostaining and the pattern of staining ( dense or faint ) ( r = 0.92 , p < 0.05 ) suggesting that colonic cancer is a highly vascular malignancy and the malignant colonic cancer cells are highly susceptible to the action of vegf . as grouped in table 1 , the present study shows that there was a gradual increase in the frequency of vegf expression in parallel with the increase in the grade of tumour ( in grade i there was 50% vegf expression , in grade ii 52% and in grade iii 54.5% ) . however , no significant differences among the three degrees of differentiation ( p > 0.05 ) were noted . this observation is supported by previous investigators who reached the same conclusion ( 26 , 27 ) . furthermore , there was a significant correlation between the stage of tumour and the intensity of vegf immunostaining ( p < 0.05 , r = 0.62 ) . this finding indicates equivocally that vegf is directly proportional to the degree of colorectal tumour spread as has been established elsewhere ( 18 , 2730 ) . there is no significant difference among the three stages of crc in relation to vegf expression ( p > 0.05 ) . these findings unfold further evidence for the role of vegf in the carcinogenesis of crc . accordingly the authors have not received any funding or benefits from industry to conduct this study .

backgroundcolorectal carcinoma ( crc ) is the seventh - most common malignancy and is the main cause of death in iraq . the incidence of this cancer has increased sharply after the invasion of iraq in 2003.aimto estimate immunohistochemical expression of vascular endothelial growth factor ( vegf ) in crc in relation to other parameters , such as grade and stage of tumour.methodsformalin fixed , paraffin - embedded blocks from 52 patients ( 27 male and 25 female ) with crc were included in this study . a group of 22 patients with non - cancerous colonic tissues were included as a control group . avidin biotin complex method was employed for immunohistochemical detection of vegf.results vegf immuno - expression was positive in 51.9% of crc , while it was 18.2% in the normal colonic tissue ( p < 0.05 ) . vegf immunostaining was positively correlated with grade of colonic malignancy ( p < 0.05).conclusionthese findings provide further evidence for the role of vegf in the carcinogenesis of crc . however , vegf could not be well correlated with stage of tumour and hence may be a poor prognostic parameter of state of malignancy of colonic carcinoma .

oral biofilm is the diverse microbial community found on the tooth surface , embedded in a matrix of polymers of bacterial and salivary origin . oral biofilm has been known to be closely related with the occurrence of oral disease.1 the formation of oral biofilm may lead to development of dental material surface biodegradation and secondary caries and periodontal inflammation , main reasons for the restoration replacement . in the process of plaque formation on solid substrate surfaces including teeth , implant and restorative materials , initial adhesion of the " early colonizers " to the surface is a critical step.1 - 3 oral streptococci have been known to bind to proteins such as alpha - amylase , proline - rich proteins and glycoproteins , and are recognized as early colonizers.4 streptococcus sanguis ( s. sanguis ) is thought to be one of the first bacterial species selectively adhere to teeth and colonize on saliva - coated teeth . this species appears in the human oral cavity after tooth eruption , and it becomes a normal inhabitant of the human mouth.4,5 numerous factors have been identified to influence oral biofilm formation such as surface roughness and surface free energy.6,7 microscopic examination of early plaque formation on teeth showed the adhesion of the initial colonizing bacteria along cracks and pits in enamel , suggesting the influence of surface structure on bacterial adhesion.3 it is evident that implant and restorative materials have different surface characteristics . various affinities of oral bacteria adhesion have been reported for different materials including titanium.7 - 10 resin composites have been widely used for operative , esthetic , and prosthodontic treatments.3,8 dental ceramic materials also applied to broad range of clinical practice . especially , zirconia has been introduced to improve esthetics for natural teeth and implant prostheses because of its biocompatibility , high resistance to wear and fracture by fatigue loading.10 - 12 the purpose of this study is to compare and characterize biofilm formation on commonly utilized restorative materials such as composite resin , titanium and zirconia . resin composite was dispensed from a syringe into a mold , and then light - cured for 30 seconds from the top and the bottom side respectively with a light curing unit ( shinwon dental , seoul , korea ) . commercially pure titanium ( cp - ti ) bars were machined into discs and 3y - tzp powder was die - pressed into disks and then isostatically pressed at 140 mpa . three kinds of 12 mm diameter disc specimens were gradually polished and finished with 1 m diamond paste to acquire mirror - like surface . the average surface roughness ( ra ) and topography were measured by the confocal laser microscope ( zeiss , germany ) . the static water contact angle of each surface of specimen was measured with a phoenix 300 contact - angle meter ( surface electro optics , korea ) at room temperature . s. sanguis 804 ( nctc 10904 ) was maintained in sterile trypticase soy broth ( bd diagnostics , md , usa ) supplemented with yeast extract ( bd diagnostics ) . prior to seeding , sterilized specimens were placed into a 24-well culture plate and were incubated with saliva for 2 hours at 37. the saliva was collected from one healthy donor who did not show any active carious lesions or periodontal diseases and sterilized by filtration devices with pore sizes 0.2 m ( millipore , ma , usa ) . after washing with pbs , bacteria were seeded onto the specimens at a density of 110 bacteria / cm . the crystal violet ( cv ) assay was performed to determine the total amount of biofilm . then , wells were washed once with pbs and the plates were air - dried . the biofilm was stained using 1% crystal violet solution ( sigma - aldrich , md , usa ) , followed by 10 minutes incubation time at room temperature . then , the excess of unbound dye was removed by washing the plates with deionized water . the bound cv was extracted with destaining solution ( 80% ethanol , 20% acetone ) . the amount of biofilm was measured at optical density of 595 nm using a microplate reader ( bio - rad laboratories , ca , usa ) . the background staining was corrected by subtracting the mean value for cv bound to negative controls . the fluorescence resazurin ( 0.75 g / ml , sigma , md , usa ) was used to determine the quantity of viable adherent bacteria . bacterial suspension with resazurin was incubated on each specimen at 37 for 150 minutes . after washing with pbs , fluorescence intensities were recorded by detection reader ( fluostar optima ; mbg labtech , offenburg , germany ) at wavelengths of 540 nm excitation and 590 nm emissions . s. sanguis attachment and morphology were also assessed after 4 hours culture by scanning electron microscopy ( jeol , tokyo , japan ) . after washing twice in the 0.1 m phosphate buffer , bacteria are post fixed with 1% oso4 in saturated hgcl2 . after dehydration in graded ethanol , bacteria are critical point dried and coated with gold by ion sputtering for 2 minutes . statistical analysis was performed using one - way analysis of variance ( anova ) and the tukey - kramer multiple comparison test was used for post hoc analysis . resin composite was dispensed from a syringe into a mold , and then light - cured for 30 seconds from the top and the bottom side respectively with a light curing unit ( shinwon dental , seoul , korea ) . commercially pure titanium ( cp - ti ) bars were machined into discs and 3y - tzp powder was die - pressed into disks and then isostatically pressed at 140 mpa . three kinds of 12 mm diameter disc specimens were gradually polished and finished with 1 m diamond paste to acquire mirror - like surface . the average surface roughness ( ra ) and topography were measured by the confocal laser microscope ( zeiss , germany ) . the static water contact angle of each surface of specimen was measured with a phoenix 300 contact - angle meter ( surface electro optics , korea ) at room temperature . s. sanguis 804 ( nctc 10904 ) was maintained in sterile trypticase soy broth ( bd diagnostics , md , usa ) supplemented with yeast extract ( bd diagnostics ) . prior to seeding , sterilized specimens were placed into a 24-well culture plate and were incubated with saliva for 2 hours at 37. the saliva was collected from one healthy donor who did not show any active carious lesions or periodontal diseases and sterilized by filtration devices with pore sizes 0.2 m ( millipore , ma , usa ) . after washing with pbs , bacteria were seeded onto the specimens at a density of 110 bacteria / cm . the crystal violet ( cv ) assay was performed to determine the total amount of biofilm . then , wells were washed once with pbs and the plates were air - dried . the biofilm was stained using 1% crystal violet solution ( sigma - aldrich , md , usa ) , followed by 10 minutes incubation time at room temperature . then , the excess of unbound dye was removed by washing the plates with deionized water . the bound cv was extracted with destaining solution ( 80% ethanol , 20% acetone ) . the amount of biofilm was measured at optical density of 595 nm using a microplate reader ( bio - rad laboratories , ca , usa ) . the background staining was corrected by subtracting the mean value for cv bound to negative controls . the fluorescence resazurin ( 0.75 g / ml , sigma , md , usa ) was used to determine the quantity of viable adherent bacteria . bacterial suspension with resazurin was incubated on each specimen at 37 for 150 minutes . after washing with pbs , fluorescence intensities were recorded by detection reader ( fluostar optima ; mbg labtech , offenburg , germany ) at wavelengths of 540 nm excitation and 590 nm emissions . s. sanguis attachment and morphology were also assessed after 4 hours culture by scanning electron microscopy ( jeol , tokyo , japan ) . after washing twice in the 0.1 m phosphate buffer , bacteria are post fixed with 1% oso4 in saturated hgcl2 . after dehydration in graded ethanol , bacteria are critical point dried and coated with gold by ion sputtering for 2 minutes . statistical analysis was performed using one - way analysis of variance ( anova ) and the tukey - kramer multiple comparison test was used for post hoc analysis . the surface of resin composite was significantly rougher than that of titanium and zirconia although final polishing condition was the same ( table 1 ) . however , all tested specimens demonstrated mean surface roughness values below 0.2 m , and were therefore classified as smooth clinically . table 1 also reports the mean values of water contact angles measurements , ranging between 54.2 and 75.1. the contact angle of titanium and zirconia were similar , however resin specimen showed significantly lower contact angle . it can be seen that there is a difference among the number of bacteria on resin , titanium and zirconia . as observed by sem , biofilm colonies were more abundant on resin in comparison with those of titanium and zirconia ( fig . 1 ) . a further analysis was performed to investigate the number of attached bacteria on each specimen quantitatively . the result of relative fluorescence intensities resulting from resazurin reduction also demonstrated that viable s. sanguis bound on each specimen . the fluorescence intensity on titanium was not significantly different from that on zirconia ( fig . oral biofilm formation on the surface of restorative and implant materials leads to secondary caries formation and affects healing process negatively . therefore , it is important to investigate the initial bacteria adhesion on the surface of biomaterials . the initial colonizers , including s. sanguis , s. gordonii and s. oralis , adhere to the pellicles on solid surfaces by several factors , namely , electrostatic and hydrodynamic interactions , thermodynamic parameters and adhesion - receptor interactions.5,11 this event leads to subsequent adhesion of cariogenic microorganisms such as s. mutans and periodontal pathogens , which may induce gingival and periodontal inflammation.13,14 the sem examination confirmed that s. sanguis can bind directly to the surface of resin , titanium and zirconia . however , adhesion of the total and viable bacteria on resin is significantly higher when evaluated by crystal violet staining and fluorometic quantification , respectively . surface roughness has been regarded as one of the most important factors . with regard to the influence of surface roughness on biofilm formation , rougher surface results to the increased adhesion of bacteria.6,7 in this study , we tried to eliminate the difference of roughness by polishing 1 m diamond paste . although , the roughness of resin showed similar level with previous report,3 it is still higher than the roughness of titanium and zirconia . previous report showed that protein adsorption and bacterial adhesion in vivo are primarily determined by a threshold surface roughness of 0.2 m , therefore ra less than threshold level appears to have a negligible effect.15,16 however , correlation of surface roughness and bacterial adhesion could be observed this in vitro study . to confirm this result when comparing between titanium and zirconia , our results demonstrated that there is no significant difference on s. sanguis adhesion . this contrasts with previous reports9,17 showing that fewer bacteria adhered to zirconia in comparison with titanium of a similar surface roughness in vivo . previous studies compared the bacterial adhesion using titanium and zirconia with rough surface which showed approximately 0.73 m of ra . to confirm , a further study will be performed to compare the bacterial adhesion according to the different level of roughness on biomaterials including titanium , zirconia and resin . surface hydrophobicity is another crucial element for influencing the bacterial adhesion.6,15 s. sanguis is highly hydrophobic microorganism . it is possible that the difference of bacterial adhesion is derived from the surface hydrophobicity . however , hydrophobicity of resin surface is lower than that of titanium and zirconia and more bacterial adhesion was observed . this observation may be the result from the difference of surface roughness even though all the specimens have surface roughness less than 0.2 m . the additional physic - chemical characteristics including crystallinity have been shown to affect biofilm formation in vivo and in vitro.7,18 it is unreasonable to conclude that difference in bacterial adhesion is entirely attributed to different surface roughness in this study . further studies are needed to investigate the bacterial adhesion on the various biomaterials not only by roughness but also by additional surface characteristics . within the limitations of this study , composite resin has a significantly higher susceptibility to adhere the initial colonizer , s. sanguis , than titanium and zirconia under the same polishing condition . the amount of both total and viable bacteria on resin was higher than those on titanium and zirconia .

purposethe aim of this in vitro study was to investigate the adhesion of initial colonizer , streptococcus sanguis , on resin , titanium and zirconia under the same surface polishing condition.materials and methodsspecimens were prepared from z-250 , cp - ti and 3y - tzp and polished with 1 m diamond paste . after coating with saliva , each specimen was incubated with streptococcus sanguis . scanning electron microscope , crystal violet staining and measurement of fluorescence intensity resulting from resazurin reduction were performed for quantifying the bacterial adhesion.resultssurface of resin composite was significantly rougher than that of titanium and zirconia , although all tested specimens are classified as smooth . the resin specimens showed lower value of contact angle compared with titanium and zirconia specimens , and had hydrophilic surfaces . the result of scanning electron microscopy demonstrated that bound bacteria were more abundant on resin in comparison with titanium and zirconia . when total biofilm mass determined by crystal violet , absorbance value of resin was significantly higher than that of titanium or zirconia . the result of relative fluorescence intensities also demonstrated that the highest fluorescence intensity was found on the surface of resin . absorbance value and fluorescence intensity on titanium was not significantly different from those on zirconia.conclusionresin specimens showed the roughest surface and have a significantly higher susceptibility to adhere streptococcus sanguis than titanium and zirconia when surfaces of each specimen were polished under same condition . there was no significant difference in bacteria adhesion between titanium and zirconia in vitro .

resin cements are composite resins developed to deliver mechanical properties and handling characteristics that are important for luting indirect restorations . due to their application under an indirect restoration , in most cases the physical activation ( photo activation ) has very limited effect . activation of the polymerization means to induce the photo initiator ( e.g. , camphorquinone ) or to break the molecule of the chemical initiator ( benzoyl peroxide ) so as to form free radicals that will initiate the polymerization . the continuous addition of monomers to a growing chain results in a polymeric chain . in general , the maximum degree of conversion ( dc ) the percentage of aliphatic c = c ( double ) bonds converted into c - c ( single ) bonds to form the polymeric network reached by resin cements is around 60% , due to the increase of cement viscosity during the polymerization reaction , hindering the mobility of the reactive species . the reaction slows down progressively up to a moment when new bonds can not be made . resin cements have been frequently employed for bonding indirect restorations to the teeth due to their mechanical behavior superior to conventional cements ( resin - free ) , possibility of adhesion to the restorative material and to the tooth structure with or without an adhesive system , and superior optical properties when compared with conventional cements . however , limitations associated with the incomplete polymerization ( low dc ) of the cement may result in higher sorption and solubility values , causing faster degradation of the cement finish line by the acids present in the oral biofilm . degradation of resin - based cements reduces the bond strength between them and the substrate and causes dissolution of the finish line at the restoration margin , which may mean the clinical loss of the restoration either by debonding , fracture or secondary caries . unreacted monomers ( not bonded to the polymeric chain ) may also irritate the pulp and generate a local inflammatory response . there are multiple factors that may interfere with the dc of resin cements and , therefore , compromise the longevity of indirect restorations . some of them are the material composition ( monomers and other components of the activation system ) , possible inadvertent interactions between the bonding system and the cement , characteristics of the restoration to be cemented ( optical properties and thickness of the restoration ) and characteristics of the photo activation step . this article aims to perform a comprehensive review of the factors involved in the dc of the resin - based luting systems and the impact of dc on luting system properties . as previously mentioned , photo - activated or light - activated resin cements are indicated for situations where the light of the curing unit may pass through the restoration , such as translucent veneers and shallow inlays . these cements are provided in a single paste with a photoinitiator system composed of a photosensitive component ( usually camphorquinone ) and a tertiary amine . the presence of light with a wavelength of 480 nm ( blue region of the visible spectrum ) activates camphorquinone , which binds to the tertiary amine and then releases two free radicals that will start the monomers conversion . photo - cured resin cements have unlimited working time , with the polymerization starting right after the exposure of the material to light . chemically cured ( self - cured ) cements are indicated under thick restorations , for luting intrarradicular posts and crowns made of materials that block the light , such as metallic copings or highly opaque ceramics , aiming to guarantee maximum properties over time in areas that light energy is unable to reach . the limitations of these systems are the reduced working time as opposed to the extended setting time and the tendency to become yellowish , due to the higher concentration of tertiary amines ( activators ) . the polymerization reaction in self - cured cements requires the components of the activation system tertiary amine and benzoyl peroxide to get in contact by the mixing of two pastes , base and catalyst . dual - cure resin cements were developed in an attempt to combine the benefits of both photo and chemically activated systems , obtaining optimized dc in the deepest locations under a restoration , controlled working time and short setting time . in such systems , there is a catalyst paste with a chemical initiator , usually benzoyl peroxide , and a base paste containing the photo - cured resin cement and the tertiary amine responsible for the activation of the self - cure reaction . when both pastes are mixed together and exposed to light , the polymerization happens by physical ( photo ) and chemical ( redox ) activation . the appropriate working time is controlled by inhibitors of the self - cure reaction or by the amount of activators of the polymerization . it is expected that in areas where there is not enough light , the interaction between the tertiary amine and benzoyl peroxide will be enough to ensure the cement polymerization . however , when not properly photo - activated , dual - cure resin cements may present reduced dc , which implicates in lower hardness , higher solubility , lower flexural and compressive strengths , and lower bond strength to dentin in comparison to directly light - cured dual cements . for instance , a self - adhesive dual cement applied in self - curing mode may show dc as low as 11% after a 10-minute setting time . considering the clinical application of the resin - based luting systems , which are used for the cementation of indirect restorations onto tooth structure , 10 min is an undesirably long time for a luting agent to obtain a great percentage of the optimal setting characteristics , without compromising the integrity of the margins and the cement layer under functional loading . in general , light - cured and dual - cured cements activated by light through a restoration thinner than 2.0 mm have higher dc than self - cured cements . when a dual cement is self - cured ( no activation by light ) , mechanical properties such as flexural strength , modulus and hardness are reduced by 68.9% , 59.2% and 91.1% , respectively , in comparison to original values presented by dual - cured samples . there are different factors that may affect the dc of self - cured luting systems , such as the relatively high concentration of polymerization inhibitors used to extend the material s shelf life and to provide a clinically viable working time , ranging from 2 to 5 minutes , which adversely inhibits polymerization during the luting procedure ; the slow rate of polymerization activation and subsequent propagation of radicals in comparison to a directly light - activated material ; and the low concentration of benzoyl peroxide incorporated into those materials . furthermore , the hand - mixing of the two pastes incorporates air bubbles that further inhibit polymerization due to the presence of oxygen and may act as stress concentrators that potentially result in cracking throughout the cement layer . although it has been demonstrated that the high incidence of air voids reduces the stress generated by the polymerization shrinkage of the cement due to a change in ratio of bonded to unbonded surfaces , the clinical benefits of the inclusion of pores have not been determined . pores are also incorporated in dual - cured cements during mixing and they may become an esthetic concern when cementing veneers . to minimize the undesired consequences of the hand - mixing procedure , some manufacturers provide cements in a self - mixing apparatus ( figure 1 ) , which eliminates the manual mixing step , generates a homogeneous mix and reduces the incorporation of bubbles . however , voids have been observed after automatic mixing as well . figure 1summary of some resin - based luting systems currently available and their characteristics based on the papers included in this review . composition may vary significantly among different materials interestingly , if light incidence on the cement layer is significantly compromised , the chemical activator of dual cements improves dc when compared to photo - activated - only systems but the efficacy of the self - curing mode is still controversial and varies from one material to another . it has been demonstrated that the absence of the self - curing component in light - activated systems negatively affects the dc of these cements when the light - curing component is not able to guarantee an acceptable degree of conversion , for example when applied underneath onlays of greater thickness . considering a clinical application in which almost no light reaches the cement layer , it is desirable to use dual resin cements that present a chemical curing mechanism as efficient as photo - curing . however , there is currently no resin luting system in the market capable of overcoming this limitation . in general , the chemical activation of dual cements does not seem enough to compensate for the absence of light under thick or opaque restorations , even 24 hours after the beginning of the activation . the dc of a self - adhesive dual cement may vary from 37% when light - cured for 20 seconds to 58% when light - cured for 40 seconds , evidencing that there is also a direct correlation between light intensity received by a photo - activated material and its dc . laboratorial studies bring evidence that the activation time generally recommended by the manufacturer ( figure 1 ) is not sufficient to result in maximum degree of conversion . therefore , when highly opaque or thicker restorations need to be employed , a prolonged light exposure time is recommended ( please read indirect restorative material below ) , since a gradual increase in light - curing time and , therefore , in light transmission , gradually increases the knoop hardness of resin - based luting systems . additionally , the use of a dual - cure system should always be considered to possibly increase the dc by means of a chemical activation of the monomeric system . with regard to post - activation time , the 24-hour dc of light - cured and dual - cured cements is directly related to the dc obtained right after light exposure . even though dc is maximized during the first 30 minutes after light activation , some cements present gradual increase in dc for up to 24 hours , mainly when used in the dual - curing mode . however , it has been speculated that a delay in light activation of dual - cured materials would enhance their properties by allowing the self - polymerization promoters to react at some extent before being entrapped by the polymeric chains as soon as the photo - activation begins . delaying the light activation for 2 min may , for instance , compensate for a lower dose of light reaching the cement layer , but no effect is observed on the bond strength of resin cements to the substrate . on the other hand , prolonged self - curing of the cement may also compromise the overall dc and increase water sorption when light activation is delayed for 10 min for the same reason , indicating that an ideal balance between self - curing and photo - activation is yet to be determined . under ideal circumstances , light - activated resin cements show higher dc than chemically cured resin cements , irrespective of brand names . however , the dc of dual - cured cements is material - related , which means that it is more associated with the brand name than with the material classification per se and some systems are significantly more dependent on light activation than others . just as an illustration , the dc of a given dual - cured cement ( relyx arc , 3 m espe , st . paul , mn , usa ) may vary from 81% to 61% when cured under light as opposed to total absence of light respectively , and from 56% to 26% when another dual - cured cement ( relyx unicem , 3 m espe , st . for instance , some resin - based cements present twice as much benzoyl peroxide than others . the lower dc may affect some critical properties of the resin - based cements . dual cements cured under a dual mode ( photo+chemical ) present lower toxicity and solubility than dual cements cured under the self - curing mechanism ( chemical only ) . dual curing also leads to a rapid increase in hardness whereas chemically cured specimens are still soft 30 minutes or even one hour after mixing . dual - curing mode also results in improved bond strength and mechanical properties such as flexural strength , modulus and hardness , in comparison to light curing or chemical curing only . adhesive and self - adhesive resin cements have functional monomers such as 10-methacryloyloxydecyl dihidrogen phosphate ( 10-mdp ) , 4-methacryloxyethyl trimellitate anhydride ( 4-meta ) and phosphoric esters . self - adhesive cements have acidic functionalities in order to demineralize tooth structure , and an acid - base reaction between the acid groups of the monomers and the glass filler of the core material or the mineralized tooth surface starts immediately after the mixing of the components and application of the cement on the tooth surface . however , those acidic monomers have been shown to negatively affect the cement degree of conversion , since they interfere with the amine initiator . this interference compromises both the self - cure and the dual - cure modes . the very low polymerization shrinkage strain of some self - adhesive cements may also be an evidence of reduced dc . indeed , there is a significant variation between the dc of different materials and increasing the light - exposure from 20 s to 40 s does not improve dc values after 6 hours as much as a temperature increase of the cement improves . however , when the absence ( self - cure ) and the presence ( dual - cure ) of photo - activation are compared , the presence of light may result in a 10-fold increase in the material degree of conversion . although another initiator system based on sodium aryl sulfate or aryl - borate salts has been proposed to compensate for the interaction between acidic monomers and the amine initiator in self - adhesive systems , no evidence has been found of any significant improvement in the dc for sodium persulfate - containing materials . another way to improve the polymerization kinetics of resin - based luting systems is to increase the temperature of the material . high viscosity cements have significantly lower degree of conversion than low viscosity cements , probably due to the reduced mobility of the monomers in viscous materials . increased temperature prior to and during polymerization leads to higher dc , due to increased free radical and monomer mobility and collision frequency of the unreacted active groups resulting from the decrease in the viscosity of the material . however , pre - heating ( 50c ) dual - cured resin cements with a higher concentration of the chemical activator ( benzoyl peroxide ) may result in significant decrease in working time , thus compromising the clinical application of the material , and still may not compensate for the absence of light . the clinical applicability of the pre - heating technique is questionable , since the tooth structure could not be possibly heated up to 50c , which would immediately result in the cement temperature decrease . therefore , any evaluation on this topic should limit the pre - heating temperature to 37c . the bonding between resin cement and the tooth structure ( or the core build - up material ) is generally made possible by the use of a self - adhesive resin cement or by the application of a bonding agent / system . the bonding agent / system may either be self - etch or total - etch ( etch - and - rinse ) . however , there are restrictions for the application of some simplified adhesive systems , more precisely two - step total - etch ( primer and adhesive in one bottle ) and all - in - one self - etch systems and resin cements with some chemical activation , either self - cured or dual - cured . it has been shown that the lower the ph of the bonding agent employed , the lower the bond strength between self - cured cement and dentin . the use of a simplified adhesive bonded to a self - cured cement results in 10 - 50% of the bond strength presented when the same adhesive is bonded to a light - cured cement . the reason for those diminished bond strength values is that when simplified - step adhesives are used together with chemical - cured cements , there is an interaction between the residual acidic monomers from the adhesive inhibition layer and the binary peroxide - amine catalytic components that are commonly employed in chemically cured resin composites . therefore , the tertiary amine of the resin cement is neutralized and does not react with the initiator , resulting in low bond strength at the adhesive - cement interface . besides that , the adhesive layer of simplified systems ( all - in - one ) is highly permeable to dentinal fluids due to incomplete polymerization , and these are then kept at the interface between the adhesive and the cement , compromising the bonding between those two substrates , which is demonstrated by exclusively adhesive failure modes . to maximize the performance of the resin cements , self - cured or dual - cure cements are to be employed only in association either with three - step total etch systems or with self - etching primer systems containing a separate bonding agent . for all of the other adhesive systems , the resin cement employed should be exclusively photo - activated . when photo - activation of a resin cement is performed , part of the visible light that reaches the crown is transmitted through the restoration , part is absorbed and part of it is reflected on the surface . consequently , the light intensity that effectively reaches the cement varies according to the optical characteristics of the restorative material , such as opacity and shade , and the final thickness of the restoration . the higher the thickness and the lower the value ( darkness ) of the restoration , the lower the light intensity reaching the cement layer , which may compromise the dc of a given cement . there are many restorative systems nowadays that may be used for the manufacturing of all - ceramic crowns ( figure 2 ) . each one of these ceramic systems has a microstructure that directly interferes with the amount of light that may be transmitted through the restoration . considering restorations with similar shade and thickness , ceramics with a higher number of light scattering centers ( interface between different microstructural phases ) are more opaque and prone to block visible light , compromising the intensity of the physical polymerization of the resin cement . pores , frequently found in feldspathic porcelains and glass - infiltrated composites due to the processing method of these materials , act as light scattering centers as well . a free of pores porcelain would be a material with no light scattering interface and would thus show transmittance , resulting in high dc for dual cements even under a 3 mm - thick layer . a multi - phase material would scatter the light because the incident light beam will change direction from one phase to another and the result will be a weaker incident light . a multi - phase structure within a material also results in light scattering and low transmittance . thereafter , glass - infiltrated alumina - zirconia ( in - ceram zirconia system , vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) is the most opaque alternative among current clinical options , due to the presence of four distinct phases with different refraction indexes ( alumina , ceria - stabilized zirconia , lantanium glass and pores ) , with a final maximum transmittance of only 6% in 0.5 mm - thick copings , and when the thickness of the same material increases to 1.5 mm the transmittance becomes as low as 1% of the initial light intensity . glass - infiltrated spinel ceramic ( in - ceram spinell , vita zahnfabrik , bad sckingen , baden - wrttemberg , germany ) presents significantly higher transmittance because it has only two phases ( glass and spinel ) , with similar refraction indexes . figure 2correlation between indirect restorative materials and the curing properties of the resin cement underneath when comparing the translucency of lithium - disilicate glass - ceramic and leucite - reinforced glass ceramic , illie , et al . ( 2008 ) observed that the first is more opaque than the latter ( figure 2 ) . lithium - disilicate glass ceramic contains a main crystalline phase of elongated crystals building a scaffold of many small interlocking needle - like crystals randomly oriented , with a second crystalline phase consisting of lithium orthophosphate . on the other hand , leucite - reinforced glass - ceramic is a less dense material , characterized by the single crystal formation of leucite crystals , indicating that lithium - disilicate ceramics scatter more light than leucite ceramics . light delivered to the cement layer through lithium - disilicate ceramic ( shade medium opacity 1 ) is reduced to 45% under 1 mm ceramic slabs , 16% under 2 mm slabs and approximately 8% under 3 mm slabs . leucite - reinforced glass ceramic slabs reduce the light transmittance to 80% , 64% and 43% under 0.7 , 1.4 and 2.0 mm thick samples , respectively . as previously mentioned , the relationship between restoration thickness and transmittance is highly dependent on the opacity of the material . however , the impact of the amount of light reaching the cement layer on its dc is controversial . dual - cure resin cements activated by light under a 1.5 mm lithium - disilicate glass ceramic ( shade a2 low translucency ) surface presented a dc similar to that of cements cured under direct light exposure , whilst samples cured through 1.4 mm - thick leucite - reinforced glass - ceramic slabs may or may not show significantly lower hardness values than groups activated with direct light exposure , depending on the luting system employed . in another study , samples light - cured under 1 or 2 mm thick lithium - disilicate slabs only showed decreased hardness when light exposure time was 20 s or less , indicating that longer exposure times may compensate for light attenuation of the indirect restorative material . a randomized clinical split - mouth study evaluating the longevity of glass - infiltrated alumina crowns cemented with three different cements ( two resin - based and one glass - ionomer ) evidenced acceptable survival rates for all groups , with dual - cured cements showing higher survival rate than glass - ionomer cement , indicating that the opacity of the crown did not affect the performance of the cement / restoration . it is important to remember that the final absolute transmittance values of a restoration would be even more compromised considering the thickness and the optical characteristics of the porcelain veneer layer . the dc of a dual - cured cement activated under glass - infiltrated alumina ( 1.2 mm thickness ) with porcelain veneer layer ( 0.8 mm thickness ) is significantly reduced when compared to feldspathic porcelain samples ( 2 mm thick ) and to the control group , activated under direct light exposure . when the impact of the shade of the ceramic system is evaluated , it can be observed that if shades with higher chroma are used , less energy reaches the cement layer , since dark pigments absorb a significant amount of light , negatively influencing the cure of light - dependant cements . dual - cured cements light - activated under 2 mm - thick samples of darker dentin shade of feldspathic porcelain present significantly lower dc than cements light - activated under lighter shades . when yellow and translucent shades of a resin cement were light - activated under the darker porcelain , only prolonged light - exposure time ( 40 seconds ) was capable of increasing the dc of the cement yellow shade , indicating that the combination of darker shades in both the cement and the indirect restorative material compromise the overall dc of the cement layer . with regard to laminate veneers , some studies show that although the bond strength between veneers and tooth structure is not affected by shade or opacity of the ceramic system , the dc of the cement may be diminished by either thicker , darker or more opaque restorations , frequently used to mask severely darkened teeth , and a lower dc of the cement layer may compromise the esthetic result due to the continuous discoloration of the material . the analysis of the dc of a light - cured resin cement after the superimposition of different veneer materials with different thicknesses indicated that the effect of light attenuation on the degree of conversion is not significant only for ceramic thicknesses of 1.0 mm or less . considering the optical properties of the indirect restorative composites , there are different factors playing a role in light transmittance , such as particle size distribution , thickness of the restoration and shade . the smaller the particles , the more interfaces will be present acting as light scattering centers , consequently increasing the opacity of the material employed , which indicates that larger particles allow for deeper activation of the cement layer by light . interestingly , the hardness of dual resin - based cements is less affected when photo - activation is performed through an indirect restorative composite either microfilled or micro - hybrid than when it is performed through an all - ceramic system lithium disilicate and glass ceramic . when the effect of thickness is evaluated , there is indeed an inverse correlation between thickness of an indirect composite resin restoration and knoop hardness of the resin based luting system . dual resin cements cured under 2 mm - thick micro - hybrid composite samples show significantly lower dc than samples cured under ideal conditions , and the dc of dual cements is 12% lower under 4 mm onlays in comparison to that measured under 2 mm thick onlays . with regard to the effect of shade on indirect composite resin light transmittance , arrais , et al . ( 2008 ) demonstrated that only 11% of light reaches the cement layer when cured through a 2 mm microhybrid composite a2 shade as opposed to 8% when a4 shade was employed , but no effect on dc was observed for dual - cured resin cements with higher concentration of benzoyl peroxide . the authors pointed out that the adhesive component also presented a chemical activator of the polymerization and could , therefore , compensate for the absence of light . it has been demonstrated that the hardness of dual - cured cements is dependent on the level of exposure to the curing light . as previously mentioned , the component responsible for the chemical activation of the material can not compensate for the total absence of light . the higher the light intensity and the longer the exposure time of the resin cement , the higher the knoop hardness of the dual - cured materials . however , even when under direct light exposure , there is a limit above which the dc of a photo or dual - cure cement can not be increased . quartz - tungsten - halogen ( qth ) light curing units ( lcu ) deliver light irradiance varying between 400 and 1360 mw / cm2 . when exposure time ( 40 s , 60 s or 120 s ) and intensity ( 1200 , 800 or 400 mw / cm ) of light exposure on dc of dual cements was evaluated , different materials showed different results , although all the associations resulted in the same amount of energy ( 48 j ) . activation of dual cements under 2 mm resin composite onlays using low light intensity for prolonged time presented a trend towards higher dc , probably due to the slow increase in the material viscosity , allowing more monomers mobility . light - emitting diodes ( led)-based units were introduced in the market in 2001 and are another option to activate photo - cured resin cements . these units generate light under a narrower spectrum ( between 450 and 490 nm ) with the peak around 468 nm , the ideal wavelength for resin - based materials using camphorquinone as the photoinitiator . when the photo - activation of a cement is performed through a ceramic system , light transmittance increases for higher wavelengths . the higher mean wavelength of led lights improves the capacity of the equipment to activate resin cements under indirect restorations . however , light - intensity is also critical , since led with relatively low light intensity ( 320 mw / cm ) results in decreased knoop hardness at the bottom of dual - cured cement samples . the effect of qth ( 905 mw / cm ) and led ( 1585 mw / cm ) curing units on knoop hardness of resin - based cements indicated that there was no effect of lcu on the hardness of dual - cure cements . samples cured under 1.4 and 2.0 mm ceramic slabs ( leucite glass ceramic , shade a3 ) showed lower hardness values than samples cured under direct light exposure and under 0.7 mm slabs . authors observed that hardness on dual - cured luting agents may not be dependent on the light source , as long as the irradiance level for the effective wavelength region to activate the photo - initiator is similar . with the application of high - power curing units in dentistry , led - based equipment with high light intensity ( 1000 - 1600 mw / cm ) are being advertised as an alternative to reduce the curing time of resin - based materials . however , the minimum time required to properly cure a dual - cured luting system is 15 s under ideal conditions , so that maximized mechanical properties can be obtained . therefore , it is not recommended to reduce the light exposure time to less than 15 seconds on each side of a restoration , irrespective of light intensity . indeed , it has been demonstrated that light - curing a dual - cure cement for 9 s with a led device ( 1100 mw / cm ) results in significantly reduced degree of conversion . the authors observed that exposing dual - cure material to high intensity light may increase its viscosity more rapidly , hindering the migration of active radical components responsible for further polymerization . similar results were obtained when led device ( 1100 mw / cm ) with different activation modes and qth ( 600 mw / cm ) were used to photoactivate resin cements between ceramic samples ( lithium disilicate ) and human dentin , and the authors found out that groups photo - activated for 10 s presented inferior bond strength . higher bond strength results were obtained when led devices under exponential mode and qth were used , and since the exponential mode was applied for twice as much time as the other led groups , the overall energy delivered was increased , which may have enhanced the dc . authors also observed that higher light intensity produces higher contraction strains during resin polymerization , which may promote debonding at the adhesive interface . therefore , prolonged exposure times are desirable not only to increase the energy delivered to the luting material , in an attempt to compensate for the attenuation of the light promoted by the indirect restorative material , but also to reduce stress generation at the cement - substrate interface , to ensure preservation of the bonding . a comparison of different light curing equipment ( qth 600 mw / cm ; led 1400 mw / cm ; argon ion laser 600 mw / cm ) used to activate resin cements under 2 mm - thick samples of composite resin indicated that the degree of conversion of the resin cements is again more related to the commercial brand and , consequently , to the material composition than to the curing device itself , with led and argon ion laser devices resulting in lower dc for one of the materials in the photo - cured mode . although the short range of the spectra peak for led devices may be advantageous when curing under ceramic systems , a wider range may be clinically interesting to photo - activate alternative photoinitiators , promoting a higher dc for qth lights even in the presence of lower light intensity . in addition to the factors presented above , there are other variables playing a role in the dc of light - activated resin - based cements , such as the distance between the tip of the curing device and the cement layer and other indirect factors reducing the light intensity being delivered . based on the results presented and the number of studies indicating that prolonged light - activation may be beneficial for the dc of dual- or photo - cured cements , increasing the light exposure time , even though this would mean a couple more minutes of clinical procedure , would be certainly beneficial for the clinical performance of an indirect restoration . the clinical success of an indirect restoration is not only attributed to the dc of the resin cement or to its mechanical properties , since there are other aspects that determine the clinical performance of dental prostheses . nonetheless , ensuring a high dc is paramount to obtain the best out of the chemical and physical properties of the resin cement , besides being a critical factor for biocompatibility . when performing a luting procedure , one should pay attention to the characteristics of the indirect restorative material to be employed , and make a conscious decision of using a cement system that would be more indicated to the clinical case necessities . curing modes and the best light - curing technique are examples of information that is to be available . it is crucial for clinicians to know and understand the cement systems they are working with .

resin - based cements have been frequently employed in clinical practice to lute indirect restorations . however , there are numerous factors that may compromise the clinical performance of those cements . the aim of this literature review is to present and discuss some of the clinical factors that may affect the performance of current resin - based luting systems . resin cements may have three different curing mechanisms : chemical curing , photo curing or a combination of both . chemically cured systems are recommended to be used under opaque or thick restorations , due to the reduced access of the light . photo - cured cements are mainly indicated for translucent veneers , due to the possibility of light transmission through the restoration . dual - cured are more versatile systems and , theoretically , can be used in either situation , since the presence of both curing mechanisms might guarantee a high degree of conversion ( dc ) under every condition . however , it has been demonstrated that clinical procedures and characteristics of the materials may have many different implications in the dc of currently available resin cements , affecting their mechanical properties , bond strength to the substrate and the esthetic results of the restoration . factors such as curing mechanism , choice of adhesive system , indirect restorative material and light - curing device may affect the degree of conversion of the cement and , therefore , have an effect on the clinical performance of resin - based cements . specific measures are to be taken to ensure a higher dc of the luting system to be used .

the annual incidence of the 2 most common neuroendocrine tumors , insulinoma and gastrinoma , is about 1 per million . although insulinomas are usually benign , 60% to 80% of gastrinomas are malignant . hepatic or nodal metastases are found in 60% of patients diagnosed with zollinger - ellison syndrome ( zes ) . long - term ( 5-year ) survival of patients with resected extrahepatic gastrinoma is 95% . this number is decreased to 85% in patients with resected hepatic metastases . however , 5-year survival in patients with unresected hepatic gastrinoma is a dismal 30% . other methods have been tried in patients with tumor locations or medical problems prohibiting major hepatic resection . these include embolization , chemoembolization , immunotherapy ( interferon alpha ) , liver transplantation , and chemotherapy . more recently , radiofrequency ablation ( rfa ) has been used in cases of unresectable hepatocellular or colorectal metastases . curley et al demonstrated a 1.8% recurrence at a mean follow - up of 15 months in a series of 169 patients . wood et al , in a series of 231 patients , cited an 18% recurrence rate at a mean follow - up of 9 months . we report a case of a 5-cm solitary hepatic gastrinoma treated with rfa with a resulting 1-cm margin on the tumor . we propose rfa as an alternative to major hepatic resection in patients with solitary hepatic gastrinoma . our patient is a 65-year - old male who was originally treated at an outside hospital in 1988 for a perforated gastric ulcer . he was referred to our institution in 1991 after a workup of his ulcer disease led to a diagnosis of zes . this diagnosis was based on elevated serum gastrin levels , a positive secretin stimulation test , and refractory ulcer disease . he underwent several localizing studies including an abdominal computed tomographic ( ct ) scan and radionucleotide scanning with octreotide . no other areas of activity were found on the octreotide scan . at exploratory laparotomy , several biopsies were taken from the following locations : left lateral lobe of the liver , right lobe of the liver ( diaphragmatic surface ) , peri - portal lymph nodes , and 2 anterior pancreatic nodules were shelled out . in addition , an anterior duodenotomy was performed from the pylorus through the third portion . he has since been managed on proton - pump inhibitors and routine medical follow - up , including repeat ct scans . in april 2001 , a ct scan revealed a 5-cm mass in the right lobe of the liver . octreotide scanning at this time demonstrated intense activity in the right lobe of the liver ; however , the left adrenal nodule remained inactive . over the course of the past 10 years , the patient has developed advanced emphysematous disease of the lungs , gained 100 lbs , and developed type ii diabetes . the location of the tumor near major branches of the portal vein would have necessitated a major right hepatic lobectomy ( figure 1 ) . after discussion with the patient and his family regarding his surgical options , the patient was given the additional option of rfa via a laparoscopic approach for his liver tumor . it was also recommended that he undergo a laparoscopic left adrenalectomy due to the enlarging left adrenal nodule . we used the aloka , reticulating laparoscopic ultrasound to examine the liver and guide rfa probe placement . the rita model 1500 radiofrequency generator was used in our patient at a frequency of 460 khz . the ablation device was the rita , starburst xl ( for 3- to 5-cm ablation ) . the electrode was first deployed at the most posterior interface , and then , after ablation , was subsequently withdrawn and redeployed at the most anterior interface in the tumor to create overlapping zones of necrosis ( figure 2 ) . power delivery was set at 50 watts , and the lesion was heated to a temperature of 90c for 10 minutes . upon withdrawing the electrode , the patient 's rfa was complicated by an intraoperative right pneumothorax , believed to be caused by a puncture to the diaphragm by the 25-gauge - seeker needle used to locate the tumor . this was treated by placement of a 22-french chest tube , which was removed on post - operative day 2 . pathologic examination of the adrenal gland revealed a 4.5 5.1 4.5-cm adrenal cortical adenoma . his follow - up ct scan ( figure 3 ) demonstrated complete destruction of the lesion seen on the preoperative ct scan with an apparent 1-cm additional margin based on the increased size of the ablative lesion to 6.0 cm 6.0 cm 5.1 cm . postoperative blood work revealed the following : gastrin 473 pg / ml , insulin 24 u / ml , serotonin 244 ng / ml , and pancreatic polypeptide 852 pg / ml . a second follow - up ct scan was done 3 months postoperatively to reevaluate this lesion ( figure 4 ) . this demonstrated a decrease in size of the ablated lesion , and no evidence of new lesions . repeat blood work at this time revealed an elevated serum gastrin of 754 pg / ml . his serum pancreatic polypeptide and serotonin , however , were decreased to 660 pg / ml and 161 ng / ml , respectively . in light of this , a radionucleotide scan with octreotide was performed . this patient will be followed with octreotide scans and ct scans on a biannual basis . nets fall into 1 of 5 categories : insulinoma , gastrinoma , glucagonoma , vipoma , and somatostatinoma . the majority of primary gastrinomas are found in the duodenum ( 40% to 50% ) , the head of the pancreas ( 30% to 50% ) , and lymph nodes ( 17% ) within the gastrinoma triangle ( defined by the junction of the cystic duct with the common bile duct , junction of the second and third portion of the duodenum , and the junction of the neck and the body of the pancreas ) . ectopic gastrinomas can be found in the stomach , parathyroids , kidney , common bile duct , ovary , lymph nodes , and liver . the presence of neuroendocrine cells in the normal bile duct explains the possible development of ectopic primary hepatic gastrinoma . although a 20% correlation exists between zes and multiple endocrine neoplasia type 1 ( men 1 ) , no correlation appears to exist between primary hepatic gastrinoma and men 1 , based on the cases reported in the literature . making a diagnosis of primary hepatic gastrinoma relies on preoperative localizing studies and a thorough intraoperative exploration to rule out an occult primary with hepatic metastases . ct can miss smaller hepatic lesions , with 1 study citing 63% sensitivity for gastrinoma . mri was found to be more sensitive ( 78% ) but can not distinguish between gastrinomas and small hepatic hemangiomas . recent studies have shown somatostatin receptor scintigraphy ( srs ) to have a sensitivity of 90% with few false positives . intraoperative ultrasound ( ious ) is the most sensitive device for detection of hepatic tumors . ious is even more sensitive than palpation , which can miss some smaller lesions located deep in the liver parenchyma . once the diagnosis of hepatic gastrinoma has been made , the next step is treatment . currently , surgery is the only effective treatment for gastrinoma . for those patients who are not candidates for surgical resection , other treatment options must be explored . in 1 prospective randomized study of 213 patients with zes , only 17 ( 8% ) had localized liver gastrinoma and were candidates for surgical resection . this has been used for treating primary hepatocellular carcinoma and hepatic colorectal metastases that were unresectable secondary to the number of lesions , anatomic location near main portal vessels , or inability of the patient to tolerate major hepatic resection . radiofrequency ablation ( rfa ) is a thermal treatment technique designed to produce localized tumor destruction by heating tumor tissue to temperatures that exceed 50c . when tumor cells are heated to temperatures above 45 to 50c for more than 3 minutes , intracellular protein denaturation and melting of lipid bilayers results in direct tumor cell death . ionic stimulation induced by the alternating current in tissue surrounding the electrode array produces gradual frictional heating , and the tissue temperatures rise to 80 to 110c , which results in coagulative necrosis of the tissue in proximity to the electrode . the basic principle is similar to that of the surgical electrocautery units used to achieve intraoperative hemostasis . novel rf needle designs have been developed with multiple - array hook electrodes that are deployed from the needle tip into the tumor . the insulated 14- to 18-gauge needle electrode shaft is placed into the tumor with the multiple - hook array retracted . using real - time ultrasonographic guidance the multiple - array hook electrodes are available in maximum diameters of 2.0 to 5.0 cm . in contrast to the small areas of tumor tissue ablation created by simple needle electrodes , multiple - array electrodes , such as the leveen electrode , can produce zones of coagulative necrosis 2.0 to 6.0 cm in diameter . for tumors less than 2.5 cm in diameter , the multiple arrays are deployed into the center of the tumor . for larger lesions , the array is first deployed at the most posterior interface ( ultrasonographically ) between tumor and normal liver parenchyma ; it is subsequently withdrawn and redeployed at 2.0- to 2.5-cm intervals in the tumor . optimal positioning of the electrode permits complete destruction of the tumor and at least a 1-cm zone of normal liver parenchyma . in our case , we describe a successful laparoscopic rfa of a 5-cm hepatic gastrinoma with a 1-cm margin . the patient was discharged from the hospital on postoperative day number 3 , and avoided major hepatic resection . as had been previously demonstrated , intraoperative ultrasound provides better resolution of the tumor for rfa treatment compared with transabdominal ultrasound or ct for percutaneous treatment . incomplete tumor destruction has been reported in up to 18% of liver cancers treated percutaneously with rfa . this underscores both the importance of careful treatment planning to overlap the zones of necrosis and the need for early post - rfa , ct , or mri to detect incompletely treated tumors after percutaneous rfa . no studies exist at this time documenting long - term follow - up of patients with hepatic gastrinoma treated with rfa . in our case , we achieved satisfactory immediate results based on postoperative ct showing adequate ablation of the tumor with a 1-cm margin . these results were supported by 3-month follow - up studies including a ct scan and an octreotide scan , both showing evidence of adequate ablation of the tumor . concern still exists about the presence of an unidentified gastrinoma primary as is evidenced by the patient 's persistent elevated serum gastrin . the slow - growing nature of this tumor , however , will make radiologic surveillance for treatable disease possible . although surgical resection remains the gold standard for treatment of hepatic gastrinomas , rfa may be a promising alternative for those patients in whom hepatic resection is not possible .

background : this is a case of a solitary hepatic gastrinoma in a 65-year - old male . the patient was diagnosed with zollinger - ellison syndrome in 1991 . he had negative radiologic and surgical explorations at that time . he was maintained on proton - pump inhibitors for the next 10 years without symptoms.methods:a computed tomographic ( ct ) scan done in april 2001 demonstrated a 5-cm right hepatic lesion . radionucleotide scanning with octreotide demonstrated intense activity in the same area in the right hepatic lobe . his serum gastrin was 317 pg / ml . he underwent laparoscopic radiofrequency ablation of the lesion.results:treatment resulted in a 6-cm ablative area giving a 1-cm margin on the tumor . one- and 3-month follow - up ct scans demonstrated adequate ablation of the tumor . an octreotide scan done 3 months postoperatively did not reveal any areas of abnormal uptake.conclusion:we report success with laparoscopic radiofrequency ablation as an alternative to major hepatic resection in patients with a solitary hepatic gastrinoma .

the surface cap - sular polysaccharide of s. pneumoniae initiates a type - specific protective immune response and provides the basis for serotyping of this organism . currently , more than 90 different pneumococcal serotypes have been identified . among the various serotypes and according to 60 - 80 percent of the worldwide reported infections , the 6 , 14 , 18 , 19 , and 23 are the most prevalent ones ( 1 ) . nasopharyngeal colonization of s. pneumoniae varies according to age and health status ( 2 ) . in the industrial world , the expected annual rate of pneumonia is about 14.5 per 10,000 in children under age 16 years old ( 3 ) . the mortality rate due to s. pneumoniae in developed countries is low ( 4 ) . on the contrary , in developing countries , respiratory tract infections are more common and more severe , accounting for more than 2 million deaths annually ; pneumonia infections are the number one killer of children in these societies(5 ) . lower socioeconomic groups have a higher prevalence of lower respiratory tract infection , which correlates best with family size , as a reflection of environmental crowding ( 6 ) . in spite of the availability of a vaccine covering 23 different serotypes , s. pneumoniae is currently the major invasive pathogen of children and adults and is a principal cause of otitis media , pneumonia , bacteremia , and meningitis(7 ) . information about the distribution of pneumococcal serotypes and demographic characteristics related with s. pneumoniae colonization , are essential for the proposing of strategies to prevent and control these infections ( 7 ) . the distribution of serotypes also varies between carriage isolates and invasive disease and antibiotic resistance is most common in pneumococcal serotypes ( types 6 , 9 , 14 , 19 , and 23 ) that are carried by children ( 8) . on the other hand , the pnenmococcal vaccination is not presently a part of the childhood immunization plan in iran , and data about the serotype distribution and prevalence of s. pneumoniae colonization , antibiotic susceptibility and risk factors of nasopharyngeal colonization of s. pneumoniae in healthy school - age children is scarce . the objectives of this study were to determine the nasopharyngeal colonization rate , serotyping , antibiotics susceptibility and risk factors for nasopharyngeal colonization with s. pneumoniae from students in kashan , iran . overall , 2100 students enrolled in this study sampled from december 2011 to november 2012 in 22 schools in kashan , iran . we excluded children showing symptoms or signs of respiratory infection , as well as those who received at least one dose of any antibiotic treatment during the previous 15 days . one of the children s parents signed informed consent and provided clinical and demographic information , such as age , sex , crowding ( two or more people sleeping in the same room ) , smokers at home , and socioeconomic status . detailed medical information such as previous diagnosis of asthma , allergic rhinitis and history of hospitalization in previous 6 months , and antibiotic treatment in previous 3 months were recorded . specimens from nasopharynx of the students were obtained by using a sterile cotton - tipped swab . swabs were plated immediately onto brain heart infusion agar plates with 5% sheep blood and 4g / ml of gentamycin . s. pneumoniae isolates were identified by gram staining , colony characteristics , susceptibility to optochin , and bile solubility . s. pneumoniae strains were investigated for the presence of the most common pneumococcal serotypes using a multiplex polymerase chain reaction ( pcr ) assay as described previously(9 ) . the susceptibility pattern of s. pneumoniae strains to rifampin ( 5 g ) , erythromycin ( 15 g ) , oxacillin ( 1 g ) , linezolid ( 30 g ) , clindamycin ( 2 g ) , levofloxacin ( 5 g ) , vancomycin ( 30 g ) , tigecycline ( 15 g ) , and tetracycline ( 30 g ) were determined by disk diffusion method according to clinical and laboratory standards institute ( clsi ) guidelines(10 ) . the study population included 2100 healthy school - age children aged 7 to 19 years ( 13.08 3.284 years ) , of which ( 1275 ) 60.7% were male . the distribution of age was as follows : 7 - 11 years : 730 ( 34.8% ) students ; 12 - 15 years : 909(43.2% ) students ; and 16 - 19 years : 461(22% ) students . the demographic and clinical characteristics of the students enrolled in this study are listed in table1 . the percentage of s. pneumoniae colonization showed a peak ( 144 out of 909 ) at the age of 12 - 15 years old ( 15.8% ) ( p < 0.009 ) and a low prevalence of 9.8% was observed in 16 - 19 years old students . as mentioned above , the highest rates of s. pneumoniae colonization were observed among children with age 12 - 15 years old ( 15.8% ) , male sex ( 18.8% ) , rhinorrhea ( 21.4% ) with previous history of respiratory infections ( 18.9% ) . the occurrence of s. pneumoniae colonization was not statistically differs between children with asthma , frequent respiratory infection , and crowding ( table 1 ) . the s. pneumoniae strains isolated in this study showed 8 different pneumococcal serotypes , of which 19f ( 30% ) , 6a / b ( 18.9% ) , 15a ( 16.5% ) , 11 ( 11.3% ) 23f ( 8.2% ) , 1(6.2% ) , 19a ( 3.4% ) and 35b ( 2.4% ) were the most common ones . the risk factors for pneumococcal carriage among students in kashan , iran the most resistance rate among isolated s. pneumoniae strains was tetracycline ( table 2 ) . the various observed serotypes among the 43 penicillin - non susceptible s. pneumoniae_strains were as follow : 6a / b ( 25.6% ) , 11(18.6% ) , 19f ( 18.6% ) , 15a ( 14% ) , 1(9.3% ) , 23f ( 4.6% ) , and non - typeable ( 9.3% ) . the most resistance rate to oxacillin observed in serotypes 11(24.2% ) , 1 ( 22.2% ) , and 6a / b ( 20% ) . the serotypes observed among the 74 tetracycline resistance s. pneumoniae strains were 19f ( 27% ) , 15a ( 18.9% ) , 6a / b ( 17.6% ) , 1(9.4% ) , 11(9.4% ) , 23f ( 8.1% ) , 19a ( 5.4% ) , 35b ( 1.4% ) and non - typeable ( 2.7% ) . the most resistance rate to tetracycline detected in serotype 19a ( 40% ) , 1(38.8% ) , 15a ( 29.2% ) , 23f ( 25% ) , 6a / b ( 23.6% ) , 19f ( 23% ) , 11(21.2% ) and 35b ( 14.3% ) . frequency rates of antimicrobial resistance among 291 s. pneumoniae strains isolated from students in kashan , iran according to serotypes in the present study , we observed a pneumococcal colonization prevalence of 13.9% in kashan , iran . the prevalence of s. pneumoniae colonization described in different parts of the world has changed widely . a variety of demographic and clinical characteristics have been explained to be related with an increase in s. pneumoniae colonization , for example age , society , family size , crowding , siblings , smoking at home , recent antibiotic use , and socioeconomic status of study population regarding variations in sampling and isolation techniques ( 2 , 11 , 12 ) . in our study previous history of upper respiratory tract infection within the last month , hospitalization , antibiotic usage during last two weeks , rhinorea , male sex and passive smoking have been determined to be risk factors for s. pneumoniae _ carriage . cigarette smoking compromises natural pulmonary defense mechanisms by disrupting both muco - ciliary function and macrophage activity . exposure to cigarette smoke , especially in smoking mothers increases the risk of pneumonia in infants younger than one year of age . we did not observe statistical differences in the prevalence of s. pneumonia colonization between children with asthma and children without respiratory distresses . s. pneumoniae _ colonizes at the upper respiratory tract and plays role as normal flora of healthy individuals . a particular serotype can be carried for many months before being eradicated or replaced by a different serotype . carriage increases in the first few months of life and the highest asymptomatic colonization rates ( > 40% ) observed in young children . factors associated with increased carriage including winter season , day care crowding , and living in crowded conditions . carriage in adults is approximately 10 - 20% and the duration of carriage is generally shorter . antibiotic resistant serotypes most frequently related to pneumococcal strains which carried by children ( types / groups 6 , 9 , 14 , 19 , and 23 ) ( 8) . resistance to tetracycline was observed in 25.4% of the strains . we did not find any s. pneumoniae_strains showing resistance to levofloxacin , linezolid , and vancomycin . therefore , exposure of these serotypes to antimicrobial drugs , may leads to a selective advantage of resistant mutants(13 ) . most of these factors have a unity in exposure to the drugs that select the resistance . macrolide resistance is also a function of exposure , particularly for a long - acting drug such as azithromycin ( 13 ) . the macrolides have been used extensively to treat community - acquired respiratory tract infections around the world , and in recent years macrolides resistance in s. pneumoniae has been raised considerably . macro - lide - resistant s. pneumoniae _ are now more common than penicillin - resistant s. pneumoniae_in many parts of the world(14 ) . the selection of resistant strains is complicated by multiple resistances where macrolides appear to be better selectors of multi - resistant strains than -lactam drugs do ( 15 ) . fluoroquinolone resistance in s. pneumoniae relatively remains low around the world ( less than1% ) , but it tends increasing , particularly in some countries . studies carried out over the past decade recommended that the prevalence of levofloxacin - resistant strains in the north america remains less than 2% ( 1618 ) . upper rates have been reported in spain ( 7% ) , sri lanka ( 9.5% ) , the philippines ( 9.1% ) , korea ( 6.5% ) and hong kong ( 15.2%)(1922 ) . the prevalence of rifampin resistance among pneumococcal isolates is low at the present time , and reported rates vary between 0.1 and 1.5% ( 23 ) . in our study the prevalence of penicillin resistance among s. pneumoniae _ isolates ranged from 18.2 to 22.1%(24 , 25 ) . reported the prevalence of antimicrobial resistance among s. pneumoniae _ strains to erythromycin 38% , penicillin 34% ( intermediate 20% and high level resistance 14% ) , tetracycline 34% , and clindamycin 29%.(1 ) . resistance to penicillin is associated with some degree of non - susceptibility to all -lactam antibiotics . resistance rates reported for amoxicillin are relatively low ( less than 5% ) ( 26 , 27 ) . tigecycline is the prototype compound of a new class of antimicrobial agents known as glycylcyclines . this derivative of minocycline provides clinicians with a novel , expanded broad - spectrum antibiotic with activity against difficult - to - treat pathogens . worldwide , serotypes 6b , 6a , 9v , 14 , 15a , 19f , 19a , and 23f have shown the highest rates of resistance to penicillin and erythromycin , with the highest rates in respiratory isolates reported in south africa ( 74% ) , asia ( 63% ) and the middle east ( 54%)(28 ) . the data from molecular typing upon numerous studies has shown that there is considerable diversity among resistant strains within most serotypes(29 ) . mcgee et al . found 30.5% of erythromycin - resistant pneumococci and showed that 83% of these isolates were associated to a serotype 19f strain originating in taiwan(30 ) . monitoring the changes in pneumococcal nasopharyngeal carriage in children is important , and may provide relevant information on the identification of serotypes that may significantly contribute to pneumococcal diseases . in this research 57% ( 166 out of 291 ) of the isolated serotypes were identified as 19f , 6a / b and 23f . though there are at this time over 90 distinct serotypes , definite serotypes commonly account for the majority of s. pneumoniae_nasopharyngeal isolates , but the circulation of serotypes differs in relation to geographic position . though serological determination by a quelling reaction of pneumococcal cells with anti - polysaccharide sera is the presently used method for pneumococcal serotyping the high cost of antiserum , the requirements for technical proficiency and bias in interpretation of results are major limitations of the procedure . multiplex pcr has the likely to overcome these problems and has been reported to have higher sensitivity and specificity than conventional quelling reaction techniques ( 9 , 3133 ) . the serotype distribution between carriage isolates is frequently used as an indicator for hypothetical vaccine coverage . vaccination could potentially reduce the carriage rate of antibiotic - resistant pneumococci . at the present time the pneumococcal vaccination is not a part of the childhood immunization program in iran . the best to our knowledge , there are few data on nasopharyngeal pneumococcal colonization and serotyping among children in iran . a high prevalence of s. pneumonia colonization in the nasopharynx in students was observed and a high rate of resistance to -lactams and tetracycline of isolates was confirmed . we found that pneumococcal serotypes 19f , 6a / b were the most common carried serotypes in our studied population . for increase protection against s. pneumonia , it is recommended to vaccinate infants and young children with pneumococcal vaccines . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

abstractbackgroundstreptococcus pneumoniae is an important problem worldwide and nasopharyngeal colonization plays significant role in pneumococcal infections . the aims of this study were to determine the nasopharyngeal colonization rate , serotyping , antibiotics susceptibility and study the risk factors for nasopharyngeal colonization with s. pneumoniae in students in kashan , iran.methodsa cross - sectional study was conducted on children aged 7 to 19 years from december 2011 to november 2012 . nasopharyngeal swabs were plated onto brain heart infusion agar plates with 5% sheep blood and 4g / ml of gentamycin . antimicrobial susceptibility profiles were determined on mueller - hinton agar in accordance with clsi . s. pneumoniae strains were investigated for the presence of the most common pneumococcal serotypes using a multiplex polymerase chain reaction.results13.9% were found to be carriers . the most prevalent serogroups were 19f ( 30% ) , 6a / b ( 18.9% ) , 15a ( 16.5% ) , 11 ( 11.3% ) , 23f ( 8.2% ) , 1 ( 6.2% ) , 19a ( 3.4% ) , and 35b ( 2.4% ) . nine strains ( 3.1% ) were non - typeable . the carrier rate was significantly higher in 12 to15 year old age group . upper respiratory tract infections within the last month ( or=1.5 , p<0.011 ) , previous hospitalization ( or=1.6 , p<0.001 ) , previous antibiotic usage last two weeks ( or=1.89 , p<0.001 ) , rhinorea ( or=1.9 p<0.001 ) , male sex ( or=3.5 p < 0.001 ) and passive smoking ( or=1.56 , p < 0.001 ) have been determined to be risk factors for s. pneumoniae carriage . the highest pneumococcal resistance was to tetracycline ( 25.4% ) . all strains were susceptible to linezolid and levofloxacin.conclusionour information leads to an important source to screen the future impact of pneumococcal vaccination on bacterial colonization .

loss of muscle mass is commonly observed in chronic diseases like cancer , chronic heart failure ( hf ) , chronic obstructive pulmonary disease , chronic kidney disease ( ckd ) , cystic fibrosis , liver cirrhosis , crohn s disease , rheumatoid arthritis ( ra ) , stroke , and many neurodegenerative diseases as well as in human immunodeficiency virus / acquired immune deficiency syndrome , malaria , and tuberculosis.13 a serious complication of these chronic illnesses is cachexia . cachexia is defined as weight loss greater than 5% of body weight in 12 months or less in the presence of chronic illness or as a body mass index ( bmi ) lower than 20 kg / m . in addition , usually three of the following five criteria are required : decreased muscle strength , fatigue , anorexia , low fat - free mass index , increase of inflammation markers such as c - reactive protein or interleukin ( il)-6 as well as anaemia or low serum albumin.4,5 loss of muscle mass and function , especially muscle strength and gait speed , associated with aging occurs in sarcopenia.6,7 indeed , sarcopenia , cachexia , and malnutrition are considered as the main causes of muscle wasting8 and affect millions of elderly people and patients.9 moreover , muscle atrophy can develop independently from diseases and age through disuse of the muscles.10 for a better classification and common language in medical science for muscle wasting disease there is a proposal to combine the concepts of muscle wasting , sarcopenia , frailty , and cachexia by disease aetiology and disease progression.8 patients with muscle atrophy show decreased muscle strength and therefore reduced quality of life , which is caused by a lower activity and increased exercise intolerance.11 in sarcopenic patients , muscle wasting is frequently associated with loss of bone , which leads to a higher risk of hip and other fractures.12 hip fracture also results in loss of musculature because of disuse atrophy.13 all these conditions lead to increased morbidity and mortality in patients,14 and therefore developments in biomarkers and treatment finding to improve patients lives is necessary ( for schematic representation of the process see figure1 ) . three major protein degradation pathways play a role in development of muscle wasting : ( 1 ) activation of the ubiquitin proteasome system ( ups ) , ( 2 ) apoptosis through caspase signalling , and ( 3 ) autophagy.15 muscle mass loss is caused by many reasons resulting in morbidity and mortality which makes it necessary to find appropriate biomarkers and treatment strategies to improve patient s quality of life . the ups pathway , which is conserved from yeast to mammals , plays a major role in degradation of most short - lived proteins . polyubiquinated proteins are subsequently degraded by the proteasome16 while monoubiquitinated substrates are eliminated in lysosomes.17 at the beginning of the reaction , ubiquitin binds to an ubiquitin activating enzyme ( e1 ) and forms a thio - ester bond . this reaction allows ubiquitin to transfer to an ubiquitin conjugating enzyme ( e2 ) followed by the formation of an isopeptide bond which finally leads to the binding of e2 to an ubiquitin ligase ( e3 ) . the ligase specifically recognizes the substrate protein and transfers ubiquitin to the target protein.18 subsequently , the target proteins are unfolded and degraded by an atp - dependent process.19 two muscle - specific e3 ubiquitin ligases named muscle atrophy f - box ( mafbx , atrogin-1 ) and muscle ring finger-1 ( murf-1 ) were first described in 2001 and are significantly up - regulated during muscle atrophy.20,21 however , the loss of these e3-ligases only leads to partial protection against muscle wasting.20 mafbx was shown to target regulatory factors for protein synthesis like myod22 and the eukaryotic initiation factor of protein synthesis elf3-f.23 murf-1 binds to titin24,25 and is targeting to myofibrillar proteins like myosin heavy chain , myosin light chain , and myosin - binding c.26 another muscle - specific ubiquitin ligase named tripartite motif 32 ( trim32 ) was discovered in 2005.27 trim32 is thought to ubiquitinate the thick myofibrillar filament as well as actin and dysbindin.27 a muscle - specific f - box protein was foun in 2007,28 which induces the ubiquitination of insulin receptor substrate 1 thereby providing a negative feedback on the igf1r / irs1/pi3k / akt pathway by early signal termination.29 in addition 2010 , tumour necrosis factor ( tnf ) receptor - associated factor has been found to play a critical role in atrophy as an e3 ubiquitin ligase.30 cancer cachexia animal models are showing significant wasting of the myocardium.3133 in one study it was shown that the heart muscle weight is decreased by 20% on average.34 in another cancer cachexia study , cardiac wasting was associated with left - ventricular ( lv)-dysfunction.35 treatment with selected agents ( bisoprolol , spirolactone , and imidapril ) used in hf resulted in placebo - treated group of ah-130 rats in a loss of 21 2% lvmass while the lvmass was stabilized by bisoprolol ( + 2 8% , p < 0.0001 ) and increased by spirolactone ( + 9 3% , p < 0.0001 ) whereas imidapril had no effect.35 moreover , a decrease in the trypsin - like activity of the ups was seen in bisoprolol and spirolactone - treated animals in contrast to imidapril which enhanced proteasome activity.35 however , under oxidative stress conditions an up - regulated expression level of the ubiquitin ligases mafbx and murf-1 in cachectic hearts leads to the induction of the ups.34 mafbx and murf-1 are elevated as well at the mrna level linked to the degradation of cardiac troponin i , -actin-2 , and myod which is leading to impaired contractility.36 furthermore , another study showed a reduced heart rate and fractional shortening using echocardiography in the myocardium of cancer cachectic mice.37 elevated levels of reactive oxygen species in cachectic skeletal muscle have been linked to an activation of the ups.38 in general , cytokines including il-1 , il-6 , tnf- , and interferon- have been shown to contribute a catabolism net in skeletal muscle and to form a state of oxidative stress.39 these cytokines lead to an activation of nuclear factor kappa - light - chain - enhancer of activated b - cells ( nfb ) and forkhead transcription factors ( foxo ) in muscle40 resulting in increased proteolysis by inducing the expression of mafbx and murf-1.15 the involvement of the nfb pathway was originally observed in a model of disuse atrophy41 where it binds directly to the murf-1.42 furthermore , increased oxidative stress activates the nfb pathway.43 surprisingly , an inhibition of nfb via the ikappab kinase complex only partially rescues the phenotype of the cachectic gastrocnemius in a murine model of cancer cachexia.44 the foxo family members consist of three isoforms as foxo1 , foxo3 , and foxo4 . it was shown that foxo1 and foxo3a are significantly up - regulated in cachectic muscles from lewis lung carcinoma45 and colon 26 tumour - bearing mice46 ; foxo1 is also up - regulated in skeletal muscle in human cancer cachexia patients.47 thus , these findings strongly support the involvement of nfb and foxo in the process of muscle atrophy . however , mitochondrial dysfunction and loss of mitochondria in skeletal muscle contribute to disrupted muscle function.48 indeed , investigations with markers of mitochondrial function and activity like the mitochondrial enzymes pyruvate dehydrogenase ( pdh ) and the cytochrome c oxidase ( cox ) showed that the protein concentrations of pdh and cox in the skeletal muscle of colon cancer patients were decreased and a lower activity of pdh was observed as well.49 despite a large number of studies , our understanding of the development of muscle wasting and the involved pathways remains very limited . for instance , in some diseases like ra muscle wasting is not well investigated yet,5052 but better understanding is imperative for designing further studies and to develop new therapies . a recent study was aimed at an evaluation of muscle atrophy in skeletal muscle in a mouse model of ra and to establish a relation between disease score and muscle wasting.53 findings implicated the existence of a progressive development of muscle wasting with an early onset , which was especially associated with increased serum levels of cytokines , e.g. il-6.53 another not well - studied muscle wasting disease is stroke , although it is known that stroke rapidly leads to an increase in muscle loss.54 hence , it is difficult to treat muscle atrophy in stroke patients . however , a large prospective stroke study with the main objectives to study changes in body composition , and metabolic and functional changes of muscle tissue in patients with acute ischemic stroke is underway.55 this study unites the knowledge of neurologists , cardiologists , and endocrinologists , and their findings might improve rehabilitation after stroke . generally , impaired feeding , reduced caloric intake , and loss of appetite lead to a negative nutritional and nitrogen balance,56,57 and immobilization causes physical inactivity and muscle atrophy after stroke.58,59 it has been shown that elevated volumes of tnf- are responsible for muscle loss and that plasma concentrations of the enzyme visfatin were significantly elevated in patients after ischemic stroke.60,61 for that , investigations to changes of inflammation parameters and its relation to body composition , insulin sensitivity , and patient s survival will be made as well.55 exact quantification of skeletal muscle mass is challenging . to better determine skeletal muscle mass , many measurement methods were developed in the last two centuries ( for a historical overview see 62 ) . since the early 1970s computed tomography , magnet resonance imaging , and dual - energy x - ray absorptiometry came into application.62 a problem of these methods is that they are all expensive and thus only available at larger institutions . moreover , these methods are only able to detect tissue wasting , but they are incapable to show the risk of developing muscle atrophy.63,64 but there is described a practical screening tool in a validated model to improve screening for low skeletal muscle mass in older adults.65 it has been suggested that the bmi is strongly associated with a low skeletal muscle mass index which could be helpful for primary care settings and treating elderly populations at risk of sarcopenia.65 however , it is imperative to find new robust biomarkers , which are cheap and easily available for diagnosis and therapy monitoring in clinics.64 potential candidates are summarized in table1 and are described in more detail below . serum creatinine may be such a reliable , cheap , and easily accessible biomarker of skeletal muscle mass in human subjects , for example in ckd patients.66 the adaption of the liquid chromatography tandem mass spectrometry based on d3-creatine dilution method from an oral dose and detection of urinary creatinine enrichment by isotope ratio mass spectrometry79 could be an accurate tool to measure total body creatine skeletal muscle mass change.80 the drawback of this method is the high cost and limited availability of the necessary machinery . furthermore , serological neoepitopes have been suggested as muscle wasting biomarkers to solve some of these problems mentioned before . neoepitopes do not reflect a condition or state like creatinine reflecting muscle mass , but a process which allows an early detection of a muscle loss in disease . in fact , neoepitope biomarkers are parent proteins that are produced through post - translational modifications , i.e. glycosylation , phosphorylation , acetylation , nitrosylation , methylation , and ubiquitination of an existing molecule and are formed by protease cleavage or addition of chemical groups in tissues of interest.67 the most common parent proteins for muscle loss biomarkers are sarcomeric proteins ( e.g. myosin , actin , troponin , and tropomyosin ) and components of the extracellular matrix ( e.g. laminins).67 that makes neoepitopes interesting to be biomarkers of muscle pathology.67 other serological biomarker candidates for muscle wasting are type vi collagen turnover - related peptides.68 in a study , blood was analysed for levels and their correlation of following biomarkers : a matrix metalloproteinase - generated degradation fragment of collagen 6 ( c6 m ) and a type vi collagen n - terminal globular domain epitope ( ic6).68 these fragments can only be considered biomarker candidates of muscle mass and change in young men but not in elderly men.68 however , circulating biomarkers like the n - terminal propeptide of type iii procollagen ( p3np ) and c - terminal agrin fragment ( caf ) respond to resistance exercise training in older adults.69 short - time resistance exercise training ( 6 weeks ) improves leg extension muscle strength , measured on a knee lift , by 29% from 39.7 16.5 to 51.1 18.3 kg in the exercise group ( p < 0.001 ) and muscle quality by 28% from 3.64 0.85 to 4.67 0.81 relative strength ( leg extension strength in kg / lean quadriceps muscle mass in kg ) in the exercise group ( p < 0.001 ) in older adults and may result in changes p3np and caf.69 indeed , caf appears to increase in response to short - time resistance exercise training in older adults in contrast to p3np where the results were less clear.69 p3np showed a positive correlation to changes in lean body mass ( r = 0.422 , p = 0.045 ) , and there was observed a positive correlation between change in circulating caf and change in cross - sectional area of the vastus lateralis ( r = 0.542 , p = 0.008).69 however , p3np is associated with subsequent changes in lean body mass and appendicular skeletal muscle mass and seems to be a useful early predictive biomarker of anabolic response to growth hormone and testosterone.81 3-methylhistidine ( 3mh ) has been proposed as a marker of myofibrillar proteolysis through post - translational methylation of specific histidine residues in actin and myosin.8285 in a clinical scenario , 3mh has to be determined quantitatively in urine or plasma collections . a major disadvantage is that meat intake for 3 days prior to sample collection of patients can disturb the analysis of 3mh . a study from 2013 used 3mh , which was labelled with an isotope by using a non - radioactive isotope - based strategy.70 the labelled methyl - d3 - 3mh ( d-3mh ) was taken orally by healthy men , and urine and plasma samples were collected next day over 56 h , and were analysed for d-3mh enrichment by gas chromatography mass spectrometry.70 the results suggest that it is possible to obtain an index of myofibrillar protein breakdown in urinary or plasma samples and that it is not necessary to quantify urine and plasma collections or to have an abstinence from meat for several days.70 growth differentiating factor-15 ( gdf-15 ) plays an important role in muscle wasting and cachexia.86 results from studies suggest that gdf-15 induces weight and muscle loss , which makes gdf-15 a promising marker of cachexia and muscle atrophy.71 myostatin is a known negative regulator of muscle growth and mass , which is associated with muscle wasting86 suggesting it as putative marker for muscle atrophy . however , this could not be confirmed in humans.87 interestingly , follistatin ( fst ) , an endogenous , strong inhibitor of myostatin - mediated muscle wasting , has been suggested as a potential biomarker in sarcopenia.72 fst binds to myostatin in the serum , thus , making myostatin often undetectable88 and moreover , fst - overexpressing transgenic mice have been shown a significant increase in muscle mass.89 therefore , fst seems to be a positive regulator of muscle growth which makes it interesting to be a biomarker . irisin , the extracellular cleaved product of fibronectin type iii domain containing protein 5 , seems to be a potential sarcopenia biomarker , because of its involvement in muscle physiology.72 plasma levels and mrna expression of irisin were found to be elevated in mice in response to exercise.90 moreover , a positive correlation between circulating irisin and fst levels has been described in healthy men and obese persons.91 other than inflammatory cytokines , like il-1 , il-6 , and tnf- , which are associated with anorexia and weight loss,92,93 hormonal factors have been postulated to play a role in development in muscle loss , especially in cachexia.94,95 such factors include for instance leptin,96 ghrelin,97 and obestatin98 which are all thought to play a major role in cancer cachexia . these emerging biomarkers were investigated in oncologic patients as diagnostic and/or predictive markers , as well as their impact on patient survival.73 the study showed that ghrelin and leptin may be promising biomarkers for the identification of cachexia related to cancer and mark survival in cancer patients.73 ghrelin serum levels were significantly higher in cancer patients in comparison to healthy subjects ( 573.31 130 vs. 320.20 66.48 ng / ml , p < 0.0001 ) and levels of leptin were significantly lower in cancer patients than in healthy controls ( 38.4 21.2 vs. 76.28 17.48 ng / ml , p < 0.0001).73 ghrelin correlated negatively with leptin ( r = 0.75 ; p < 0.0001 ) and inverse as well.73 by kaplan meier analysis , the survival prediction was tested , and it was shown that patients with the best profile were those with low levels of ghrelin associated with high levels of leptin and on the contrary patients with high ghrelin levels and low levels of leptin had a minor survival probability ( log - rank ( 2 ) 8.02 ; p = 0.004).73 furthermore , in a recently published study , in which a large number of putative biomarker candidates were tested , with a cohort of upper gastrointestinal cancer patients , -dystroglycan was identified as a potential biomarker for weight - loss and myosin heavy chain or dystrophin as survival biomarkers.74 as mentioned before , inflammatory cytokines are used for prognosis of cancer cachexia . a new promising chronic inflammatory marker was found recently and is suggested to play a prognostic role in cancer cachexia : the tartrate - resistant acid phosphatase 5a ( tracp5a).75 moreover , it is already known that serum tracp5a is elevated in patients with ra76 and that the protein level of tracp5a is reflected in cardiovascular diseases and sarcoidosis.77,78 emerging candidates for biomarkers for cachexia and sarcopenia mmp , matrix metalloproteinase . in 2013 and 2014 , many new biomarkers , as described before , were investigated in different diseases and models . but although many researchers and pharmaceutical companies tried to find therapies for muscle atrophy , including cachexia and sarcopenia , no solution has been established until now.71,99,100 interestingly , morley et al . discussed if we are closer to having drugs for treatment muscle wasting disease and therefore drugs were highlighted , which showed current advances in therapy for sarcopenia and cachexia ( e.g. ghrelin agonists , selective androgen receptor molecules , megestrol acetate , activin receptor antagonists , espindolol , and fast skeletal muscle troponin inhibitors).101 indeed , morley et al . postulated that there is a remarkable increase in the knowledge of muscle wasting diseases because of new studies . however , a general strategy to avoid muscle mass loss and function is exercise.102,103 evidence of positive effects on fraility and sarcopenia through exercising are emerging.102,104108 recently , an exercise investigation focused on muscle quality in men and women aged 50 years and older suggested that long - term exercise , especially resistance exercise , is beneficial for muscle quality.109 interestingly , people over 60 years , who perform aerobic exercise once a week , also show positive association to muscle quality.109 in rat skeletal muscle , an example for a successful result of exercising was postulated for glycogen synthase kinase-3 ( gsk-3 ) , which has a big therapeutical potential when it is inhibited.110 an inhibition of the constitutively active kinase gsk-3 is considered to be beneficial , as it is involved in the regulatory inactivation of many anabolic pathways often leading to muscle wasting.111116 in detail , it has been demonstrated that physical exercise significantly decreases gsk-3 activity in rat skeletal muscle within 10 min of exercise and remained depressed with 30 and 60 min of exercise.110 in the following part current treatment substances discoveries will be listed ( see for detailed therapies117,118 ) . ghrelin , which was delineated as a good biomarker before,73 and its analogues bim-28125 and bim-28131 seem to have a beneficial effect after administration in a rat hf study.119 in that animal model it was shown that the expression of myostatin and the tnf- concentration are significantly reduced in the gastrocnemius after treatment.119 moreover in a pre - clinical study , treatment with a ghrelin receptor agonist , named anamorelin , showed a significant and dose - dependent increased food intake as well as body weight compared with the vehicle control in healthy rats.120 thereby , with a treatment of anamorelin , a significant increase of growth hormone and insulin - like growth factor-1 plasma levels was observed in healthy female pigs in comparison to the placebos,120 which makes anamorelin a potential drug for treatment of cancer anorexia cachexia syndrome . espindolol , an anabolic catabolic transforming agent , was used in a sarcopenia rat study.121 espindolol itself is a non - specific -1 and -2 adrenergic receptor blocker with intrinsic sympathomimetic activity on the -2 adrenergic receptor which results in reduced catabolism and increased anabolism , and espindolol is a highly potent antagonist of 5-ht1a receptors which has an effect on food - intake as well as reduced fatigue and thermogenesis.122 a recent study , the act - one trial , which was a multicentre , randomized , double - blind , and placebo - controlled study for dose - finding of espindolol in cachectic patients with non - small cell lung cancer and colorectal cancer in stages iii and iv was started.122 at the 7th cachexia conference , the first results of the act - one trial with 87 patients from 17 centers were presented.86 patients were treated with two doses of espindolol twice daily for 16 weeks . the results showed that the higher dose improves lean and fat mass , and the that handgrip strength is significantly increased at both doses.86 interestingly , in a rat sarcopenia model , espindolol treatment only had a small effect on overall body weight , but did significantly increase lean body mass , while at the same time reducing fat mass . this makes espindolol an attractive candidate for treating sarcopenic patients , as these patients are often obese.121 a highly potent -2 adrenoceptor - selective agonist , formoterol , was used as a drug in a cancer cachexia rat model , and it significantly reduced muscle wasting but had no influence on heart weight and function as often described in literature.123 epigallocatechin-3-gallate ( egcg ) , which is a component of green tea , was published to be an effective inhibitor of increased protein degradation and depressed protein synthesis in an in vitro study by using murine c2c12 myotubes.124 egcg is not an approved drug but acts as a nutritional support and has been shown to attenuate skeletal muscle wasting in the lewis lung carcinoma model of cancer cachexia.125 muscle loss arises from a dysbalance of catabolism and anabolism , i.e. protein degradation and protein synthesis . despite a large number of studies , knowledge of disease related but investigations in the last two years like studies focused on ra53 and stroke55 bring us one step ahead in understanding processes of muscle wasting because of those diseases . cachectic and sarcopenic patients often suffer from quality of life including appetite loss and lower muscle strength , which makes finding appropriate biomarkers for diagnosis of muscle wasting associated diseases a timely matter . although an ideal marker has not yet been identified , the development of some emerging candidates ( table1 ) promise much potential . neoepitopes67 as biomarkers could be the solution for early diagnosis of a potential muscle mass loss allowing earlier detection and treatment to prevent morbidity and mortality in patients . in addition , finding new treatment strategies and drugs has to be developed to treat patient s symptoms . there are some very promising investigational drugs in studies related to cachexia and sarcopenia , but further research is necessary for a transition into the clinic . maybe there is the need to combine existing treatment strategies with further novel approaches to treat muscle mass loss . cathleen drescher , masaaki konishi , nicole ebner , and jochen springer declare that they have no conflict of interest .

loss of muscle mass arises from an imbalance of protein synthesis and protein degradation . potential triggers of muscle wasting and function are immobilization , loss of appetite , dystrophies , and chronic diseases as well as aging . all these conditions lead to increased morbidity and mortality in patients , which makes it a timely matter to find new biomarkers to get a fast clinical diagnosis and to develop new therapies . this mini - review covers current developments in the field of biomarkers and drugs on cachexia and sarcopenia . here , we reported about promising markers , e.g. tartate - resistant acid phosphatase 5a , and novel substances like epigallocatechin-3-gallate . in summary , the progress to combat muscle wasting is in full swing , and perhaps diagnosis of muscle atrophy and of course patient treatments could be soon support by improved and more helpful strategies .

bariatric surgery for weight loss in the morbidly obese is a common , major upper abdominal operations currently performed in north america . following surgery patients are often concerned to know how their weight loss compares to that of other patients . various factors are known to influence weight loss after surgery but their relative importance is unknown . from our data we developed a curve predicting weight loss after surgery as a percent of initial weight . by adding and subtracting factors known to influence weight loss and examining how they affected variability around the predicted weight loss curve we were able to assess the contribution of each factor to the predictability of the model . the medical records of 1551 patients maintained in microsoft access / excel databases ( excel versions 11 and 12 ) who had had roux - en - y gastric bypass from 1989 to 2008 for morbid obesity were reviewed . data collection included age , sex , initial weight , height , type of operation ( open or laparoscopic ) , and subsequent weights obtained on outpatient visits . follow - up appointments were made for patients approximately once a month for the first three months and once every two to three months thereafter for the first year and beyond . the operations were performed by one surgeon ( mlo ) in nine community hospitals using standard methods previously described [ 2 , 3 ] . to understand the relation of weight loss after gastric bypass to preoperative variables we use three alternate but relatively equivalent methods . the first method groups patients by the month after surgery and compares data in a vertical fashion ; the second uses the same time groupings but looks at the linear regression between initial weight and weight for a given month ; the final method creates a model based on time as a continuous variable and can be used to look at variance about the curve . in order to compare patients with different initial weights we used percent initial weight . initial weights ( iws ) were collected at or near time of surgery and all subsequent weight points were divided by iw . only the last weight entry was taken for patients following up multiple times in a given month and used in analysis . for comparison of continuous variables such as age and height , the two outermost quartiles of patients were compared . height , age , gender , operative approach , and race were compared at three , six , nine , and twelve months using a two - tailed student 's t - test . percent iws for caucasian , hispanic , and african americans were compared using the f - test . differences were considered statistically significant when the probability of the occurrence by chance was less than 0.05 . as in traditional methods , patient data was grouped into month intervals using the latest weight data point for each patient . the patient 's initial weight was plotted on the x - axis and weights for a particular month were plotted on the y - axis . linear regression was then performed using excel ( version 12 published by the microsoft ) . a plot of the data suggested that weight loss approximately followed an exponential decay , which was supported by existing literature . we took the natural log of the percent initial weight ( % iw ) and fit a linear function ( this is equivalent to % iw = e ) . the data still had significant curvature , suggesting that a quadratic fit was more appropriate ( % iw = ebt of the 1551 roux - en - y gastric bypass operations , 314 were performed laparoscopically and 1237 were preformed via a midline laparotomy . the open operations occurred during the early years of the study with only laparoscopic procedures done in the last eight years . the mean age for all patients was 40.5 years ( range 15.4 to 71.8 years ) and the initial mean weight was 133.5 kg at a bmi of 48.1 kg / m ( range 79.9 to 315.1 kg ; bmi 29.9 to 98.3 kg / m ) . the initial mean weight was less for women 127.8 kg with bmi at 47.4 kg / m ( range 79.9 to 267.9 kg ; bmi 29.9 to 98.3 kg / m ) . the racial background of the patients was primarily caucasian ( 940 ) , latino ( 200 ) , african - american ( 137 ) , and asian ( 7 ) , other ( 29 ) , and not reported for 238 patients . figure 1 shows that 72.3% , 58.0% , 47.3% , and 37.3% of patients were still following up at three , six , nine , and twelve month intervals , respectively . this is not the same as the number of patients following up in each month interval ( not every patient followed up at every month ) . the percentage of patients following up in a given month is also provided in figure 1 . the variables reaching statistical significance were age , height , race , and type of operation . extremes in age at 12 months postoperatively had a mean difference of 6% ( p < 0.05 ) . zero the slope of the regression line will equal one with a perfect fit and an intercept at zero . the subsequent months have an intercept very close to zero with decreasing slopes , indicating that the greater the initial weight is the more weight that is lost and that actual weight is the same percent of initial weight at any given time after surgery . the slopes at four , eight , and twelve months are 0.81 , 0.64 , and 0.58 , respectively . data show increasing variation in percent weight over time ( see standard deviations in table 1 ) . the model predicts weight loss as a percent of initial weight and fits with an r squared value of 0.93 for the first 12 months ( figure 3 ) . constants a and b were calculated to be 3.43 0.04 10 and 2.57 0.01 10 , respectively . initial weight is the most significant factor influencing weight loss and accounts for more than 93% of the variability . anova results suggest race , height , gender , and type of operation ( laparoscopic versus open ) account for less than 1% of variability . variability in weight loss after one year increases over time and is probably due to influence of other factors . the positive results measured by reduction in various morbidities experienced by many patients electing bariatric surgery in recent years have reinforced the use of gastric bypass as a weight loss treatment . for clinical reasons the rate of weight loss following surgery is of interest . also of interest are the relative contributions of different factors known to influence the rate of weight loss , not the least important of these being time after surgery . recent studies investigating weight loss predictability and rate factors examined different variables individually and in combination , with results ranging from single variable positive correlations to specific profiles for postoperative success . due to the contrasting or inconclusive findings reported in the bariatric literature , further analysis seems useful to predict weight loss following surgery . previous work by two groups constructed a polynomial fit for the absolute weight or bmi after surgery [ 4 , 5 ] . using this model dallal et al . , showed that patients lost similar weight followed by an upward rebound regardless of which experienced surgeon preformed the operation . we followed a model similar to livingston et al . who used an exponential decay model . the formula developed by their team in 2001 was dependent on the initial fat and lean compartment mass in order to accurately predict weight loss . in 2007 , chevallier et al . predicted successful procedures involving patients less than 40 years old who had an initial bmi less than 50 kg / m , combined with a willingness to change their lifestyle , and supported by an experienced bariatric surgical team . initial attempts to compare and reconcile these various bariatric surgery outcomes , however , have been complicated by discrepancies in nomenclature , study methods , statistical detail , and definitions of weight - loss success and comorbid disease resolution . in our study , we observed the influence of initial weight to far exceed other factors in predicting weight loss up to one year following gastric bypass . variability in the amount of weight lost increases over time , suggesting that continued success is dependent on additional factors . the challenge of identifying the dominant predictors of postoperative weight loss and rate of loss stems from the range of factors influencing the patient 's postoperative condition . a literature investigation conducted by picot et al . in 2009 revealed gastric bypass as a more effective weight loss procedure than vertical banded gastroplasty and adjustable gastric banding , with open versus laparoscopic surgeries producing similar results in each group . furthermore , a 2010 study recommends the application of gastric bypass over adjustable gastric banding for diabetes treatment . all of our patients had gastric bypass removing the type of procedure as a variable . although our laparoscopic approach produced significantly better weight loss , the addition of this and similar variables to the weight loss prediction curve increased explanation of variability only very slightly . most of the laparoscopic procedures were done in the second half of the study period but a separate analysis of weight loss by date of surgery showed no influence due to the year in which surgery was done . report that non - hispanic whites and hispanic adolescents respond positively with significant weight loss to both gastric bypass and banding surgery . we did identify age and ethnicity as statistically significant variables in rate of weight loss for the first year after operation . this is similar to other studies suggesting that african american women are more resistant to weight loss than their white counter parts [ 1315 ] . with respect to gender , report that males lose significantly more weight than females do at 6 and 12 months post operation . this correlation was weakened by the fact that the male group was significantly taller and heavier than the female group pre - op . greater preoperative weight results in increased weight loss postoperatively as is shown by our model . our weighted model for method 3 shows an adjusted r value of 0.9017 between initial weight and weight loss within the first year post - op . the effect of gender on prediction of weight loss in the first year postoperation accounts for 0.51% of the variability suggesting that neither gender nor height is of much overall importance . other studies suggest that psychological factors also contribute to postoperative long - term success . in 2004 , tsushima et al . observed an association between the revised minnesota multiphasic personality inventory-2 and weight loss outcome one year following gastric bypass . 's 2009 study , in which the assessment of mental health factors as the sum of psychiatric conditions rather than the presence of a specific condition improved the prediction of postoperative weight loss . pursuing the potential contribution of a patient 's motivational characteristics , kaiser et al . presented evidence supporting the beneficial impact of support group attendance on the percentage of excess weight loss . although our patients had psychological evaluations before and after surgery , and they attended support groups , we did not have objective data with which to measure the effect . alternatively , the predictive methodology itself is under scrutiny . in a 2009 study , lee et al . demonstrated greater predictive accuracy with the decision tree model as opposed to the traditional logistical regression and discriminant analysis classification models . from our perspective regression in the period one year after gastric bypass provides a continuous model allowing patients to see the average weight decline and is more easily understood as the major variables are time and weight as related to initial weight . decision trees are preferred when using multiple variables to predict outcomes at a single point in time . potential weaknesses in our model include the fact that procedures were done by one surgeon and the variability due to multiple surgeons was thereby eliminated . it follows that we were not able to assess the effect that having different surgeons might have had . we examined weight loss following gastric bypass as a single procedure and although it is tempting to think that preoperative weight is the major predictor of weight loss following other procedures we do not have data in support . increasingly large differences in weight loss after one year make predictions based on initial weight increasingly inaccurate . low percent followup in our patients one year after gastric bypass may have led to a lower percent iw calculation as patients that drop out early may not lose as much weight . in summary , we find that weight loss and thus predictability of outcome for the first postoperative year is primarily dependent on initial weight . beyond one year , weight loss varies and is likely related to factors other than the operation such as dietary adaptation , psychiatric conditions , and physical activity . our information may be helpful to patients and clinicians in deciding how much weight loss to expect after gastric bypass . similar analyses for gastric banding , duodenal switch , and sleeve gastrectomy would be useful in the future .

factors postulated to predict weight loss after gastric bypass surgery , include race , age , gender , technique , height , and initial weight . this paper contained 1551 gastric bypass patients ( 85.9% female ) . operations were performed by one surgeon ( mlo ) at community hospitals in southern california from 1989 to 2008 with 314 being laparoscopic and 1237 open . we created the following equation : in[percent weight ] = at2 bt , where t was the time after operation ( days ) and a and b are constants . analysis was completed on r - software . the model fits with r2 value 0.93 and gives patients a realistic mean target weight with a confidence interval of 95% for the first year . conclusion . we created a curve predicting weight loss after surgery as a percentage of initial weight . initial weight was the single most important predictor of weight loss after surgery . other recorded variables accounted for less than 1% of variability . unknown factors account for the remaining 6 - 7% .

we investigated the patterns of illegal drug and alcohol consumption in young people , focusing in particular on binge drinking behaviours , a known important current public health issue . this phenomenon has been investigated in other studies , especially those focused on the us , but to date , to our knowledge , no european studies have been conducted in the natural setting of a large cohort of young people . the ten - d survey confirmed that the prevalence of binge drinking is higher among younger people ( 20 - 24 years old ) and may be related to socio - economic variables ( such as living with friend and colleagues or lower educational level ) . we found a significant association between the consumption of illegal drugs and binge drinking ( poly - drug consumption ) . these findings should be considered when addressing specific prevention campaigns that focus on the groups with the highest likelihood of unhealthy behaviours . during the last two decades , an increase has been reported in the consumption of psychoactive substances ( alcohol and illegal and medical drugs ) among young people in europe . a common theme of many studies is the dynamic nature of problematic alcohol and illegal drug use . currently , the members of the european community show a growing interest in these issues and demonstrate a desire and a shared determination to improve their understanding of this complex social issue . the attention given to this topic is also based on evidence that the consumption of psychoactive substances is one of the major risk factors for road crash accidents , the leading cause of death among people younger than 30 years old . as reported in a previous study , almost 73,500 young people aged 16 - 24 died in road crash accidents in 14 european countries during the decade of 1995 - 2004 . road crash accidents occur more frequently on weekend nights , when youths are more likely to consume large quantities of alcohol , a phenomenon known as binge drinking ( defined as 5 drinks on one occasion for men and 4 drinks on one occasion for women ) . knowledge of these patterns of psychoactive substance consumption among young people is therefore crucial for planning effective prevention programmes . in the past , several studies have examined the prevalence of illegal drugs use , especially in the us , whereas other studies have reported on specific drug use in small samples of young people in regional settings . to our knowledge , however , no study has investigated binge drinking and poly - drug use in large samples directly recruited in a natural setting , such as the recreational sites where youths spend their weekend nights ( in our case , clubs and discotheques ) . the ten - d ( dark , dance , disco , dose , drugs , drive , danger , damage , disability , death ) by night project is an international , multicentre , cross - sectional survey conducted in six european countries . this study was designed to investigate psychoactive substance consumption ( alcohol , cocaine , methamphetamine / mdma , thc , amphetamine , opiates , benzodiazepines ) and the relationship between this risk behaviour and driving performance in a large sample of young drivers , who were enrolled in this study at typical locations of consumption . using data provided by the ten - d survey , this study aims to examine the patterns of alcohol and illegal drug use among european youths , focusing on the prevalence of binge drinking and poly - drug use . details about the methodology of the study protocol have been described in a previous paper . briefly , ten - d by night is an international , multicentre , cross - sectional portal survey funded by the european commission . the required sample size was calculated using the schlesselmann formula for known population sizes , assuming a two - sided alpha = 0.05 , a statistical power = 90% , losses or missing = 25% and taking into account the need to stratify for each country and adjust for several potential confounders in the multivariate analysis . based on these parameters , the overall sample consisted of 4695 young drivers who were enrolled in the study on weekend nights at recreational meeting places in six european countries [ italy ( 18% ) , spain ( 21% ) , belgium / netherlands ( 19% ) , poland ( 21% ) and bulgaria ( 21% ) ] . in each country , a map of the recreational meeting places was produced based on official regional lists , and an opportunistic selection was made according to the willingness of the clubs owners . in each country , the interventions were conducted by survey staff composed of at least six operators who had been specifically trained . the staff included a survey manager ( responsible for supervising all field activities ) , one technician for a driving simulation test , at least two interviewers ( responsible for the administration of questionnaires and two psychoactive substance detection tests ) and two hostesses for approaching the young people . the scheduled activities were conducted at both the entry and exit of the recreational sites . at the entry , after the voluntary recruitment of the participants based on eligibility criteria ( possession of a driver s license and age between 16 and 35 years old ) , the staff explained the study s protocol , collected informed consent , and assigned a personal , anonymous code to each subject . each participant was asked to complete a structured questionnaire and a test to assess his / her bac ( breath alcohol concentration , a proxy for blood alcohol concentration ) and to participate in a driving simulation ( using simunomad3 ecrans ) . at the exit , a second questionnaire and a saliva test26 for several illegal drugs were administered to the participants in addition to the same bac test and the same driving simulation performed at entry . the ten - d by night intervention is based on the portal survey technique12,15 and was applied at selected youth recreational locations on weekend nights by survey staff . the questionnaires were tested for comprehensiveness and translated into the official language of each participating country . a total of 27 closed- and open - ended questions investigated the following topics : demographic and socio - economic status ( gender , age , education , occupational status , living situation ) ; medical drug consumption in the last seven days ; driving habits ( licensing age , driving frequency ) ; alcohol and illegal drug consumption ; driving under the influence of psychoactive substances ( self - reported driving after heavy drinking or illegal drug use or having had penalties , car crashes , or license revocation when drunk or doped in the last year ) ; alcohol consumption during the event ( number of shots , glasses or bottles consumed for each different alcohol type ) ; illegal drug consumption during the event ( each substance ) ; binge drinking during the event ; intent to drive upon exit ; opinions about the intervention . the results of the analyses on the predictors of reaction time were published previously , including descriptive statistics for males and females and the categorical variables based on relative frequencies . a multivariate logistic regression model was fitted through a backward stepwise elimination analysis to predict the probability of binge drinking . finally , the goodness of fit was evaluated using the brier score , which quantifies the overall accuracy of the predictions ( for sensible models , the brier score ranges from 0 , perfect , to 0.25 , worthless ) , and the c index ( the closer to the absolute value of one , the better ) , which represents the concordance between the predicted and observed responses . in the absence of an external data source , all goodness - of - fit indices were computed using a bootstrap method to account for the degree of optimism in the model accuracy evaluations induced by the use of the same data source for training and testing purposes . a two - tailed p - value of 0.05 was considered significant for all analyses , which were conducted using the r statistical package version 2.9 . finally , based on the multivariate logistic model , a nomogram was produced to estimate the likelihood of engaging in binge drinking behaviour for each subject with specific socio - demographic characteristics . nomograms are graphs that allow for the approximate calculation of output from mathematical functions . to compute the probability of engaging in binge drinking , each covariate in our nomogram is represented by a horizontal line , with tick marks indicating the categories of an explanatory variable . given a risk profile , that is , a set of covariates ( age , living situation , education and marijuana and cocaine consumption ) , the likelihood is derived as follows . a vertical line is drawn from each covariate s category to the points axis at the top of the nomogram . the sum of all of the partial risk scores is reported on the total points axis , and the corresponding probability is obtained by drawing a vertical line to the lower predicted value . the required sample size was calculated using the schlesselmann formula for known population sizes , assuming a two - sided alpha = 0.05 , a statistical power = 90% , losses or missing = 25% and taking into account the need to stratify for each country and adjust for several potential confounders in the multivariate analysis . based on these parameters , the overall sample consisted of 4695 young drivers who were enrolled in the study on weekend nights at recreational meeting places in six european countries [ italy ( 18% ) , spain ( 21% ) , belgium / netherlands ( 19% ) , poland ( 21% ) and bulgaria ( 21% ) ] . in each country , a map of the recreational meeting places was produced based on official regional lists , and an opportunistic selection was made according to the willingness of the clubs owners . in each country , the interventions were conducted by survey staff composed of at least six operators who had been specifically trained . the staff included a survey manager ( responsible for supervising all field activities ) , one technician for a driving simulation test , at least two interviewers ( responsible for the administration of questionnaires and two psychoactive substance detection tests ) and two hostesses for approaching the young people . the scheduled activities were conducted at both the entry and exit of the recreational sites . at the entry , after the voluntary recruitment of the participants based on eligibility criteria ( possession of a driver s license and age between 16 and 35 years old ) , the staff explained the study s protocol , collected informed consent , and assigned a personal , anonymous code to each subject . each participant was asked to complete a structured questionnaire and a test to assess his / her bac ( breath alcohol concentration , a proxy for blood alcohol concentration ) and to participate in a driving simulation ( using simunomad3 ecrans ) . at the exit , a second questionnaire and a saliva test26 for several illegal drugs were administered to the participants in addition to the same bac test and the same driving simulation performed at entry . the ten - d by night intervention is based on the portal survey technique12,15 and was applied at selected youth recreational locations on weekend nights by survey staff . the questionnaires were tested for comprehensiveness and translated into the official language of each participating country . a total of 27 closed- and open - ended questions investigated the following topics : demographic and socio - economic status ( gender , age , education , occupational status , living situation ) ; medical drug consumption in the last seven days ; driving habits ( licensing age , driving frequency ) ; alcohol and illegal drug consumption ; driving under the influence of psychoactive substances ( self - reported driving after heavy drinking or illegal drug use or having had penalties , car crashes , or license revocation when drunk or doped in the last year ) ; alcohol consumption during the event ( number of shots , glasses or bottles consumed for each different alcohol type ) ; illegal drug consumption during the event ( each substance ) ; binge drinking during the event ; intent to drive upon exit ; opinions about the intervention . the results of the analyses on the predictors of reaction time were published previously , including descriptive statistics for males and females and the categorical variables based on relative frequencies . a multivariate logistic regression model was fitted through a backward stepwise elimination analysis to predict the probability of binge drinking . finally , the goodness of fit was evaluated using the brier score , which quantifies the overall accuracy of the predictions ( for sensible models , the brier score ranges from 0 , perfect , to 0.25 , worthless ) , and the c index ( the closer to the absolute value of one , the better ) , which represents the concordance between the predicted and observed responses . in the absence of an external data source , all goodness - of - fit indices were computed using a bootstrap method to account for the degree of optimism in the model accuracy evaluations induced by the use of the same data source for training and testing purposes . a two - tailed p - value of 0.05 was considered significant for all analyses , which were conducted using the r statistical package version 2.9 . finally , based on the multivariate logistic model , a nomogram was produced to estimate the likelihood of engaging in binge drinking behaviour for each subject with specific socio - demographic characteristics . nomograms are graphs that allow for the approximate calculation of output from mathematical functions . to compute the probability of engaging in binge drinking , each covariate in our nomogram is represented by a horizontal line , with tick marks indicating the categories of an explanatory variable . given a risk profile , that is , a set of covariates ( age , living situation , education and marijuana and cocaine consumption ) , the likelihood is derived as follows . a vertical line is drawn from each covariate s category to the points axis at the top of the nomogram . the sum of all of the partial risk scores is reported on the total points axis , and the corresponding probability is obtained by drawing a vertical line to the lower predicted value . 67.9% were male , and the most represented age class was the 20- to 24-year - olds . more than 80% of the subjects had at least a high school education , and the majority of the participants lived with their parents . the characteristics of the sample , according to the socio - demographic variables and psychoactive substance consumption , are reported in table 1 . only 7.0% of the males and 10.5% of the females reported no alcohol consumption , whereas approximately 40% of both males and females reported drinking once per week and 36.8% of the males and 19.1% of the females reported drinking regularly several times per week . approximately half of the participants , both males and females , reported being drunk at least once in the past month ( 56.5% and 45.0% , respectively ) . routine illegal drug use was also evaluated ( illegal drugs consumed in the last year ) . most of the participants denied illegal drug consumption ( 63.9% of the males , 69.7% of the females ) . among the illegal drug users , the most frequent pattern was occasional ( 15.3% of the males , 15.7% of the females ) , followed by multi - week ( 9.7% and 6.4% , respectively ) . table 2 shows the distribution of specific illegal drugs consumed in the last year by gender . 31.9% of the males and 26.9% of the females reported marijuana consumption in the last year , whereas 10.1% of the males and 5.8% of the females reported cocaine consumption . in the case of other psychoactive drugs , the proportion of users was slightly higher among males than females , with benzodiazepines as the only exception . the differences observed between male and female illegal drug consumption were significant for all types of drugs except for opiates ( p=0.28 ) . table 3 describes the use of alcohol and illegal drugs reported by the participants prior to entering the club and during the evening of the intervention . approximately half of the respondents ( 47.1% of the males and 51.1% of the females ) reported being completely sober before entering the club , whereas more than one - third of the participants ( 40.7% of males and 36.0% of females ) reported that they had consumed two or more drinks . the proportion of subjects that self - reported binge drinking ( more than 4 drinks ) prior to entering the recreational sites was 19.6% of the males and 12.8% of the females ( p=0.001 ) . we found that 79.8% of the males and 69.7% of the females reported alcohol consumption in the club , and 18.0% of the males and 11.0% of the females reported binge drinking ( p<0.001 ) . with regard to the type of drink consumed during the night ( data not shown ) , male drinkers reported consuming beer and distilled spirits ( approximately 35% and 25% , respectively ) , whereas females reported consuming alcoholpops ( 22.2% ) , distilled spirits and beer ( both 19.1% ) . psychoactive drug use prior to entry was reported by 8.5% of the males and 6.0% of the females ( p=0.002 ) , and consumption of these drugs inside the recreational site was confirmed by 5.5% and 3.9% of the male and female respondents , respectively ( p=0.059 ) . the questionnaire also investigated the consumption of more than one illegal drug within the recreational site by self - reported psychoactive substance users ( poly - drug consumption , alcohol excluded ) . poly - drug use was reported by 71.2% of the male users and 66.6% of the female users ( p=0.733 ) . socio - economic variables and illegal consumption of marijuana and cocaine were analysed in the univariate analysis to obtain crude ors for inclusion in the multivariate logistic model to predict the probability of binge drinking . table 4 shows the crude and adjusted ors of the variables that remained statistically significant after the stepwise backward elimination analysis . the univariate analysis found that 20- to 24-year - olds were the age group with the highest likelihood of binge drinking behaviour ( crude or 1.34 , 95% ci 1.08 - 1.66 ) . however , when the model was adjusted for the main confounding factors , the association between age and binge drinking was attenuated ( adjusted or 1.14 , p=0.3 ) . after controlling for the main confounding factors , living with a partner ( adjusted or 1.57 , 95% ci 1.13 - 2.19 , p=0.01 ) and seeking employment ( adjusted or 1.66 , 95% ci 1.17 - 2.36 , p=0.005 ) were associated with a higher likelihood of alcohol abuse . the univariate analysis suggested a protective role for the highest educational levels , but after adjustment , only those who attended high school had a lower likelihood of alcohol abuse behaviour ( adjusted or 0.38 , 95% ci 0.25 - 0.58 , p<0.001 ) . an increased likelihood of binge drinking was evident in cannabis consumers , among whom the risk of binge drinking increased with the frequency of consumption ( once , but not anymore : crude or 1.52 ; p=0.01 ; sometimes : crude or 2.23 , p<0.001 ; several times a month : crude or 2.61 , p<0.001 ; several times a week : crude or 4.29 p<0.001 ) , and among cocaine users ( once but not anymore : crude or 2.92 , p=0.01 ; sometimes : crude or 2.98 , p<0.001 ; several times a month : crude or 4.18 , p=0.004 ) . the brier score ( the lower the score , the better the model performance ) and the discrimination index c were 0.09 and 0.73 , respectively . even after adjusting for age , education and socio - economic variables , the relationship between engaging in binge drinking and the frequency of marijuana use was apparent ( once but not anymore : adjusted or 1.45 , p=0.03 ; sometimes : adjusted or 1.96 , p<0.001 ; several times a month : adjusted or 1.94 , p=0.007 ; several times a week : adjusted or 3.66 , p<0.001 ) . the nomogram of the logistic regression model is shown in figure 1 . following the indications reported in the methods , it was easy to identify the profiles that were most at risk for binge drinking ( for each variable ) . the characteristics most related to binge drinking were age 20 - 24 years , living with friends , lack of education , employment and frequent marijuana and cocaine use . 67.9% were male , and the most represented age class was the 20- to 24-year - olds . more than 80% of the subjects had at least a high school education , and the majority of the participants lived with their parents . the characteristics of the sample , according to the socio - demographic variables and psychoactive substance consumption , are reported in table 1 . only 7.0% of the males and 10.5% of the females reported no alcohol consumption , whereas approximately 40% of both males and females reported drinking once per week and 36.8% of the males and 19.1% of the females reported drinking regularly several times per week . approximately half of the participants , both males and females , reported being drunk at least once in the past month ( 56.5% and 45.0% , respectively ) . routine illegal drug use was also evaluated ( illegal drugs consumed in the last year ) . most of the participants denied illegal drug consumption ( 63.9% of the males , 69.7% of the females ) . among the illegal drug users , the most frequent pattern was occasional ( 15.3% of the males , 15.7% of the females ) , followed by multi - week ( 9.7% and 6.4% , respectively ) . table 2 shows the distribution of specific illegal drugs consumed in the last year by gender . overall , 31.9% of the males and 26.9% of the females reported marijuana consumption in the last year , whereas 10.1% of the males and 5.8% of the females reported cocaine consumption . in the case of other psychoactive drugs , the proportion of users was slightly higher among males than females , with benzodiazepines as the only exception . the differences observed between male and female illegal drug consumption were significant for all types of drugs except for opiates ( p=0.28 ) . table 3 describes the use of alcohol and illegal drugs reported by the participants prior to entering the club and during the evening of the intervention . approximately half of the respondents ( 47.1% of the males and 51.1% of the females ) reported being completely sober before entering the club , whereas more than one - third of the participants ( 40.7% of males and 36.0% of females ) reported that they had consumed two or more drinks . the proportion of subjects that self - reported binge drinking ( more than 4 drinks ) prior to entering the recreational sites was 19.6% of the males and 12.8% of the females ( p=0.001 ) . we found that 79.8% of the males and 69.7% of the females reported alcohol consumption in the club , and 18.0% of the males and 11.0% of the females reported binge drinking ( p<0.001 ) . with regard to the type of drink consumed during the night ( data not shown ) , male drinkers reported consuming beer and distilled spirits ( approximately 35% and 25% , respectively ) , whereas females reported consuming alcoholpops ( 22.2% ) , distilled spirits and beer ( both 19.1% ) . psychoactive drug use prior to entry was reported by 8.5% of the males and 6.0% of the females ( p=0.002 ) , and consumption of these drugs inside the recreational site was confirmed by 5.5% and 3.9% of the male and female respondents , respectively ( p=0.059 ) . the questionnaire also investigated the consumption of more than one illegal drug within the recreational site by self - reported psychoactive substance users ( poly - drug consumption , alcohol excluded ) . poly - drug use was reported by 71.2% of the male users and 66.6% of the female users ( p=0.733 ) . socio - economic variables and illegal consumption of marijuana and cocaine were analysed in the univariate analysis to obtain crude ors for inclusion in the multivariate logistic model to predict the probability of binge drinking . table 4 shows the crude and adjusted ors of the variables that remained statistically significant after the stepwise backward elimination analysis . the univariate analysis found that 20- to 24-year - olds were the age group with the highest likelihood of binge drinking behaviour ( crude or 1.34 , 95% ci 1.08 - 1.66 ) . however , when the model was adjusted for the main confounding factors , the association between age and binge drinking was attenuated ( adjusted or 1.14 , p=0.3 ) . after controlling for the main confounding factors , living with a partner ( adjusted or 1.57 , 95% ci 1.13 - 2.19 , p=0.01 ) and seeking employment ( adjusted or 1.66 , 95% ci 1.17 - 2.36 , p=0.005 ) were associated with a higher likelihood of alcohol abuse . the univariate analysis suggested a protective role for the highest educational levels , but after adjustment , only those who attended high school had a lower likelihood of alcohol abuse behaviour ( adjusted or 0.38 , 95% ci 0.25 - 0.58 , p<0.001 ) . an increased likelihood of binge drinking was evident in cannabis consumers , among whom the risk of binge drinking increased with the frequency of consumption ( once , but not anymore : crude or 1.52 ; p=0.01 ; sometimes : crude or 2.23 , p<0.001 ; several times a month : crude or 2.61 , p<0.001 ; several times a week : crude or 4.29 p<0.001 ) , and among cocaine users ( once but not anymore : crude or 2.92 , p=0.01 ; sometimes : crude or 2.98 , p<0.001 ; several times a month : crude or 4.18 , p=0.004 ) . the brier score ( the lower the score , the better the model performance ) and the discrimination index c were 0.09 and 0.73 , respectively . even after adjusting for age , education and socio - economic variables , the relationship between engaging in binge drinking and the frequency of marijuana use was apparent ( once but not anymore : adjusted or 1.45 , p=0.03 ; sometimes : adjusted or 1.96 , p<0.001 ; several times a month : adjusted or 1.94 , p=0.007 ; several times a week : adjusted or 3.66 , p<0.001 ) . the nomogram of the logistic regression model is shown in figure 1 . following the indications reported in the methods , it was easy to identify the profiles that were most at risk for binge drinking ( for each variable ) . the characteristics most related to binge drinking were age 20 - 24 years , living with friends , lack of education , employment and frequent marijuana and cocaine use . this paper reports results from the ten - d by night survey , which aimed to investigate the patterns of psychoactive substance consumption and the prevalence of binge drinking and poly - drug use in a large sample of young europeans . the limitations and strengths of our study have been extensively discussed in a previous paper . in brief , the ten - d survey has some strengths compared with other surveys on the same topic . most studies examining alcohol and illegal drug consumption among young people in the nightlife context have used only self - reported measures . in the limited number of studies in which an objective measure of alcohol level was obtained , by contrast , our survey combined self - reported alcohol and illegal drug use with objective testing , thus reducing the potential for bias . moreover , our sample was recruited at typical recreational sites , which were not considered settings for major risk ( such as rave parties ) . the portal survey technique conducts a temporal evaluation of substance use and provides pre- and post - assessment of consumption , ensuring the most reliable measure of substance effects . first , the study design is cross - sectional ; therefore , we were able to document only the correlations between variables , not the presence of a causal relationship . second , the voluntary recruitment of the participants and the opportunistic selection of the recreational sites may have introduced a selection bias , limiting the representativeness of the sample and the generalisation of the results . however , the guarantee of anonymity reduced the number of young people who refused to participate . the data on illegal drug use should be interpreted with extra caution because consumption may be underestimated due to underreporting . however , we evaluated this assumption using the saliva test , which provided the opportunity to estimate the number of false declarations in both contexts . our findings confirm the previously reported pattern of alcohol consumption among young people , known as the weekend drinking culture , especially among those in the 20- to 24-year - old age group . moreover , the results from our analysis confirm that females usually drink less than males , and a higher percentage of females reported being teetotalers . interestingly , the portal survey technique showed that a very large proportion of subjects had already consumed alcohol prior to entering the recreational site . this finding suggests that alcohol misuse is not limited to clubs . the survey on alcohol consumption our results also confirm that binge drinking is a common practice among young people who visit clubs . with regard to this issue , there were no significant differences between males and females . the ten - d survey confirmed the narrowing of the gap between males and females in terms of unhealthy lifestyles . furthermore , binge drinking appeared to be linked to other independent risk factors , including age ( with 20-to 24-year - olds at highest risk ) , living situation ( living with friends ) , education ( none or low ) , and illegal drug consumption ( marijuana and cocaine ) . this finding confirms the results of other studies analysing the growing phenomenon of concurrent alcohol and psychoactive substance use in young men and women . other studies , primarily conducted in united states , have found that unhealthy lifestyles are associated with certain risk factors , including the influence of college students distance from family , the level of education , and the protective role of marriage . although binge drinking and the correlation between alcohol consumption and illegal drug use have been described extensively in the literature , the contribution of our european survey is the use of data collected directly from a location in which psychoactive substances are usually consumed . regarding illegal drug use , our study found that many youths reported having tried one or more substances in the last year , most frequently marijuana ( one - third of the sample ) and cocaine . these data are in agreement with those reported in the literature , but our study had a larger sample size than previous surveys because of the specific setting and timing of our study intervention , which was conducted at recreational locations on weekend nights . the illegal drug consumption at the recreational sites was lower than that reported by other surveys , especially for methamphetamines , hallucinogens and opiates . it is possible that the particular setting of these surveys , which were usually conducted in the context of rave parties , captured the behaviours of a different population at an increased risk of use . more than 70% of those who reported substance use during the visit to the club reported consuming more than one drug . these data confirm previous literature showing that a majority of substance users are also poly - drug users . the ten - d by night survey showed that it is possible to identify individuals and groups at high risk . this identification allows for a focus on specific targets and facilitates the reshaping of prevention programmes . the increasing use of psychoactive substances among younger generations should be studied extensively to promote successful intervention campaigns . these interventions should consider locations , in addition to discos and clubs , where young people consume alcohol and use illicit drugs . b. alamar , f. alonso , c. calatayud , f. carena , s. chiad piat , t. chiapusso , consepi group , a. druart , m.t . kalitowicz , m. kedzia , g. manassero , e. pignata , r. papalia , i. pudule , l. snchez , r. siliquini , v. siliquini , r. sroka , d. vankov , s. vanzino , a. villerusa . b. alamar , f. alonso , c. calatayud , f. carena , s. chiad piat , t. chiapusso , consepi group , a. druart , m.t . kalitowicz , m. kedzia , g. manassero , e. pignata , r. papalia , i. pudule , l. snchez , r. siliquini , v. siliquini , r. sroka , d. vankov , s. vanzino , a. villerusa .

backgroundten - d by night is an international , multicentre , cross - sectional portal survey conducted on a large sample of young people in six european countries . this paper aims to investigate the alcohol and psychoactive drug consumption of this sample , with a focus on the prevalence of binge drinking and the poly - drug habits of the ten - d cohort.design and methodsthe study population consisted of 4695 young people attending recreational sites on weekend nights . the intervention included two questionnaires and two psychoactive substance detection tests performed at the entry and exit of the recreational sites . a multivariate logistic regression model was used to predict the probability of binge drinking.resultsbinge drinking was reported by 20% of the males and 13% of the females ( p=0.001 ) before entry into the recreational sites and by 18% of the males and 11% of the females before entry into the clubs ( p<0.001 ) . poly - drug use was reported by 71% of the males and 66% of the females . living with a parent ( or 1.57 ; p=0.01 ) , seeking employment ( or 1.66 ; p=0.005 ) and cannabis consumption ( several times per month and several times per week , or 1.94 and 3.66 , respectively , p<0.05 ) were associated with binge drinking.conclusionsour survey showed that it is possible to identify individuals and groups at higher risk of binge drinking . this identification would allow for a focus on specific targets and would facilitate the redesign of prevention programmes . the increased use of psychoactive substances among youths should be studied extensively to promote successful prevention campaigns .

it is well established that active life style is associated with improved quality of life . however , skin wound is one of the most common causes of inactivity ( lack of movement ) . the skin wound healing is a complicated process requiring coordination of different tissues and cells , to ensure successful healing . adipose mesenchymal stem cells ( amscs ) have the ability to repair skin damage and promote wound healing it is believed that the secretome of amscs plays an important role in skin wound healing [ 2 , 3 ] . the conditioned medium of amscs ( amscs - cm ) accelerated wound closure with increased reepithelialization , cell infiltration , and angiogenesis . recent research showed that the low oxygen concentration could improve the effects of paracrine of the bone mesenchymal stem cells on murine skin wound healing . in this study , we performed experiments using concentrated hypoxia - preconditioned amscs - cm ( conc . hypo - amscs - cm ) to evaluate the effects of concentration and nonconcentration of hypo - amscs - cm on the rat 's full - thickness skin defect model . the abdominal subcutaneous adipose tissue was collected from the female sprague - dawley rats ( 250300 g ) . the adipose tissue was minced and digested with collagenase ( 0.12 u / ml , gibco , usa ) at 37c for 40 min under constant shaking . after centrifugation ( 400 g for 10 min ) , the cellular pellet was resuspended in dulbecco 's modified eagle medium/10% fetal bovine serum placed in concentration 24 10 cells / cm . the analysis ( fluorescence - activated cell sorting , facs ) was performed using cd29 , cd34 , cd71 , and cd90 as primary fluorescent antibodies and using igg - fitc and igg - pe as secondary antibodies . the negative control was cells without primary antibodies . to induce osteogenic and adipogenic differentiation , the medium was replaced with adipogenic or osteogenic differentiation medium , as described before ( when amscs as passage 3 grew to approximately 90% confluence ) . the differentiated amscs were stained with oil red o for analysis of lipid droplet in adipogenic induction or alizarin red for calcium in osteogenic induction . as described previously , cells were cultured in the sealed box with an anaeropack , a disposable oxygen - absorbed and co2 generator , for 24 h at 37c . the anaeropack starts to absorb oxygen within 1 min ; oxygen tension inside the box drops to 1 mm hg within 1 h. the final concentration of oxygen was less than 1% , and the concentration of carbon dioxide was about 20% . the amscs culture dishes were washed three times with pbs and cultured overnight in culture medium consisting of dmem / f-12 , 0.4% rat serum . after 24 hours of incubation , the cultured medium was collected and then the fresh low - serum medium was added . the cultured medium of amscs was filtered using a 0.22 m filter and then centrifuged at amicon - ultra-15 ml ( mwco 3 kd , millipore ) at 4c 4000 g for 30 minutes . the medium was concentrated about 5 times using ultrafiltration membrane of 3.5 kd with polyethylene glycol at 4c . freezing - dried powder was prepared in the sterilized penicillin bottle using the lyophilizer ( boyikang corp . , the concentration of protein in conditioned medium was measured by nanodrop 2000 ( thermo , ma , usa ) . all animal experiments were conducted with the approval of nanjing medical university ethics committee and were performed in accordance with the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication number 86 - 23 , revised 1996 ) . the full - thickness skin defect model used in this study is identical to the model previously described . thirty adult female sprague - dawley rats , weighing 200 g , were used . full - thickness skin incisions , 30 mm in diameter , were made on the back of each rat ( figure 2(a)(1 ) ) . thirty rats were randomly divided into 3 groups : ( 1 ) concentrated hypoxia - preconditioned adipose mesenchymal stem cells - conditioned medium group ( conc . hypo - amscs - cm group ) ; ( 2 ) asc - conditioned medium group ( hypo - amscs - cm group ) ; ( 3 ) control group ( 0.4% rat serum medium ) . the location of skin injury in all experimental groups was covered with concentrated and normal hypo - amscs - cm , which contains 1% hyaluronic acid ( ha ) . all medium was attached to the surface of wound completely and fixed on the skin with sterile transparent patches . statistical analysis was performed using analysis of variance ( anova ) test by spss17.0 , as appropriate . the t - test analysis was applied for measuring the data , and the regression analysis was used for the relationship between the time and the percentage of wound healing . the average number of adipose mesenchymal stem cells isolated was 1.4 0.5 10/ml from abdominal fat . primary amscs were cultured for 7 days ( figure 1(a)(1 ) ) and showed a typical fibroblast - like morphology at passage 3 after 27 days ( figure 1(a)(2 ) ) . the result of flow cytometry confirmed that amscs expressed cd29 ( figure 1(b)(1 ) ) , cd90 ( figure 1(b)(3 ) ) , and cd105 ( figure 1(b)(4 ) ) , but not cd34 ( figure 1(b)(2 ) ) . oil red o staining showed the lipid vacuoles appearance in cells ( figure 1(c)(2 ) ) and alizarin red staining obviously revealed calcium in cells , as observed from red staining ( figure 1(c)(4 ) ) . meanwhile , the oil red o staining of noninduced cells ( before staining , ( c)(5 ) ; after staining , ( c)(6 ) ) and the alizarin red staining of noninduced cells were also shown in figure 1 ( before staining , ( c)(7 ) ; after staining , ( c)(8 ) ) . the hypo - amscs - cm was collected and lyophilized in the sterilized penicillin bottle . in application , hypo - amscs - cm was diluted with 1 ml sterilized water or hyaluronic acid ( figure 2(a)(2 ) ) . the protein concentration of hypo - amscs - cm is 0.389 0.04 mg / ml , the conc . hypo - amscs - cm is 5.989 0.07 mg / ml , and the 0.4% rat serum medium is 0.350 0.04 mg / ml . the average healing time of the three groups was measured , of which the conc . hypo - amscs - cm group was 16.2 0.98 days , hypo - amscs - cm group was 17.7 0.78 days , and medium control group was 21.3 1.10 days ( figure 3(a ) ) . hypo - amscs - cm and hypo - amscs - cm groups ( p = 0.002 ) and between hypo - amscs - cm and control groups ( p < 0.001 ) . percentage of wound healing was evaluated by measurement of healing area / initial wound area 100% on 7 , 14 , and 21 days after treatment in three groups ( figure 3(b ) ) . in the regression analysis , the equation of relationship between the time and the percentage of wound healing in conc . hypo - amscs - cm group was y = 0.0455x + 0.127 ( r = 0.9043 , p < 0.01 ) ; that in hypo - amscs - cm group was y = 0.0444x + 0.1108 ( r = 0.9122 , p < 0.01 ) ; and that in the control group was y = 0.0442x + 0.0688 ( r = 0.9589 , p < 0.01 ) . hypo - amscs - cm and hypo - amscs - cm groups ( p = 0.299 ) . the conc . hypo - amscs - cm treatment improved wound healing when compared with that of the hypo - amscs - cm group at 7 ( p < 0.001 ) and 14 ( p < 0.001 ) days ( figures 2(b ) and 3(b ) ) . the hypo - amscs - cm group 's wound healing was also significantly different from that of the control groups at 7 ( p < 0.001 ) , 14 ( p < 0.001 ) , and 21 days ( p = 0.0005 ) ( figures 2(b ) and 3(b ) ) . for hematoxylin and eosin stain , biopsies of three groups were harvested from center of wound at 21 days after wound . hypo - amscs - cm showed well organized epidermis , thick cuticular layer , and increased collagen content ( figure 2(b ) ) , whereas wound areas treated with hypo - amscs - cm showed immature epidermal regeneration , weak cuticular layer , and less collagen content ( figure 2(b ) ) . hypo - amscs - cm treatment has better effects on wound repair than hypo - amscs - cm treatment . it has been shown that amscs can differentiate into adipogenic and osteogenic lineages in the particular medium . in this study , amscs showed the same immune - phenotypes as described previously [ 6 , 10 ] . they expressed the mesenchymal stem cell markers cd29 , cd90 , and cd105 , but not hematopoietic marker cd34 . the applications of culture medium of other cells have also been widely used in wound healing . we searched the medline and embase databases , using the conditioned medium and skin wound as search terms . all eligible studies were parallel - controlled trials about conditioned medium of stem cell in skin wound healing ( see supp . we first freeze - dried the conditioned medium of amscs , so the multiple concentration of conditioned medium could be made for clinical application . however , the umbilical cord wharton 's jelly cells and bone marrow mesenchymal cells were used frequently , and the adipose mesenchymal stem cells were selected in this research not only because of extensive sources , but also because of the fact that the fat tissue plays an important role in reepithelialization of damaged tissue and reconstruction of skin appendage . subcutaneous adipose cells , which demonstrate the dynamic regeneration parallel to the activation of skin stem cell , are necessary and sufficient to drive follicular stem cell activation and support lanugo growth . the hypo - amscs - cm has been shown to promote hair growth in c(3)h / neh mice . amscs were located in perivascular niches , close to vascular structure in fat . in vivo the lower oxygen environment is an important factor for maintaining the self - renewal and plastic characterization of amscs . in vitro , the oxygen concentration is considered a driver of cell function , and it helps maintain stem cell pluripotency , induces angiogenesis , and regulates the signaling of stem cell . the hypo - amscs - cm could enhance the wound healing via angiogenesis , increasing the collagen 1 expression , migration of fibroblasts , vascular endothelial cell and keratinocytes , and recruiting the circulating stem cells . therefore , response of hypoxia - preconditioned amscs may be invaluable for the development of novel wound healing and skin regeneration strategies . skin is the first line of body 's defense in the protection of foreign invasion , as the skin is exposed to pathogens in the macroenvironment . the unexpected skin injury may affect circulative exercise training , thus disrupting normal training rhythm . serious injury could potentially end athlete 's whole sports career . according to the skin wound severity , the healing takes around 2 weeks generally . many treatments have been proven to accelerate the healing , such as tissue engineering , stem cell , prp , and gene therapy . however , the cost and safety are major challenges . the results from this study showed that there are no statistical differences between the concentrated and nonconcentrated groups . the process of concentration may be important for wound healing because of difference in day 7 and day 14 . additionally , the hypo - amscs - cm has several advantages in skin wound healing : ( 1 ) being without nucleic acid and suitable for allogeneic application , ( 2 ) terminal sterilization by filtration , ( 3 ) easiness in producing and maintaining , and ( 4 ) a variety of concentrations of amscs - cm that could be obtained from freezing - dried powder . the hypo - amscs - cm could be powdered through the concentrated and lyophilized process , and the high concentrated hypo - amscs - cm , made by the powder , may be more effective in the skin wound treatment of the rat model .

in recent years , the bioactive factors were utilized in exercise and athletic skin injuries . in this research , the concentrated conditioned medium of hypoxia - preconditioned adipose mesenchymal stem cells , which is rich in bioactive factor , is applied in full - thickness skin defect model to evaluate the therapeutic efficacy . adipose mesenchymal stem cells were harvested from the abdominal subcutaneous adipose tissues . the surface markers and the potential of differentiation were analyzed . the conditioned medium of hypoxia - preconditioned stem cells was collected and freeze - dried and then applied on the rat full - thickness skin defect model , and the healing time of each group was recorded . haematoxylin and eosin staining of skin was assessed by microscope . the characteristics of adipose mesenchymal stem cells were similar to those of other mesenchymal stem cells . the concentration of protein in freeze - dried conditioned medium in 1 ml water was about 15 times higher than in the normal condition medium . in vivo , the concentrated hypoxia - preconditioned conditioned medium can reduce the wound size and accelerate the skin wound healing . the concentrated hypoxia - preconditioned adipose mesenchymal stem cell - conditioned medium has great effect on rat model of wound healing , and it would be an ideal agent for wound care in clinical application .

the pulmonary lymphatic system plays an important role in the lung perfusion homeostasis . congenital errors of lymphatic vessel development lead to primary pulmonary lymphatic disorders , including lymphangiomas , lymphangiectasis , lymphangiomatosis , and lymphatic dysplasia syndromes ( 1 ) . among them , pulmonary lymphangioma is a rare benign lesion thought to result from the development of abnormally proliferating lymphatic vessels . the abnormal vessels may be capillary , cavernous or cystic in type . the basic defect has been considered to be an abnormality in the developmental lymphangiogenesis ( 2 ) . we present a case of intrapulmonary cystic lymphangioma involving the upper lobe of the right lung , which presented with dyspnea in a 2-month - old female infant , and review the literature on its pathogenesis , clinicopathologic features , and radiographic findings . a 2-month - old korean baby girl developed dyspnea and irritability about a week before admission to our hospital . on physical examination at admission , her temperature was 36.2 , blood pressure 80/60 mmhg , pulse 145/min , respiratory rate 28/min , and weight 4,200 g. breathing sound decreased in the right lung field and was accompanied by chest wall retraction . otherwise physical examination and laboratory results proved unremarkable . on birth history , delivery occurred spontaneously at 37 weeks gestation and her birth weight was 3,000 g. a chest radiograph at admission showed a solitary cystic mass , entirely occupying the upper lobe of the right lung . high - resolution computed tomography ( hrct ) of the chest depicted a well - circumscribed , multiseptate , air - filled cystic lesion in the upper lobe of the right lung , mimicking the feature of type i congenital cystic adenomatoid malformation ( fig . grossly , the large part of the upper lobe was replaced by multiple cystic spaces that contained some serous fluid and varied in size from 2 - 3 mm to 3.5 cm histopathological examination of the upper lobe revealed a relatively circumscribed intrapulmonary cystic lesion composed of multiple cysts or cavities ( fig . lung parenchyma outside the cystic lesion exhibited emphysematous change and some lymphangiectasia in the connective tissues of interlobular septa and subpleural zones . in the immunohistochemical staining the lining cells of the cystic walls were not reactive for epithelial membrane antigen and cytokeratin , but weakly positive for factor viii - related antigen ( fig . lymphangiomas are focal proliferation of well differentiated lymphatic tissue that presents as multicystic or sponge - like appearance . they are generally subdivided into three pathologic categories : capillary lymphangiomas describe thin - walled lymphatic channels that occur as small well circumscribed cutaneous lesions ; cavernous lymphangiomas are microscopic thin - walled lymphatic channels with associated stroma ; and cystic lymphangiomas are large , well - circumscribed , multiloculated cystic spaces lined by endothelium that contain a significant connective tissue component ( 2 ) . their pathogenesis is still uncertain , but they may present as congenital or acquired forms . congenital lymphangiomas probably represent embryologic remnants of lymphatic tissues that either failed to connect to efferent channels or arose from portions of lymph sacs that were sequestered during development ( 3 ) . acquired or secondary lymphangiomas develop in areas of chronic lymphatic obstruction related to surgery , chronic infection , or radiation . lymphangioma can occur in any region of the body in which there is lymphatic drainage . the single most common site of cystic occurrence is in the neck , where the lesion is referred to as a cystic hygroma . in the chest , lymphangiomas are most commonly found in the mediastinum , where they account for 0.7% to 4.5% of all mediastinal tumors ( 4 ) . in a regard to intrapulmonary lymphangiomas they have been described but are extremely rare : approximately a dozen cases , with patient ages ranging from 6 months to 54 yr , are described in the english literature ( 1 ) . they frequently occur in the lower lobe of the lung , which is suspected to be associated with a rich lymphatic supply noted in the part of the lung . but we also found two case reports developed in the upper lobe ( 5 , 6 ) , as in our case . in a review of 20 patients with thoracic lymphangioma ( including mediastinal ) , shaffer et al . wilson et al . ( 5 ) suggested that all adult patients with intrapulmonary lymphangioma were men , including their case . symptoms are known to vary widely depending on the age of the patient and the extent of disease . in neonates and infants , cystic lymphangiomas often present with pneumothorax and respiratory distress ( 4 ) . in our case radiologically plain radiographs , ultrasound , ct scan , and magnetic resonance imaging ( mri ) have proven useful in determining the number and extent of lesions . the most common ct appearance of lymphangiomas is that of a cystic mass with smooth margins , but in the childhood the ct and/or mri findings do not appear to be specific to differentiate lymphangiomas from other intrapulmonary cystic lesions , including congenital cystic adenomatoid malformation ( ccam ) , pleuropulmonary blastoma ( ppb ) , bronchial atresia , bronchogenic cyst , and congenital lobar emphysema ( 7 ) . therefore histological and roentgenographic correlation may be necessary for establishing a definite diagnosis . among the list of differential diagnosis , ccam is a disorder of embryonic bronchopulmonary development , characterized by a multicystic mass of pulmonary tissue with an abnormal proliferation of bronchial structures . there are three types : type i consists of a large single cyst ; type ii of numerous small cysts ; type iii appears as a solid mass . the prognosis is variable and depends on the size and the histologic type ( 8) . ppb is a rare malignant dysontogenetic neoplasm affecting children , and is characterized histologically by a variably mixed blastematous and sarcomatous patterns . ppb has also known to have three pathological varieties , including predominantly cystic type i , cystic and solid type ii , and predominantly solid type iii ( 9 ) . the solid areas of the types ii and iii ppb are composed of undifferentiated blastemal tissue which may overlap with spindle cell sarcomatous , rhabdomyosarcomatous , anaplastic , and chondrosarcomatous foci . bronchial atresia may be present as an asymptomatic mass in the newborn period or the lung may appear as a fluid - filled , pulmonary mass at birth . later the fluid is replaced by air and the lobe becomes emphysematous ( 10 ) . though the middle and posterior mediastinum are generally regarded as the commonest sites , the cysts can unusually show the intrapulmonary localization ( 11 ) . when a communication persists between the tracheobronchial tree and the cyst , an air - fluid level is present as well as a history of multiple infections . a pseudostratifed , ciliated columnar epithelium , submucous glands , and isolated islands of cartilage are the composite of histologic findings for an unequivocal diagnosis of bronchogenic cyst . congenital lobar emphysema or congenital lobar hyperinflation affects only one of the upper lobes or the right middle lobe of the lung . the main pathologic change consists of massive overdistention of the alveolar spaces , not accompanied by the destruction of the tissues . in addition , intrapulmonary lymphangiomas need to be differentiated from other primary pulmonary lymphatic disorders , including lymphangiectasis and lymphangiomatosis ( 1 ) . secondary forms of lymphangiectasis result from a variety of processes that impair lymph drainage and increase lymph production . pathologically the lesion shows multifocal lymphatic dilations along the lymphatic routes of the interlobular septa and the bronchovascular bundles . but it does not show the localized proliferation of anastomosing lymphatic channels which is characteristic of lymphangiomas . it is frequently associated with other lymphatic - related abnormalities and usually ( in 75% of cases ) involves multiple organs . the histology of lymphangiomatosis resembles a lymphangioma , but can appear to infiltrate tissues , raising concern for a more aggressive lesion . clinically it presents at a later age than solitary lymphangiomas either because of an influence of hormonal factors or because a more subtle , albeit more widespread , defect requires a longer period for growth . the natural history of intrapulmonary lymphangiomas in infants is not well - known , but surgical resection should be considered in the light of the neoplastic nature of these lesions and of the difficulty in clinically differentiating them from other cystic lesions occurring in the age class ( 12 ) . if the lesion is small and localized , the application of thoracoscopic surgery is becoming increasingly popular . incomplete resection , however , can result in recurrence at sites of surgical resection , because of their autonomous growth and also by their reaccumulation of lymph fluid ( 1 ) . prior to the surgical exploration and excision , it is prudent to investigate for other lymphangiomatous lesions and associated congenital anomalies . in summary , we present a case of an intrapulmonary cystic lymphangioma causing dyspnea in a 2-month - old infant . to the best of our knowledge this case is the second report in the korean literature since 1997 ( 13 ) . despite of the scarcity , this lesion

lymphangioma is an abnormal collection of lymphatics that are developmentally isolated from the normal lymphatic system . lymphangioma rarely presents as a solitary pulmonary lesion . we report a rare case of intrapulmonary cystic lymphangioma involving the upper lobe of the right lung , which presented with dyspnea in a 2-month - old infant . high - resolution computed tomography ( hrct ) of the chest demonstrated a well - circumscribed , multiseptate , cystic lesion in the upper lobe of the right lung , mimicking the feature of type i congenital cystic adenomatoid malformation . the tumor was removed by bilobectomy of the upper and middle lobes of the right lung , and its pathologic examination confirmed the diagnosis of an intrapulmonary cystic lymphangioma .

ms is a highly polymorphic disease characterized by diverse neurological signs and symptoms ( e.g. , optical neuritis , dizziness , disturbances in bladder control , peripheral sensory neuropathies , and/or limb weakness ) . in most patients ( 80% ) ms has a relapse - remitting pattern , whereas in a minority it can be primary progressive ( 6% ) , secondary progressive ( 10% ) , or progressive relapsing ( 4% ) . ms incidence rates vary significantly depending on geographic location and ethnic origin , ranging from 1 : 500 in northern europe to 1 : 100,000 in tropical countries . individuals migrating from a tropical region to northern europe seem to maintain a low risk . although a predisposition to develop ms may be hereditary , environmental factors display a significant interaction with genetic factors to help determine disease outcome . an effect of shared environment has not been yet proven , and little is known about potential environmental triggers ( e.g. , viral infections , including epstein - barr or varicella zoster virus ) . genes associated with a predisposition to develop ms are being actively sought following two main approaches : broad genomic screenings and targeted gene searches . over 15 research groups have reported linkages of chromosomal regions with ms : chromosomes 6p ( on which hla is located ) , 5p , 5q , 17q , and 19q [ 35 ] . however more than half of the human genome has been linked to ms to varying degrees . an association with the hla - drb11501-dqb10602 haplotype has been repeatedly found in high - risk , northern european populations . about 25% to 30% of monozygotic twins of an affected individual develop ms compared with only 3% to 5% of dizygotic twins and other siblings [ 4 , 5 ] . the games consortium recently examined 6,000 microsatellite markers among thousands of samples . although regions in chromosome 1q43 could be consistently associated with ms , other findings were difficult to replicate . independent genome - screening results also point to the 1q43 chromosomal region ( lod score > 3 using nonparametric multipoint analysis ) as a genetic risk factor for ms , as reported by the us and french ms genetics groups after analyzing data sets from 186 multiplex families . gene polymorphisms in the apolipoprotein e ( apoe ) gene of patients with ms have been reported by most as not associated with an increased risk of developing ms , but different groups found a discordant effect of this locus on the severity and/or disease progression in individuals already affected with ms [ 1 , 4 ] . reviewing epidemiological literature can help us identify the main demographic and sociocultural features of ms among ethnic groups . although ms has been observed in the three main racial groups worldwide ( white caucasian , oriental , and black ) , the disease tends to be unequally distributed . two hypotheses have been put forth to help explain the unequal susceptibility of ms among races . the first hypothesis states that whereas the highest ms rates are found in regions of the world inhabited largely by white populations , the lowest rates tend to be found in those areas where nonwhites live . the second hypothesis suggests that racially different groups living in the same geographical area tend to have different rates of ms , although there is a tendency for whites to display ms more often than nonwhites . data associated with the first and second hypotheses support a uniformly higher and lower risk for ms among whites and nonwhites , respectively . if these findings are in fact valid , these studies indicate that racial ( genetic ) factors may play an important role in the distribution of ms . another view on the geographic diversity of ms indicates the existence of protective alleles that help against to develop autoimmune disorders [ 7 , 8 ] . the latter view seems to indicate ms is an acquired , exogenous ( environmental ) disease and confirms the importance of an environmental agent in disease causality that seems to be independent of ethnicity . we carried out an on - site epidemiological study among lacandonians , a pure ethnic group in mexico to describe the presence or absence of clinical data compatible with ms or nmo . we carried out an on - site epidemiological study to confirm if lacandonians , a pure amerindian ethnic group in mexico , had clinical signs and symptoms compatible with ms or with other demyelinating diseases . our research team visited the lacandonian forest located in the southern state of chiapas , a south - west mexican state with nearly 4 million of habitants ; 52% lives in rural area . 12 of 62 indigenous populations in mexico live there , tseltal , tsotsil , chol , tojol - abal , zoque , chuj , kanjobal , mam , jacalteco , moch , cakchiquel y lacandn o maya caribe ; all these people conform near 1 million habitants ; lacandonians are nearly 50,000 people divided in different areas in lacandonian forest ; the main communities where lacandonians live are nah and metzaboc in the north and lacan h chan sayab , bethel and el tumbo in the south , a region throughout all year has 2029c , humid tropical weather . we interviewed the community leader and we explained the nature of the study and requested informed consent . we also sought help from local general practitioners working in different communities ( nah and metzaboc , two communities located in the northern part and lacan h chan sayab , bethel and el tumbo , in the south ) . our study instrument included a survey with basic socio - demographic characteristics , a structured interview , and a comprehensive neurological examination . the research team concentrated on two strategic points in the north and south areas of the state where lacandonians often gather ( nah and lacan h chan sayab ) . a group of local translators helped the research team to administer the study instrument , and during the face - to - face interview , we mention that near of 85% of all lacandonians speak mother language and spanish , so the translator was used only in few cases . the ad hoc clinical questionnaire was designed by senior members of our team adapted from the standardized instrument routinely used at the national institute of neurology and neurosurgery , a tertiary care referral center in mexico city . the study instrument was administered by a group of 50 general local practitioners previously trained in study protocol with the help of community translators . each participant gave oral or written informed consent with prior authorization from the community leader . we explained to all study participants we were interested in exploring the presence or absence of clinical data suggestive of a previous demyelinating event . all study participants were given a thorough neurological exam performed by senior neurologists specializing in demyelinating disorders . the exam emphasized the presence of any clinical sign suggestive of a demyelinating disease . during the - face - to face interview , and after consent for biological samples from participants was obtained , we collected blood samples ( 30 mls by venopuncture ) to perform further genetic analysis that would substantiate our epidemiological findings . by the nature of study we collected clinical and demographic data from 5,372 lacandonians living in five communities in chiapas , mexico . we collected 350 dna blood samples for future analysis and during our first screening identified that 99% of samples were o+ blood type . we were not able to identify by family background or clinical history the presence of any demyelinating disease in our sample . the main three causes of disease among our sample were acute respiratory disease , gastrointestinal disease , and conjunctivitis . we did not identify patients with symptoms of ms or neuromyelitis optica ( nmo ) . ms prevalence is higher among northern europeans and americans who report european ancestry or have a predominantly caucasian extraction than among native americans living in the same latitudes . consensus from the latin american committee for treatment and research in multiple sclerosis ( lactrims ) [ 9 , 10 ] suggests that nonmixed native americans or amerindians are seldom affected by ms . the prevalence of ms continues to increase among mestizos , who have a complex mixture of caucasian and mongoloid genes and constitute the core people living in latin america . studiesamong ethnic groups considered pure such as the tarahumara , pima , mazahua , and quarijo indian groups [ 911 ] in chihuahua , a northern state in mexico , or among ethnic groups living in the central regions , of mexico such as the nahuatl , mexica , huastecos , or otomies do not seem to report ms . the disease has not been reported among other american indian groups such as the aymars in per , the xingu or among the yanomamis from brazil , or the mapuche from chile [ 9 , 10 ] . it seems plausible that american indians are protected against ms or other demyelinating disorders like neuromelitis optica ( nmo ) in part due to their ancestral genetic profile . in our study using a large sample of lacandonian subjects who underwent comprehensive clinical and neurological testing , other studies done among mestizos in mexico have shown that ms mestizo ( mixture of indigenous and spanish descent ) patients share an hla - drb1 profile similar to the one found among european groups at high risk for ms . this finding was not corroborated in our study among ethnically pure groups such as the lacandonians who display a low frequency of these haplotypes thereby pointing towards a genetic admixture between caucasian and amerindians , a mixture that took place over the last five centuries when spain conquered most of the region . not only is the genetic background of latin america singular , but also the exposure to certain infectious agents may be a critical factor that may help prevent or ameliorate some autoimmune diseases such as ms . a dichotomous association has been shown to exist between the global distribution of ms and the presence of parasitic infections [ 13 , 14 ] . in the case of our study participants , lacandonians displayed a high frequency of gastrointestinal disorders that may indicate that parasitic diseases could be an epidemiological finding that stands in contrast with developing any type of demyelinating disorders . we did not find clinical evidence of ms or nmo among our sample of lacandonians , a finding that may indicate a likely environmental and genetic background that influences the incidence of these types of autoimmune disorders .

multiple sclerosis ( ms ) is a highly polymorphic disease characterized by different neurologic signs and symptoms . in ms , racial and genetic factors may play an important role in the geographic distribution of this disease . studies have reported the presence of several protective alleles against the development of autoimmune disorders . in the case of ms , however , they help define ms as a complex disease , and confirm the importance of environmental agents as an independent variable not associated with ethnicity . we carried out an on - site epidemiological study to confirm the absence of ms or nmo among lacandonians , a pure amerindian ethnic group in mexico . we administered a structured interview to 5,372 lacandonians to assess by family background any clinical data consistent with the presence of a prior demyelinating event . every participating subject underwent a comprehensive neurological examination by a group of three members of the research team with experience in the diagnosis and treatment of demyelinating disorders to detect clinical signs compatible with a demyelinating disease . we did not find any clinical signs compatible with multiple sclerosis among study participants .

the extension of a renal staghorn stone could be into a few calyces or it may involve all the calyces . the standard of care for renal staghorn stones is percutaneous nephrolithotomy ( pcnl ) monotherapy . the distribution of staghorn stone burden in the pelvi calyceal system ( pcs ) is an important determinant of the complexity of pcnl . in the era of pcnl monotherapy , there is a need for standardized nomenclature to quantitate differences in the staghorn complexity . this demands a detailed description of stone volume or surface area , distribution of the stone in the pcs and also the pcs anatomy . the historical literature is deficient in terms of detailed description of the staghorn stone and , therefore , series across the world are not comparable . various authors have proposed a uniform objective stone volume or surface area data that would make the assessment in a given scenario more objective . there is also a need for such an objective data with respect to pcnl monotherapy as a standard of care for managing staghorn stones . pcnl monotherapy is varied with respect to different approaches for renal staghorn stones in terms of patient position , access , number of tracts , size of the tracts and intracorporeal energy sources utilized . in the context of surgical recommendations , the treatment pattern is mostly affected by the anatomical attribute of the staghorn calculus . a staghorn stone that one clinician deems inappropriately complex due to its branching pattern or calyceal anatomy , morphometry - based classification is a natural evolution and attempt to objectively define the clinical complexity of renal staghorn stones . staghorn stone classification and staghorn morphometry. there were a total of 43 abstracts , of which 19 were chosen for review . a total of four publications are present in the contemporary literature regarding staghorn morphometry . in view of the paucity of adequate data , other classifications of staghorn an attempt was made to rationalize the importance of morphometry - based classification in the contemporary management of staghorn calculus . several groups have proposed classification schemes to better define staghorn calculi taking into account size , morphology and composition . most of the initial classification schemes have involved morphological and composition based classification . from a contemporary clinician 's perspective stone composition is important because a soft stone would break easily during intracorporeal lithotripsy . struvite staghorn stones have a higher content of organic matrix , rendering these stones fragile . secondary staghorn stones are seen in cushing 's syndrome , hyperparathyroidism and renal tubular acidosis . there is no specific method of treating a particular staghorn in a given renal unit . the traditional anatomical definition of staghorn calculus is a renal pelvic stone with extension into the renal calyces . rocco et al . in 1984 suggested renal calculus classification according to topography and morphology . + and being used for the presence or absence of dilatation and e and i for extra- and intrarenal position of the pelvis . the letter griffith et al . subsequently suggested another classification based on complexity , burden and distribution of stone in the pelvi calyceal system . a scoring diagram was used to notify the complexity and burden of stone in the pelvi calyceal system . the stone burden was defined as the sum of the longest axial diameter of all stones . the use of such a scoring diagram allowed a concise description of the complexity and burden of stone in each renal system and facilitated computerized stratification of the upper tract stones . di silverio classified staghorn stones into : borderline , when stones cover the renal pelvis and one calyx ; partial , when beside the pelvis , two calices are occupied ; complete , when stones are in the whole collecting system or 80% of it ; and gigantic , when the whole collecting system has stones and a dilation of the system occurs . partial staghorn calculi were defined as renal pelvic calculi extending into two calyceal groups and complete staghorn stones were defined as renal pelvic calculi extending into all major calyceal groups , filling at least 80% of the collecting system . but , this type of classification does not put any light on the management planning of staghorn stones and is also not based on any specific volume criteria . guy 's stone score was developed through a combination of expert opinion and published data review to test the association of stone with stone - free rates achievable . it comprised four grades : grade i , solitary stone in mid / lower pole or solitary stone in the pelvis with simple anatomy ; grade ii , solitary stone in upper pole or multiple stones in a patient with simple anatomy or a solitary stone in a patient with abnormal anatomy ; grade iii , multiple stones in a patient with abnormal anatomy or stones in a calyceal diverticulum or partial staghorn calculus ; and grade iv , staghorn calculus or any stone in a patient with spina bifida or spinal injury . guy 's stone score was the only factor that significantly and independently predicted the stone - free rate . the system was a generalized one and was not exclusively formulated for staghorn stones . with the advent of widespread ct scan , stone area and volume could be measured easily using software . stone volume is an important predictor of stone - free rates following shock wave lithotripsy . advocated the use of stone surface area by 3d ct for more accurate reporting of treatment results . the difficulty in accurately assessing stone burden explains the wide range of reported stone - free rates for shock wave lithotripsy ( swl ) monotherapy from 22% to 85% . lam and lingeman et al . suggested that stone surface area correlates well with stone volume , whereas maximal stone length does not . stone surface area determination enables more accurate reporting of treatment results , and thus recommendation based on stone burden . the european association of urology recommends stone volume measurement by using maximum diameter , and using it on a scalene ellipsoid formula . studied the correlation of 3d - reconstructed stone volume with respect to the accuracy of scalene , oblate and prolate ellipsoid volume equations . therefore , staghorn stones having a hallmark of large stone volume are less accurately measured with ct diameter . as the maximum diameter increases , the calculated stone volume becomes less accurate , suggesting that larger stones have more asymmetric shapes . the best way to measure stone volume , therefore , is from 3d - reconstructed stone volumes . construction of a 3d model of renal stones can also potentially minimize the risks of percutaneous procedures and achieve higher one - stage stone - free rates . 3d reconstruction of the renal stones by many types of software can establish a virtual safe and reliable percutaneous renal access route on the 3d model of renal stones . performed pcnl with the assistance of the 3d model and found it to be feasible and highly effective in achieving a single - stage clearance rate of 93.3% . nephrometry score for predicting the outcome of pcnl , which varies from four to 11 . this score is determined by five parameters by a pre - operative ct scan : stone size ( s ) , tract length ( t ) , obstruction ( o ) , number of involved calices ( n ) and essence or stone density ( e ) . as the score increases , blood loss , complication and hospital stay increases and the clearance rate decreases . this classification is based on subjective criteria of obstruction that can be measured as no , mild or severe hydronephrosis ; therefore , this score may vary for the subjective interpretation of the degree of hydronephrosis . the aua nephrolithiasis guidelines panel has demonstrated superior stone - free rates , improved complication rates and reduced need for secondary procedures in those patients treated with pcnl monotherapy for staghorn calculus . standardized reporting of renal staghorn is essential for consistent decision making and effective comparisons , particularly as data emerge suggesting a relation between staghorn stone burden and pcnl monotherapy outcomes . therefore , a classification of staghorn based on actual stone burden and integrative with pcnl monotherapy is required . mishra et al . attempted to classify staghorn based on volumetric burden distribution within the pelvi calyceal system . herein , the objective was to afford standardized communication regarding the anatomical features of the staghorn . in the preliminary retrospective study , they performed an extensive volumetric burden data assessment correlating with the tracts and stages required in pcnl monotherapy for staghorn stone . the classification was made according to the total stone volume ( tsv ) and unfavorable calyx stone percentile volume ( ucspv ) . tsv was defined as the stone volume calculated by reconstructing the entire stone image on the software . in order to quantitate the stone volume , ct urography was performed and stone volume was assessed using a ct scan volumetric assessment software ( 3d - doctor ; able software corporation , lexington , ma , usa ) . the assessment of favorable and unfavorable calyx was performed on the image plane view of the software . a favorable calyx was defined as a calyx - containing stone that is at an obtuse angle to the entry calyx and has an infundibular width > 8 mm . the stones were classified in the following groups : type 1 staghorn < 5000 mm tsv and < 5% ufcspv ; type 2a 5000 - 20,000 mm tsv and < 5% ufcspv ; type 2b < 20,000 mm tsv and > 5% ufcspv ; and type 3 > 20,000 mm and any ufcspv . multivariate analysis revealed that the tract depends on the ufcspv while the stages required depend on the tsv in pcnl monotherapy . the combination of tsv and ucspv predicted the complexity of staghorn . in the odds ratio calculation , they found that the odds ratio increased adversely for multiple tracts as the ucspv increased . quantitating stone volume paul et al . performed a retrospective study including a total of 170 renal units in 163 patients who underwent treatment for staghorn calculi . there was no significant difference in tsv when single or two tracts were compared , but there was a significant increase for more tracts . pelvic , pelvic and entry caylx percentile volume were significantly lower in the multiple tract group . ufcspv were significantly less in single and two tract ( s ) than in the multiple tracts group . however , it has opened a pandora of future work to be done on classifying staghorn stones . what may seem complex today could become easy with the development of dedicated stone software . there should also be a prospective study to account for the clinical benefit of classifying staghorn stones . we presume that the higher is the complexity of the staghorn morphometry , more is the hemoglobin drop , hospital stay , complications and treatment costs . several groups have proposed classification schemes to better define staghorn calculi taking into account size , morphology and composition . most of the initial classification schemes have involved morphological and composition based classification . from a contemporary clinician 's perspective stone composition is important because a soft stone would break easily during intracorporeal lithotripsy . struvite staghorn stones have a higher content of organic matrix , rendering these stones fragile . secondary staghorn stones are seen in cushing 's syndrome , hyperparathyroidism and renal tubular acidosis . there is no specific method of treating a particular staghorn in a given renal unit . the traditional anatomical definition of staghorn calculus is a renal pelvic stone with extension into the renal calyces . rocco et al . in 1984 suggested renal calculus classification according to topography and morphology . + and being used for the presence or absence of dilatation and e and i for extra- and intrarenal position of the pelvis . the letter griffith et al . subsequently suggested another classification based on complexity , burden and distribution of stone in the pelvi calyceal system . a scoring diagram was used to notify the complexity and burden of stone in the pelvi calyceal system . the stone burden was defined as the sum of the longest axial diameter of all stones . the use of such a scoring diagram allowed a concise description of the complexity and burden of stone in each renal system and facilitated computerized stratification of the upper tract stones . when stones cover the renal pelvis and one calyx ; partial , when beside the pelvis , two calices are occupied ; complete , when stones are in the whole collecting system or 80% of it ; and gigantic , when the whole collecting system has stones and a dilation of the system occurs . partial staghorn calculi were defined as renal pelvic calculi extending into two calyceal groups and complete staghorn stones were defined as renal pelvic calculi extending into all major calyceal groups , filling at least 80% of the collecting system . but , this type of classification does not put any light on the management planning of staghorn stones and is also not based on any specific volume criteria . guy 's stone score was developed through a combination of expert opinion and published data review to test the association of stone with stone - free rates achievable . it comprised four grades : grade i , solitary stone in mid / lower pole or solitary stone in the pelvis with simple anatomy ; grade ii , solitary stone in upper pole or multiple stones in a patient with simple anatomy or a solitary stone in a patient with abnormal anatomy ; grade iii , multiple stones in a patient with abnormal anatomy or stones in a calyceal diverticulum or partial staghorn calculus ; and grade iv , staghorn calculus or any stone in a patient with spina bifida or spinal injury . guy 's stone score was the only factor that significantly and independently predicted the stone - free rate . the system was a generalized one and was not exclusively formulated for staghorn stones . with the advent of widespread ct scan , stone area and volume could be measured easily using software . stone volume is an important predictor of stone - free rates following shock wave lithotripsy . advocated the use of stone surface area by 3d ct for more accurate reporting of treatment results . the difficulty in accurately assessing stone burden explains the wide range of reported stone - free rates for shock wave lithotripsy ( swl ) monotherapy from 22% to 85% . lam and lingeman et al . suggested that stone surface area correlates well with stone volume , whereas maximal stone length does not . stone surface area determination enables more accurate reporting of treatment results , and thus recommendation based on stone burden . the european association of urology recommends stone volume measurement by using maximum diameter , and using it on a scalene ellipsoid formula . studied the correlation of 3d - reconstructed stone volume with respect to the accuracy of scalene , oblate and prolate ellipsoid volume equations . therefore , staghorn stones having a hallmark of large stone volume are less accurately measured with ct diameter . as the maximum diameter increases , the calculated stone volume becomes less accurate , suggesting that larger stones have more asymmetric shapes . the best way to measure stone volume , construction of a 3d model of renal stones can also potentially minimize the risks of percutaneous procedures and achieve higher one - stage stone - free rates . 3d reconstruction of the renal stones by many types of software can establish a virtual safe and reliable percutaneous renal access route on the 3d model of renal stones . performed pcnl with the assistance of the 3d model and found it to be feasible and highly effective in achieving a single - stage clearance rate of 93.3% . nephrometry score for predicting the outcome of pcnl , which varies from four to 11 . this score is determined by five parameters by a pre - operative ct scan : stone size ( s ) , tract length ( t ) , obstruction ( o ) , number of involved calices ( n ) and essence or stone density ( e ) . as the score increases , blood loss , complication and hospital stay increases and the clearance rate decreases . this classification is based on subjective criteria of obstruction that can be measured as no , mild or severe hydronephrosis ; therefore , this score may vary for the subjective interpretation of the degree of hydronephrosis . the aua nephrolithiasis guidelines panel has demonstrated superior stone - free rates , improved complication rates and reduced need for secondary procedures in those patients treated with pcnl monotherapy for staghorn calculus . standardized reporting of renal staghorn is essential for consistent decision making and effective comparisons , particularly as data emerge suggesting a relation between staghorn stone burden and pcnl monotherapy outcomes . therefore , a classification of staghorn based on actual stone burden and integrative with pcnl monotherapy is required . mishra et al . attempted to classify staghorn based on volumetric burden distribution within the pelvi calyceal system . herein , the objective was to afford standardized communication regarding the anatomical features of the staghorn . in the preliminary retrospective study , they performed an extensive volumetric burden data assessment correlating with the tracts and stages required in pcnl monotherapy for staghorn stone . the classification was made according to the total stone volume ( tsv ) and unfavorable calyx stone percentile volume ( ucspv ) . tsv was defined as the stone volume calculated by reconstructing the entire stone image on the software . in order to quantitate the stone volume , ct urography was performed and stone volume was assessed using a ct scan volumetric assessment software ( 3d - doctor ; able software corporation , lexington , ma , usa ) . the assessment of favorable and unfavorable calyx was performed on the image plane view of the software . a favorable calyx was defined as a calyx - containing stone that is at an obtuse angle to the entry calyx and has an infundibular width > 8 mm . the stones were classified in the following groups : type 1 staghorn < 5000 mm tsv and < 5% ufcspv ; type 2a 5000 - 20,000 mm tsv and < 5% ufcspv ; type 2b < 20,000 mm tsv and > 5% ufcspv ; and type 3 > 20,000 mm and any ufcspv . multivariate analysis revealed that the tract depends on the ufcspv while the stages required depend on the tsv in pcnl monotherapy . in the odds ratio calculation , they found that the odds ratio increased adversely for multiple tracts as the ucspv increased . quantitating stone volume may be standardized universally if ct - assisted stone volumetric assessment is performed . paul et al . performed a retrospective study including a total of 170 renal units in 163 patients who underwent treatment for staghorn calculi . there was no significant difference in tsv when single or two tracts were compared , but there was a significant increase for more tracts . pelvic , pelvic and entry caylx percentile volume were significantly lower in the multiple tract group . ufcspv were significantly less in single and two tract ( s ) than in the multiple tracts group . however , it has opened a pandora of future work to be done on classifying staghorn stones . what may seem complex today could become easy with the development of dedicated stone software . there should also be a prospective study to account for the clinical benefit of classifying staghorn stones . we presume that the higher is the complexity of the staghorn morphometry , more is the hemoglobin drop , hospital stay , complications and treatment costs . several groups have proposed classification schemes to better define staghorn calculi taking into account size , morphology and composition . most of the initial classification schemes have involved morphological and composition based classification . from a contemporary clinician 's perspective stone composition is important because a soft stone would break easily during intracorporeal lithotripsy . struvite staghorn stones have a higher content of organic matrix , rendering these stones fragile . secondary staghorn stones are seen in cushing 's syndrome , hyperparathyroidism and renal tubular acidosis . there is no specific method of treating a particular staghorn in a given renal unit . the traditional anatomical definition of staghorn calculus is a renal pelvic stone with extension into the renal calyces . rocco et al . in 1984 suggested renal calculus classification according to topography and morphology . + and being used for the presence or absence of dilatation and e and i for extra- and intrarenal position of the pelvis . the letter r referred to clinical recurrences . finally , the letter griffith et al . subsequently suggested another classification based on complexity , burden and distribution of stone in the pelvi calyceal system . a scoring diagram was used to notify the complexity and burden of stone in the pelvi calyceal system . the stone burden was defined as the sum of the longest axial diameter of all stones . the use of such a scoring diagram allowed a concise description of the complexity and burden of stone in each renal system and facilitated computerized stratification of the upper tract stones . when stones cover the renal pelvis and one calyx ; partial , when beside the pelvis , two calices are occupied ; complete , when stones are in the whole collecting system or 80% of it ; and gigantic , when the whole collecting system has stones and a dilation of the system occurs . partial staghorn calculi were defined as renal pelvic calculi extending into two calyceal groups and complete staghorn stones were defined as renal pelvic calculi extending into all major calyceal groups , filling at least 80% of the collecting system . but , this type of classification does not put any light on the management planning of staghorn stones and is also not based on any specific volume criteria . guy 's stone score was developed through a combination of expert opinion and published data review to test the association of stone with stone - free rates achievable . it comprised four grades : grade i , solitary stone in mid / lower pole or solitary stone in the pelvis with simple anatomy ; grade ii , solitary stone in upper pole or multiple stones in a patient with simple anatomy or a solitary stone in a patient with abnormal anatomy ; grade iii , multiple stones in a patient with abnormal anatomy or stones in a calyceal diverticulum or partial staghorn calculus ; and grade iv , staghorn calculus or any stone in a patient with spina bifida or spinal injury . guy 's stone score was the only factor that significantly and independently predicted the stone - free rate . with the advent of widespread ct scan , stone area and volume could be measured easily using software . stone volume is an important predictor of stone - free rates following shock wave lithotripsy . advocated the use of stone surface area by 3d ct for more accurate reporting of treatment results . the difficulty in accurately assessing stone burden explains the wide range of reported stone - free rates for shock wave lithotripsy ( swl ) monotherapy from 22% to 85% . lam and lingeman et al . suggested that stone surface area correlates well with stone volume , whereas maximal stone length does not . stone surface area determination enables more accurate reporting of treatment results , and thus recommendation based on stone burden . the european association of urology recommends stone volume measurement by using maximum diameter , and using it on a scalene ellipsoid formula . studied the correlation of 3d - reconstructed stone volume with respect to the accuracy of scalene , oblate and prolate ellipsoid volume equations . they concluded that the average shape of renal stones changes with diameter . as the stone diameter increases , the accuracy of determining stone volume decreases . therefore , staghorn stones having a hallmark of large stone volume are less accurately measured with ct diameter . as the maximum diameter increases , the calculated stone volume becomes less accurate , suggesting that larger stones have more asymmetric shapes . the best way to measure stone volume , construction of a 3d model of renal stones can also potentially minimize the risks of percutaneous procedures and achieve higher one - stage stone - free rates . 3d reconstruction of the renal stones by many types of software can establish a virtual safe and reliable percutaneous renal access route on the 3d model of renal stones . performed pcnl with the assistance of the 3d model and found it to be feasible and highly effective in achieving a single - stage clearance rate of 93.3% . nephrometry score for predicting the outcome of pcnl , which varies from four to 11 . this score is determined by five parameters by a pre - operative ct scan : stone size ( s ) , tract length ( t ) , obstruction ( o ) , number of involved calices ( n ) and essence or stone density ( e ) . as the score increases , blood loss , complication and hospital stay increases and the clearance rate decreases . this classification is based on subjective criteria of obstruction that can be measured as no , mild or severe hydronephrosis ; therefore , this score may vary for the subjective interpretation of the degree of hydronephrosis . the aua nephrolithiasis guidelines panel has demonstrated superior stone - free rates , improved complication rates and reduced need for secondary procedures in those patients treated with pcnl monotherapy for staghorn calculus . standardized reporting of renal staghorn is essential for consistent decision making and effective comparisons , particularly as data emerge suggesting a relation between staghorn stone burden and pcnl monotherapy outcomes . therefore , a classification of staghorn based on actual stone burden and integrative with pcnl monotherapy is required . mishra et al . attempted to classify staghorn based on volumetric burden distribution within the pelvi calyceal system . herein , the objective was to afford standardized communication regarding the anatomical features of the staghorn . in the preliminary retrospective study , they performed an extensive volumetric burden data assessment correlating with the tracts and stages required in pcnl monotherapy for staghorn stone . the classification was made according to the total stone volume ( tsv ) and unfavorable calyx stone percentile volume ( ucspv ) . tsv was defined as the stone volume calculated by reconstructing the entire stone image on the software . in order to quantitate the stone volume , ct urography was performed and stone volume was assessed using a ct scan volumetric assessment software ( 3d - doctor ; able software corporation , lexington , ma , usa ) . the assessment of favorable and unfavorable calyx was performed on the image plane view of the software . a favorable calyx was defined as a calyx - containing stone that is at an obtuse angle to the entry calyx and has an infundibular width > 8 mm . the stones were classified in the following groups : type 1 staghorn < 5000 mm tsv and < 5% ufcspv ; type 2a 5000 - 20,000 mm tsv and < 5% ufcspv ; type 2b < 20,000 mm tsv and > 5% ufcspv ; and type 3 > 20,000 mm and any ufcspv . multivariate analysis revealed that the tract depends on the ufcspv while the stages required depend on the tsv in pcnl monotherapy . the combination of tsv and ucspv predicted the complexity of staghorn . in the odds ratio calculation , they found that the odds ratio increased adversely for multiple tracts as the ucspv increased . quantitating stone volume may be standardized universally if ct - assisted stone volumetric assessment is performed . paul et al . performed a retrospective study including a total of 170 renal units in 163 patients who underwent treatment for staghorn calculi . there was no significant difference in tsv when single or two tracts were compared , but there was a significant increase for more tracts . pelvic , pelvic and entry caylx percentile volume were significantly lower in the multiple tract group . ufcspv were significantly less in single and two tract ( s ) than in the multiple tracts group . however , it has opened a pandora of future work to be done on classifying staghorn stones . what may seem complex today could become easy with the development of dedicated stone software . there should also be a prospective study to account for the clinical benefit of classifying staghorn stones . we presume that the higher is the complexity of the staghorn morphometry , more is the hemoglobin drop , hospital stay , complications and treatment costs . the outcome parameters that are most relevant are stone clearance , complications , hospital stay and auxiliary procedures . as the staghorn stone morphometry increases , there is a possibility of a lower stone - free rate . in addition ,

introduction : the majority of staghorn classifications do not incorporate volumetric stone burden assessment . accurate volumetric data can easily be acquired with the ever - increasingly available computerized tomography ( ct ) scan . this manuscript reviews the available staghorn stone classifications and rationalizes the morphometry - based classification.materials and methods : a pubmed search was performed for articles concerning staghorn classification and morphometry . twenty abstracts were shortlisted from a total of 43 published abstracts . in view of the paucity of manuscripts on staghorn morphometry ( 4 ) , older staghorn classifications were analyzed with the aim to determine the most optimum one having relevance to the percutaneous nephrolithotomy ( pcnl ) monotherapy outcome.results:all available staghorn classifications are limited with non - widespread applicability . the traditional partial and complete staghorn are limited due to non - descript stone volumetric data and considerable overlap of the intermediate ones in either group . a lack of standardized definition limits intergroup comparison as well . staghorn morphometry is a recent addition to the clinical classification profiling of a staghorn calculus . it comprises extensive ct volumetric stone distribution assessment of a staghorn in a given pelvi calyceal anatomy . it allowsmeaningful clinical classification of staghorn stones from a contemporary pcnl monotherapy perspective.conclusions:morphometry-based classification affords clinically relevant nomenclature in predicting the outcome of pcnl for staghorn stones . further research is required to reduce the complexity associated with measuring the volumetric stone distribution in a given calyceal system .

a cruciform is a structure based on a dna inverted repeat , and characterized by the presence of a four - way junction in which two of the branches are hairpin structures formed on each strand of the inverted repeat ( figure 1a ) . the bases located between the inverted repeats do not self - pair and instead form the apical loops of the hairpins . although the four - way junction and the apical loops render the cruciform less stable than b - form dna , negative dna supercoiling has long been known to stabilize cruciform structures ( 14 ) . a single dna molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead . a pair of magnets serves to control the vertical extending force , f , applied to the dna and the number of rotations , n , imposed on the bead and hence the dna supercoiling . the dna end - to - end extension , l , is determined by measuring the position of the bead above the surface in real - time by computer - aided videomicroscopy . when the inverted repeat extrudes into a cruciform , unwinding of b - dna titrates out negative supercoils and an increase , l , in dna extension is observed . ( b ) real - time data showing abrupt , reversible , changes in extension for charomid 9 - 5 kb dna ( f 0.45 pn , n = 16 turns ) . green points correspond to real - time data acquisition ( 60 hz ) while red points correspond to raw signal averaged over 1 s. both structural ( l ) and kinetic ( twait and tcruciform ) features of the reaction can be obtained from these time - traces . the dashed line indicates the mean < l > = 241 3 nm ( n = 74 points ) . ( d ) histograms of twait ( blue ) and tcruciform ( red ) and single - exponential fits to data . mean lifetimes are < twait > = 20.2 1.2 s ( n = 490 events ) and < tcruciform > = 13.5 0.9 s ( n = 489 events ) . single - molecule detection of cruciform extrusion . ( a ) sketch of the experiment . a single dna molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead . a pair of magnets serves to control the vertical extending force , f , applied to the dna and the number of rotations , n , imposed on the bead and hence the dna supercoiling . the dna end - to - end extension , l , is determined by measuring the position of the bead above the surface in real - time by computer - aided videomicroscopy . when the inverted repeat extrudes into a cruciform , unwinding of b - dna titrates out negative supercoils and an increase , l , in dna extension is observed . ( b ) real - time data showing abrupt , reversible , changes in extension for charomid 9 - 5 kb dna ( f 0.45 pn , n = 16 turns ) . green points correspond to real - time data acquisition ( 60 hz ) while red points correspond to raw signal averaged over 1 s. both structural ( l ) and kinetic ( twait and tcruciform ) features of the reaction can be obtained from these time - traces . the dashed line indicates the mean < l > = 241 3 nm ( n = 74 points ) . ( d ) histograms of twait ( blue ) and tcruciform ( red ) and single - exponential fits to data . mean lifetimes are < twait > = 20.2 1.2 s ( n = 490 events ) and < tcruciform > = 13.5 0.9 s ( n = 489 events ) . from the experimental standpoint , cruciform dna has been extensively studied in vitro over the course of the past decades ( 5 ) using for instance gel - shift analysis of supercoiled inverted repeats to determine the equilibrium , and even the interconversion rates , between the b - dna and extruded cruciform states ( 68 ) . in such measurements , the b - dna and cruciform states can be distinguished by their overall topology ; indeed as discussed below , formation of the cruciform titrates out negative supercoils , typically causing the cruciform dna to migrate more slowly than b - dna during gel electrophoresis . in parallel , in vivo studies have suggested that the more structurally stable a cruciform , the more likely it is to be genetically unstable , for instance subject to excision as in bacteria ( 7,9 ) or even responsible for genetic rearrangements in eukaryotes ( 1014 ) . theoretical , numerical and experimental studies ( 15,16 ) further indicate that negative dna supercoiling in vivo may participate in modulating gene expression levels by driving dna unwinding transitions in gene regulatory regions , and cruciform extrusion could participate in such processes . on the other hand , many inverted repeats are obviously genetically stable ( consider the dna encoding trnas , microrna precursors or riboswitches ) , and only a few clear examples of regulatory roles have been identified [ e.g. promoters of the n4 virion ( 17,18 ) ] . thus , the role of cruciform extrusion in vivo remains elusive ( 19 ) . to better understand how inverted repeats could behave under in vivo conditions of negative supercoiling , it is interesting to determine how their sequence and structural features contribute to the stability or instability of a cruciform structure . here , we describe a single - molecule nanomanipulation procedure that allows us to directly monitor both cruciform extrusion and rewinding in real - time , and thus analyze cruciform stability in terms of the balance between the rates of extrusion and rewinding . this allows us to explore in detail how structural and sequence determinants of cruciforms contribute to their overall stability . in these experiments , an individual dna molecule is torsionally constrained by attaching both strands at one end of the dna to a treated glass coverslip , and by attaching both strands at the other end to a 1-m magnetic bead , as previously described ( 20,21 ) . to accomplish this , the dna is multiply labeled at one end with digoxigenin and at the other end with biotin . the labeled dna is then allowed to bind to 1-m streptavidin - coated magnetic beads . the bead - dna solution is then deposited on an antidigoxigenin - coated glass surface , and when the dna binds to the glass surface a tethered dna - bead system forms . the sample is placed on an inverted microscope through which bead images can be viewed and captured by videomicroscopy . the position of the bead in the three spatial directions is tracked at 30 hz and with 5 nm accuracy using the picotwist software package . from the position of the bead above the surface , we determine the end - to - end extension , l , of the dna . a pair of permanent magnets mounted on translation and rotation stages is located above the sample and generates a magnetic field that imposes a vertical extending force on the bead and sets the bead s orientation . the extending force is increased ( decreased ) by translating the magnets closer to ( farther away from ) the sample . rotating the magnets causes the bead to rotate in a perfectly synchronous fashion about the axis defined by the extended dna , allowing for quantitative supercoiling of the dna . both extending force and dna supercoiling can therefore be controlled in a fully reversible and quantitative fashion ( 20 ) . the extending force is calibrated using brownian motion analysis of the tethered bead , and determination of the dnas elastic behavior confirms that a single dna molecule tethers the bead to the surface ( 20 ) . indeed , the mechanical and topological properties of a single supercoiled dna molecule have been extensively studied experimentally ( 20,2225 ) and theoretically ( 2628 ) . briefly , dna topology is described by three numbers that reflect the number of times the two strands of dna wind about one another ( 29 ) . the twist number , tw , counts the number of times the two strands wrap one around the other in the double - helix structure ; in b - dna , there is one unit of twist for every 10.5 bp . the writhe , wr , counts the number of times the axis of the dna crosses itself , in particular in the looped structures ( also termed plectonemes ) that arise as a result of dna supercoiling and that resemble the tangles observed on a twisted phone cord . finally , the linking number lk is the sum total of crossings in the dna , either by twist or by writhe : lk = tw + wr . in a topologically constrained system ( i.e. a closed circular plasmid or a dna torsionally constrained in a magnetic trap ) , lk is a constant , and any change in tw must be compensated by an equal but opposite change in wr . by rotating the magnets and thus the bead , we directly modify lk . supercoiling a dna molecule subjected to a very low extending force ( f = 0.45 pn ) rapidly causes it to form loops , or writhe , as a means of storing the topological constraint ( 20 ) . it thereby adopts a compact conformation with low extension ( see sketch in leftmost panel of figure 1a ) . loops , and therefore writhe , can be calibrated by measuring the changes in dna extension that take place upon supercoiling of the molecule ( 30,31 ) ( see supplementary figure s1 for an example of such calibration curves ) . from these curves , we determine that adding or removing a supercoil changes the dna extension by an amount = 74 nm for the experimental conditions used here . thus for a negatively supercoiled dna containing an inverted repeat , cruciform extrusion is expected to result in a large increase in dna end - to - end extension ( figure 1a ) . this strategy is formally identical to that used in standard electrophoresis assays to measure cruciform extrusion , and has previously been used in this single - molecule assay to study dna unwinding in real - time , for instance during transcription by escherichia coli rna polymerase ( 32,33 ) . indeed the change in twist tw that occurs during cruciform extrusion must be compensated by an equal but opposite change in the writhing number , wr , of the dna : wr = tw ( 34 ) . conversion of ncruciform bp of b - dna to cruciform dna leads to a decrease in twist tw = ncruciform / h = ncruciform and an equal increase in wr ( h , the dna pitch , is 10.5 bp / turn ) . for negatively supercoiled dna , this results in a loss of negatively signed loops , and thus an increase in dna extension of ncruciform or 7 nm / bp in the cruciform . it is important to note that this is a very large increase , far greater than the 2 decrease in dna extension that results from removal of the base pair from the end - to - end extension of the dna so that it may participate in the cruciform ( lbp , the extension of a base pair of dna at this low extending force , is only 2 ) . the dna constructs studied in this article include the charomid 9 - 5 kb plasmid ( 35 ) ( for studies of the charomid x sequence , see results section ) and the 3.7 kb pcolir315 plasmid ( 36 ) ( for studies of the cole1 inverted repeat and its derivatives ) . experiments with charomid dna were carried out in sb solution containing 10 mm potassium phosphate buffer at ph 8.0 , 0.1% tween-20 and 0.1 mg / ml bsa , and for experiments with pcolir315 the potassium phosphate concentration was 11 mm . the extending force acting on the dna was f = 0.5 pn for experiments performed in 10 or 11 mm phosphate buffer and f = 0.8 pn for experiments performed in the presence of 50 mm nacl . the experimental system was thermally regulated to 26 0.1c using pelletier modules as the rates of cruciform extrusion and rewinding were found to be highly temperature sensitive . raw data traces were filtered at 12 s and analyzed using in - house software to determine where transitions between the b - dna and extruded cruciform state occurred . the low signal - to - noise ratio of the data , which results from the weak stiffness of supercoiled dna under the experimental conditions used , can limit the effectiveness of automatic analysis , and so results of this processing were then manually verified . typically , the mean rates were determined from analysis of at least 300 transitions ( providing for a standard error of about 6% on the mean rates ) , and transition amplitude histograms contain at least 50 points ( smaller error was achieved here due to the lower variance of the observed distributions ) . raw data traces were filtered at 12 s and analyzed using in - house software to determine where transitions between the b - dna and extruded cruciform state occurred . the low signal - to - noise ratio of the data , which results from the weak stiffness of supercoiled dna under the experimental conditions used , can limit the effectiveness of automatic analysis , and so results of this processing were then manually verified . typically , the mean rates were determined from analysis of at least 300 transitions ( providing for a standard error of about 6% on the mean rates ) , and transition amplitude histograms contain at least 50 points ( smaller error was achieved here due to the lower variance of the observed distributions ) . in figure 1b , we present a real - time measurement of the change in dna extension resulting from reversible extrusion of the charomid x imperfect inverted repeat under conditions of negative supercoiling . the time - trace shows the 5-kb dna undergoing abrupt transitions ( hopping ) between a low - extension state and a high - extension state . the charomid x sequence responsible for these fluctuations was identified by selectively removing different regions of the charomid 9 - 5 kb dna and screening the resulting constructs for the slow , large - scale fluctuations ( figure 2 ) . the low- and high - extension states we observe are separated by a remarkably large change in dna extension of < l > = 241 3 nm ( figure 1c ) , nearly 15% of the full contour length of the dna . no such slow , large - scale fluctuations are observed if the charomid x sequence is positively supercoiled ( supplementary figure s1 ) . we thus estimate that when the dna hops from the low - extension state to the high - extension state , < l>/ = 3.3 units of writhe are titrated out , consistent with formation of a cruciform involving ncruciform = 34 bp . this is in agreement with the expected size of the cruciform based on the charomid x quasipalindromic sequence ( 33 bp , see figure 2c ) . the charomid x sequence still undergoes these fluctuations if separated from its original flanking sequences ( supplementary figure s2 ) . figure 2.single-molecule screen of charomid 9 - 5 kb is used to identify the charomid x quasipalindrome . charomid 9 - 5 kb is derived from charomid 9 - 11 kb by removing the 6 kb region of the plasmid , corresponding to three consecutive repeats of a 2-kb fragment ( 35 ) . then , five constructs are derived from charomid 9 - 5 kb using the pcr reaction , each construct lacking a different 1 kb segment of the plasmid . ( b ) time - traces for the five constructs described above : first level of screening . dna constructs were unwound by n = 12 turns using the magnetic trap , and their extension monitored in real - time . the charomid 9 - 5 kb displays the same behavior as the charomid 9 - 11 kb plasmid , indicating that the repeat region is not responsible for the observed fluctuations . the charo - s3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid , indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of dna , which is specifically lacking in the construct . fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid ( c ) screen results . the above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations . the 33-bp charomid x sequence responsible for the observed fluctuations is a quasipalindrome , which extrudes to form a complex and unstable cruciform . a putative minimum energy fold derived from mfold for high - salt conditions ( 0.5 m nacl ) is presented ; in the lower salt conditions initially used here , the 3-bp separating the two apical loops are unlikely to be stable at room temperature , implying that the apical loop is in fact the sum of the two smaller loops . this is consistent with the extensive amount of unwinding estimated to be present in the transition state . the mutation made to increase the loop size involved converting the g circled in red into a t , disrupting a crucial base pair for high - salt conditions but not low - salt conditions ( table 1 ) . single - molecule screen of charomid 9 - 5 kb is used to identify the charomid x quasipalindrome . charomid 9 - 5 kb is derived from charomid 9 - 11 kb by removing the 6 kb region of the plasmid , corresponding to three consecutive repeats of a 2-kb fragment ( 35 ) . then , five constructs are derived from charomid 9 - 5 kb using the pcr reaction , each construct lacking a different 1 kb segment of the plasmid . ( b ) time - traces for the five constructs described above : first level of screening . dna constructs were unwound by n = 12 turns using the magnetic trap , and their extension monitored in real - time . the charomid 9 - 5 kb displays the same behavior as the charomid 9 - 11 kb plasmid , indicating that the repeat region is not responsible for the observed fluctuations . the charo - s3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid , indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of dna , which is specifically lacking in the construct . fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid ( c ) screen results . the above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations . the 33-bp charomid x sequence responsible for the observed fluctuations is a quasipalindrome , which extrudes to form a complex and unstable cruciform . a putative minimum energy fold derived from mfold for high - salt conditions ( 0.5 m nacl ) is presented ; in the lower salt conditions initially used here , the 3-bp separating the two apical loops are unlikely to be stable at room temperature , implying that the apical loop is in fact the sum of the two smaller loops . this is consistent with the extensive amount of unwinding estimated to be present in the transition state . the mutation made to increase the loop size involved converting the g circled in red into a t , disrupting a crucial base pair for high - salt conditions but not low - salt conditions ( table 1 ) . the time elapsed before an extrusion event , twait , and the lifetime of the extruded state , tcruciform , can be directly determined from the time - traces ( figure 1b ) . histograms of twait and tcruciform display single - exponential lifetime distributions ( figure 1d ) , suggesting that at the temporal resolution of the experiment ( 1 s ) a single energy barrier dominates the transition between the b - dna and cruciform states . these measurements were then repeated on dna for different levels of negative supercoiling ( figure 3a ) . the data show that as the dna is progressively unwound , the cruciform state becomes more likely as a result of an increase in the rate of cruciform extrusion , kf = 1/<twait > , and a concomitant decrease in the rate of cruciform rewinding , kr = 1/<tcruciform>. more precisely , kf increases exponentially while kr decreases exponentially with negative supercoiling as per an arrhenius law ( figure 3b ) . at the same time , we find that ncruciform remains essentially constant , indicating that the size of the cruciform does not increase with negative supercoiling ( supplementary figure s3 ) . these results imply that the cruciform is fully formed upon extrusion , and that the hairpin branches can not be extended beyond the inverted repeats . instead , the cruciform becomes progressively more stable with negative supercoiling , consistent with mechanical stabilization of the extruded cruciform by the torque generated by negative supercoiling . figure 3.kinetic analysis of cruciform extrusion and rewinding for charomid 9 - 5 kb dna . ( a ) as negative dna supercoiling increases , progressively less time is spent in the b - dna state and more time is spent in the extruded - cruciform state . ( b ) free energy barrier to cruciform extrusion ( blue ) and rewinding ( red ) as a function of negative supercoiling . the barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 0.015 kbt / turn ( b - dna is destabilized by unwinding ) , while the barrier to rewinding increases linearly by 0.71 0.014 kbt / turn ( cruciform dna is stabilized by unwinding ) . at least 300 events were measured at each rotation point to determine each mean transition rate . kinetic analysis of cruciform extrusion and rewinding for charomid 9 - 5 kb dna . ( a ) as negative dna supercoiling increases , progressively less time is spent in the b - dna state and more time is spent in the extruded - cruciform state . ( b ) free energy barrier to cruciform extrusion ( blue ) and rewinding ( red ) as a function of negative supercoiling . the barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 0.015 kbt / turn ( b - dna is destabilized by unwinding ) , while the barrier to rewinding increases linearly by 0.71 0.014 kbt / turn ( cruciform dna is stabilized by unwinding ) . at least 300 events were measured at each rotation point to determine each mean transition rate . this can be seen by calculating the difference in free energy between the two states , g = kbt ln kf / kr , as a function of supercoiling . in figure 3b , this corresponds to the difference between the blue and red curves : thus g grows linearly with negative dna supercoiling at a rate of about 1 kbt / turn . we conclude that in this regime torque increases slowly and linearly with negative supercoiling , even after the buckling transition . indeed , g = gcruciform + gsupercoiling and gcruciform is constant . thus , the increase in free energy between the b - dna and cruciform states can only be due to the increase in mechanical work done on the system by torque during the unwinding of ncruciform turns of the double helix : gsupercoiling = 2ncruciform . as ncruciform does not depend on supercoiling , this implies that increases with supercoiling . note that this increase in torque is quite small , on the order of 0.05 kbt for every turn added . although this slow increase in torque after the formation of dna plectonemes has already been observed ( 25 ) , there are currently no theoretical models of dna statistical mechanics to explain this weak increase in torque occurring after the formation of dna writhe ( 24 ) . as can be done with force spectroscopy ( 37,38 ) , the supercoiling dependence of kf and kr can be quantitatively analyzed to determine the ratio nr / nf , where nf is the amount of dna unwinding separating the b - dna state from the transition state , nr is the amount of dna unwinding required to complete extrusion , i.e. to go from the transition state to the cruciform state and nf + nr = ncruciform is well - determined experimentally ( for details , see supplementary data and figure 5e ) . we thus find for the charomid x quasipalindrome nr / nf = 2.3 0.1 . using nr + nf = ncruciform = 34 1 bp , we interpret this as meaning that in the transition state 10 1 bp of dna are unwound . note that the charomid x cruciform is predicted to have a large , weakly structured and presumably degenerate 12 base loop region at its apex ( figure 2c ) . [ we further note that this result is independent of the extending force used during the measurement , as in the range of forces tested increasing the force leaves the relative position of the transition state essentially unchanged ( supplementary figure s4 ) . ] based on the simple two - state model the data permit for , these experiments suggest that the transition - state intermediate to cruciform extrusion corresponds to a state where the dna is unwound only in the apical loop region of the cruciform . to test this , we determined the effect of a single base mutation designed to disrupt the stem and increase the size of the loop ( see figure 2c ) . however in the salt conditions previously used ( 10 mm potassium phosphate buffer , ph 8.0 ) , we do not find any effect for this mutation ( table 1 ) , suggesting that under these salt conditions the loop is already large enough to encompass the targeted base . upon addition of 50 mm nacl , however , analysis of the mutant becomes possible ( table 1 ) . first , we find that the wild - type cruciform becomes slightly more structured . indeed here the ratio nr / nf increases , implying proportionally less unwinding in the transition state and consistent with an increase in stem length at the cost of a decrease in loop length , for a constant sum of stem and loop length . this is expected for ionic conditions that stabilize base pairing . in these higher salt conditions , the mutation now causes a large decrease in the ratio nr / nf , consistent with an increase in loop size and a decrease in stem length resulting from this disruptive mutation . these experiments support the notion that disrupting base pairs involved in closing the loop affects the transition state intermediate . table 1.compilation of transition amplitudes and torque dependency of cruciform extrusion and refolding rates for the inverted repeats studied in this articlednaionic conditionsncruciform ( bp)d(ln kr)/dn ( kbt / turn)d(ln kf)/dn ( kbt / turn)d(ln kr)/d ( ln kf ) = nr / nfdna unwinding in ts ( bp)wt charomid x10 mm phosphate buffer34 1 ( 74)0.71 0.0140.31 0.0152.3 0.110 110 mm phosphate buffer + 50 mm nacl36 1 ( 58)0.30 0.010.10 0.012.9 0.69 1mutated charomid x10 mm phosphate buffer34 1 ( 51)0.91 0.020.43 0.022.1 0.1111 110 mm phosphate buffer + 50 mm nacl36 1 ( 65)0.11 0.020.11 0.021.0 0.2518 3cole1 - 5b11 mm phosphate buffer30 10.39 0.051.68 0.054.3 0.55.8 0.8cole1 - 8b11 mm phosphate buffer32 10.61 0.131.57 0.132.6 0.59.1 1.5for unwinding amplitudes , the number in parenthesis is the number of events used to calculate the average . compilation of transition amplitudes and torque dependency of cruciform extrusion and refolding rates for the inverted repeats studied in this article for unwinding amplitudes , the number in parenthesis is the number of events used to calculate the average . to further test our model , we performed another series of experiments investigating the kinetics of extrusion and rewinding for the canonical 31 bp cole1 perfect inverted repeat , a cruciform characterized by a 5-base apical loop and perfectly paired 13 bp stem ( figure 4a ) ( 1 ) . we selected this sequence based on its perfect pairing and its reported propensity for irreversible extrusion , as it could potentially provide a basis on which to test modifications leading to reversible cruciform extrusion . single - molecule analysis of the wtcole1 inverted repeat subjected to negative supercoiling indeed showed that cruciform extrusion was essentially irreversible , with a large increase in dna extension during cruciform extrusion which never fully reversed . nevertheless , the data indicate that the extruded cruciform can make attempts at refolding ( figure 4b ) . during such attempts , the extension of the system can nearly return to that of the native low - extension state . however , these attempts are unsuccessful and do not correspond to full rewinding of the cruciform , as their lifetime , on the order of seconds , is much shorter than the lifetime of the true b - dna state ( 410 100 s , see supplementary figure s5 ) . in the context of our two - state model describing the relative position of the transition state , these results suggest that the transition state to rewinding lies very close to the native state for this perfect inverted repeat . further experiments ( supplementary figure s6 ) show that rewinding of the cruciform can occur when the dna is returned to n = 9 turns but that this is still a slow process . thus under the conditions in which this perfect inverted repeat extrudes , it is thereafter kinetically trapped and thus not a good thermodynamic system . figure 4.irreversible and reversible cruciform extrusion on the wtcole1 inverted repeat and its derivative mutcole1 - 5b . ( a ) the wtcole1 inverted repeat forms a perfect cruciform ( b ) in an irreversible fashion under conditions of negative supercoiling ( n = 21 ) . immediately after the extrusion event , supercoiling is returned to n = 19 rotations to favor rewinding . despite this no complete rewinding is observed , although unsuccessful attempts at refolding are regularly observed ( down arrows ) . ( c ) the mutcole1 - 5b inverted repeat extrudes into an imperfect cruciform ( d ) in a reversible fashion , allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding . the increased level of noise in the extension of the b - dna state is due to the presence of a + t - rich regions flanking the inverted repeat ( see supplementary figure s7 ) . irreversible and reversible cruciform extrusion on the wtcole1 inverted repeat and its derivative mutcole1 - 5b . ( a ) the wtcole1 inverted repeat forms a perfect cruciform ( b ) in an irreversible fashion under conditions of negative supercoiling ( n = 21 ) . immediately after the extrusion event , supercoiling is returned to n = 19 rotations to favor rewinding . despite this no complete rewinding is observed , although unsuccessful attempts at refolding are regularly observed ( down arrows ) . ( c ) the mutcole1 - 5b inverted repeat extrudes into an imperfect cruciform ( d ) in a reversible fashion , allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding . the increased level of noise in the extension of the b - dna state is due to the presence of a + t - rich regions flanking the inverted repeat ( see supplementary figure s7 ) . so as to perform kinetic and structural analysis of this inverted repeat , we first sought to introduce limited changes to the sequence that would convert the cruciform into a thermodynamically reversible system . we accomplished this by removing three bases from each stem so as to shorten each stem by a full at base pair and generate a single base adenine or thymine bulge on each stem ( figure 4c ) . thanks to these modifications in the stem region , the resulting inverted repeat , mutcole1 - 5b , was now observed to extrude and rewind reversibly ( figure 4d ) . structural analysis of the extrusion transition for this inverted repeat gives < l > = 213 4 nm , corresponding to ncruciform = 30 1 bp ( supplementary figure s7 ) , in close agreement with the expected length of the inverted repeat ( 28 bp ) . from a kinetic standpoint , with this cruciform kr is again significantly more sensitive to changes in supercoiling than kf , and analysis of the rates as above gives nr / nf = 4.2 0.6 ( figure 5c and table 1 ) . we infer that in the transition state the inverted repeat is unwound by 5.8 0.8 bp . in agreement with the simple two - state model for cruciform extrusion , this amount of unwinding in the transition state is just enough to accommodate formation of the five base apical loop of the cruciform . note that , in agreement with previous findings , this family of inverted repeats requires the presence of a + t - rich flanking regions to extrude ( see supplementary figure s8 ) ; transient unwinding in these regions is the cause of the increase in noise level in the b - dna state and presumably allows for attempts at cruciform folding . figure 5.kinetic and structural analysis of cruciform extrusion in mutcole1 inverted repeats bearing ( a c ) a 5 base loop or ( d f ) an 8 base loop . ( a ) the mutcole1 - 5b inverted repeat is seen ( b ) to extrude reversibly as shown previously in figure 4 . ( c ) the free energy barrier to cruciform rewinding ( red ) is more sensitive to torque than the free energy barrier to cruciform extrusion ( blue ) , d ln kf / d ln kr = 4.2 0.6 . ( d ) the mutcole1 - 8b inverted repeat with an 8 base loop is seen ( e ) to extrude with slower kinetics than the 5-base loop . ( f ) the free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion , but less so : d ln kf / d ln kr = 2.6 0.5 . the transition - state intermediate to cruciform extrusion is formed by unwinding b - dna by nf turns , and for complete formation of the cruciform the b - dna must thereafter be unwound by an additional nr turns , with nf + nr = ncruciform . the relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states , d ln kf / d ln kr = nf / nr . kinetic and structural analysis of cruciform extrusion in mutcole1 inverted repeats bearing ( a c ) a 5 base loop or ( d f ) an 8 base loop . ( a ) the mutcole1 - 5b inverted repeat is seen ( b ) to extrude reversibly as shown previously in figure 4 . ( c ) the free energy barrier to cruciform rewinding ( red ) is more sensitive to torque than the free energy barrier to cruciform extrusion ( blue ) , d ln kf / d ln kr = 4.2 0.6 . ( d ) the mutcole1 - 8b inverted repeat with an 8 base loop is seen ( e ) to extrude with slower kinetics than the 5-base loop . ( f ) the free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion , but less so : d ln kf / d ln kr = 2.6 0.5 . the transition - state intermediate to cruciform extrusion is formed by unwinding b - dna by nf turns , and for complete formation of the cruciform the b - dna must thereafter be unwound by an additional nr turns , with nf + nr = ncruciform . the relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states , d ln kf / d ln kr = nf / nr . furthermore , using a second cruciform made reversible by a separate set of destabilizing changes in the stem region , we again find the same position for the transition state intermediate ( supplementary figure s9 ) . thus in further agreement with the simple two - state model , we find that the position of the transition state is unaffected by the position of destabilizing changes in the stem . to see if we could quantitatively predict the structure of the transition state intermediate from the minimal two - state model , we derived the mutcole1 - 8b inverted repeat , for which the apical loop is three bases larger than for the mutcole1 - 5b inverted repeat ( figure 5d f ) . structural analysis of the unwinding transition gives < l > = 233 3 nm ( supplementary figure s7b ) , corresponding to ncruciform = 32 1 bp , again in close agreement with the expected length of the inverted repeat ( 31 bp ) . for this construct , torque spectroscopy yielded nr / nf = 2.6 0.5 ( figure 5f and table 1 ) . we infer that in the transition state , 9.1 1 bases of the inverted repeat are unwound , in agreement with the predicted size of the cruciform s apical loop . we conclude that in the transition state to cruciform extrusion only the apical loop is unwound ; presumably , the stem is then poised to zip up via base pairing as in the holliday junction ( 39,40 ) . although single - molecule approaches to studying dna cruciforms have already been described ( 41,39 ) , these prior studies focused on the behavior of a preexisting cruciform and did not analyze the nucleation process itself . here , we show that it is also possible to observe in real - time the process of cruciform extrusion , as well as the reverse rewinding process . cruciform size can be measured to within base pair accuracy , and kinetics determined to 1 s. the cole1 and charomid x cruciforms presumably begin extrusion by different mechanisms : the cole1 cruciform requires flanking a + t - rich regions to initiate cruciform extrusion , but not the charomid x cruciform ( supplementary figures s8 and s3 , respectively ) . thus , extrusion of the cole1 cruciform likely depends on unwinding fluctuations in the flanking a + t - rich regions , while the a + t - rich content of the charomid x cruciform loop region suggests that unwinding fluctuations required to nucleate the apical loop are localized within the apical loop itself . this is consistent with past studies of cruciform extrusion ( 8) in which the cole1 cruciform was classified as c - type , i.e. requiring unwinding of a large flanking region to initiate extrusion ( 42 ) . we here propose that the charomid x behaves more like the s - type cruciform , as it does not require unwinding of a large flanking region to initiate extrusion . despite the difference in how dna unwinding in the apical loop initiates , we find that a similar transition state rate - limits the extrusion of both charomid x and cole1 cruciforms ( figure 5 g ) . although these cruciforms may depend on dna unwinding fluctuations to initiate extrusion , it appears that in negatively supercoiled dna this is not the rate - limiting step of extrusion . for instance , although the cole1 palindrome requires large - scale unwinding to extrude ( as evidenced by its dependence on a + t - rich flanking regions ) , the rate - limiting step of extrusion is the formation of an intermediate with unwinding only equal to that of the final apical loop . this supports the proposal that after initial unwinding most of the dna reanneals but for the apical loop , and it is the subsequent formation of a four - way junction intermediate that is rate - limiting and which obligatorily precedes zipping of the stem ( 6,43 ) . indeed in the high - energy state of the four - way junction intermediate , bases are unstacked and little or no base pairing takes place in the stem yet . once the bases between the inverted repeats are unwound and the apical loop stabilized by pairing at its base , pairing in the stem can proceed and supercoiling torque drives full extrusion of the cruciform . according to this model , the rate of cruciform extrusion is set principally by the size of the unpaired loop , while the rate of cruciform rewinding is set principally by the size and structure of the stem . for instance , the position of the transition state intermediate close to the native b - dna state helps to explain the apparently irreversible extrusion of perfect stem loop structures with small loops ( 1 ) : these are in fact kinetically trapped . in particular , these results help explain why certain inverted repeats can lead to genetic instability ( 10,11,14 ) , while those that underlie functional rna structures such as microrna precursors or riboswitches do not . genetically unstable inverted repeats are characterized by short loops and long stems ( 10,11 ) , making their extrusion frequent and their rewinding highly unlikely . the high probability of the resulting cruciform facilitates its targeting by excision and repair machineries . on the other hand , the apical loops in , for instance , structured rna precursors are typically ( but not always ) quite large , on the order of > 10 bases . for the dsdna encoding the precursor , this corresponds to a large amount of dna to unwind , making the barrier to cruciform extrusion too high and too far along the reaction coordinate to be crossed via thermal fluctuations . this is important as the long length of the dna inverted repeats encoding mirna precursors ( typically 2025 bp ) is likely to forbid rewinding of an extruded cruciform in the underlying dsdna . in agreement with this , we were unable to detect cruciform extrusion in dna inverted repeats encoding the mirna precursors brf1 - 1 , brf1 - 2 , hsa - mir-379 and hsa - mir-1225b ( data not shown ) . finally , we note that the inverted repeats studied in this work underwent cruciform extrusion at levels of negative supercoiling no more than half of what is expected in vivo . as we find that the rates of cruciform extrusion and rewinding vary exponentially with supercoiling , it is therefore likely that supercoiling in vivo may in certain cases regulate cruciform extrusion . thus , these results may help better understand the genome rules that dictate the way in which ubiquitous inverted repeats in dsdna can evolve to generate a novel function rather than be targeted for repair . cold spring harbor fellows program ; cnrs atip program ; university of paris 7 diderot ; euryi grant from the european science foundation ( to t.r.s . ) ; r.s . acknowledges the support of cold spring harbor laboratory .

during cruciform extrusion , a dna inverted repeat unwinds and forms a four - way junction in which two of the branches consist of hairpin structures obtained by self - pairing of the inverted repeats . here , we use single - molecule dna nanomanipulation to monitor in real - time cruciform extrusion and rewinding . this allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second - scale time resolution . we present data obtained with two different inverted repeats , one perfect and one imperfect , and extend single - molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics . using mutational analysis and a simple two - state model , we find that in the transition state intermediate only the b - dna located between the inverted repeats ( and corresponding to the unpaired apical loop ) is unwound , implying that initial stabilization of the four - way ( or holliday ) junction is rate - limiting . we thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop , while rewinding is kinetically regulated by features of the stem . these results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and b - form states .

their sequences are too long to memorize , their structures are too complex to draw , their motions are too convoluted to animate and their folding processes are too hard to explain . to deal with these fine grain complexities we must often resort to describing proteins in terms of their coarse grain physical or chemical properties . these coarse - grain properties include such features as radius of gyration , molecular weight , isoelectric point , hydrophobicity , secondary structure , contact order ( 1 ) , order parameters ( 2 ) and folding rates ( 1,3 ) . in many cases , these physico - chemical properties can be accurately calculated or predicted directly from the protein sequence or the protein 's 3d structure ( 4,47 ) . some properties , such as radius of gyration , molecular weight and isoelectric point can be easily calculated using simple formulas or tables ( 6 ) , while other properties , such as secondary structure , order parameters and disulfide connectivity are non - trivial to predict or calculate ( 7,2,5 ) . the challenges faced in accurately predicting or calculating non - trivial protein properties has attracted the interest of many protein chemists , structural biologists and computational biologists for a very long time . indeed , protein property prediction is one of the oldest disciplines in bioinformatics , with secondary structure prediction being perhaps the earliest ( 8) and most frequently attempted kind of protein property prediction . since the 1960s many other kinds of protein properties and property prediction methods have emerged , including methods to predict or identify beta turns ( 9 ) , membrane helices ( 10 ) , transmembrane barrel proteins ( 11 ) , signal peptides ( 12 ) , disulfide pairings ( 7 ) edge or central beta strands ( 13 ) , beta hairpins ( 14 ) , coiled - coils ( 15 ) , accessible surface area ( 16 ) and flexibility ( 17)to name just a few . key to the development of all of these property prediction methods has been the creation or compilation of databases that contain the properties of interest that are to be predicted . historically these databases served as the raw material from which to derive statistical or heuristic rules about certain protein or amino acid properties ( 5,18 ) . more recently these databases have served as the testing , training and validation sets for more advanced machine - learning methods ( such as neural nets , hidden markov models and support vector machines ) aimed at improving the accuracy of older protein property prediction methods ( 917 ) . in almost all cases , the accuracy of the prediction method is directly dependent on the size , completeness and accuracy of the testing / training database . in other words , unfortunately , the importance of these databases is somewhat underplayed in the bioinformatics community . with the exception of a small number of database resources such as eva ( 19 ) , tmh - benchmark ( 20 ) , scratch ( 7 ) and spdb ( 21 ) , very few protein property databases are publicly available or routinely updated . in many cases , protein property databases that were painstakingly assembled by a graduate student or post - doctoral fellow to train their particular predictor in other cases , if the database is available , it is so woefully out of date or its format is so obscure that it is often more efficient to regenerate a new database from scratch . still in other cases , the precise origin , method of data generation or the quality of the data is too uncertain to allow the database to be used . even if a high quality , continuously updated protein property database is available , it is often difficult to locate or access such a resource as there is no common repository for these kinds of databases . as a result most labs that wish to develop , refine or improve upon a given protein property prediction method must resort to re - inventing the wheel and generate their own database in their own format . while limited access to high - quality protein property databases is certainly a concern for many data miners and software developers , this limited access also has negative consequence for a large community of users ( i.e. scientists wanting predictions ) . what is not widely appreciated is the fact that protein property databases can also be used as property predictors on their own , especially through the use of homology - based property prediction . this simple method of property prediction is based on the well - known fact that both protein structure and protein dynamic properties are highly conserved between homologous proteins ( 22 ) . in homology - based property prediction the sequence of interest is aligned against a database of sequences with known properties , features or coordinates . the properties for the highest scoring homolog(s ) are then mapped to the query sequence to create a prediction. certainly the success of homology or comparative modeling has clearly shown how coordinate mapping from the pdb can be exploited to accurately predict 3d structures for a large number of query proteins . similar success has been achieved in chemical shift prediction and torsion angle prediction ( 23,24 ) . more recently , the use of sequence alignments against large databases of proteins of known secondary structure has been shown to improve the quality of secondary structure prediction by a substantial margin ( 22 ) . obviously these kinds of homology - based predictions are limited to query proteins that exhibit some degree of sequence identity to proteins in the database(s ) . however , with the size of these protein property databases getting so large , the probability of finding a match is often > 60% ( 22 ) . given the importance of protein property predictions and the critical need for high - quality protein property databases , we decided to create an open - access , continuously updated , comprehensive protein property database called the protein property prediction and testing database ( ppt - db ) . the intent of this database is to facilitate software development in protein property prediction and to facilitate property prediction by homology . the ppt - db currently contains nearly 30 carefully curated databases , each of which contains non - trivial protein property information . these properties include both structural and dynamic features that have been measured , derived or tabulated from a variety of sources ( table 1 ) . first , it is intended to serve as a centralized , up - to - date , freely downloadable and easily queried repository of predictable or derived protein property data . in this role , ppt - db can serve as a one - stop , standardized ( i.e. uniformly formatted and carefully validated ) repository for software developers to obtain the required training , testing and validation data needed for almost any kind of protein property prediction program they may wish to create . the second role that ppt - db can play is as a tool for homology - based protein property prediction . users may query ppt - db with a sequence of interest and have a specific property predicted using a sequence similarity search against ppt - db 's collection of proteins with known properties . in many cases these homology - derived property predictions are substantially better than those that would be obtained using conventional or ab initio predictors . a more detailed description of the ppt - db along with all of its contents and capabilities follows . table 1.summary of the content and description of different ppt - db databasesdatabasedescriptiondatabasedescription2 structure ( cytoplasmic ) 15 002 sequences3-state 2 structure assignments obtained by vadar ( 25 ) for non - membrane proteinssignal peptide ( eukaryotic , gram+ , gram ) 23 067 sequences2-state signal peptide assignments obtained from swissprot comment fields grouped via organism typeeva 2 structure test set 7117 sequences3-state 2 structure assignments via vadar ( 25 ) for eva 's sequence - unique proteins ( 19)accessible surface area ( integerized ) 14 871 sequencesresidue - specific accessible surface area obtained via vadar ( 25 ) and scaled to values between 0 and 92 structure ( membrane helix ) 254 sequences2-state 2 structure assignments obtained via vadar ( 25 ) for helical membrane proteinsaccessible surface area ( % ) 14871 sequencesresidue - specific accessible surface area obtained via vadar ( 25 ) and converted to percentage valuestmh benchmark test set 2247 sequences2-state 2 structure assignments for transmembrane helices from tmh benchmark ( 20)b - factor ( integerized ) 10 332 sequencesresidue - specific b - factors obtained directly from pdb files of x - ray structures and scaled to values from 0 to 92 structure ( membrane barrel ) 41 sequences2-state 2 structure assignments obtained by vadar ( 25 ) for trans - membrane barrel proteinsb - factor 10 332 sequencesresidue - specific b - factors obtained directly from pdb ( 26 ) files of non - membrane x - ray structures% 2 structure ( cytoplasmic ) 15 002 sequences3-state 2 structure content obtained by vadar ( 25 ) for non - membrane proteinsrmsf ( integerized ) 2134 sequencesscaled ( 09 ) residue - specific rmsf values determined from nmr structures via superpose ( 29)beta turns 14 571 sequences5-state beta - turn assignments obtained via vadar ( 25 ) for non - membrane proteinsrmsf 2134 sequencesresidue - specific root mean square fluctuation ( rmsf ) determined from nmr structures via superpose ( 29)coiled - coil 824 sequences2-state , positional assignments for coiled coil regions from the paircoil2 training set ( 15)order parameter ( integerized ) 9800 sequencesscaled ( 09 ) residue - specific order parameter ( model free ) determined using contact model method ( 2)edge / central beta strands 13 255 sequences2-state beta strand type assignments obtained by vadar ( 25 ) and pattern recognition programsorder parameter 9800 sequencesresidue - specific order parameter ( model free ) determined using contact model method ( 2)beta hairpins 8600 sequences2-state beta hairpin assignments obtained by vadar ( 25 ) and pattern recognition programscontact order 14 769 sequencescontact order calculated using method of plaxco et al . ( 1 ) directly from pdb coordinatesdisulfide bonds 2785 sequencesdisulfide bond pairings obtained by vadar ( 25 ) and pdb comment fieldsfolding rate 83 sequencesexperimentally measured folding rates ( ln[kf ] ) obtained from multiple sources ( 1,3)spdb ( eukaryotic , gram+ , gram ) 2590 sequencesexperimentally verified 2-state signal peptide assignments obtained from the spdb ( 21 ) grouped via organism type3d folding decoys 52 sequencespdb coordinates for misfolded or improperly folded proteins generated via different 3d prediction tools summary of the content and description of different ppt - db databases as seen in table 1 , ppt - db consists of 29 smaller sequence databases , each of which contains between 41 and 23 067 sequences . altogether the most recent version of the ppt database consists of 234 787 sequences ( of which 40 254 are unique ) , occupying a total of 245 mb of disk space . so far as we are aware , ppt - db is the largest and most complete collection of protein property data that has ever been assembled . the protein properties covered in the current release of ppt - db fall into two broad categories : ( i ) structural properties and ( ii ) dynamic properties . the structural properties consist of features that define some aspect of the secondary or tertiary structure of a protein such as secondary structure content , helix location , beta strand location , random coil location , beta turn location , coil coil location , disulfide bond patterns , signal peptides , contact order , etc . the dynamic properties consist of features that define some aspect of the motion , flexibility or rate processes for a protein , such as root - mean square fluctuation ( rmsf ) , b - factors , folding rate ( ln[k ] ) or order parameters . some of these properties are global , meaning that one or two numbers describe the property for the entire protein ( such as folding rate or contact order ) . other properties are local or residue - specific , meaning that individual residues are assigned a value or property ( such as secondary structure , b - factors or disulfide pairings ) . every protein property database in ppt - db , with the exception of the folding decoy database , consists of sequences in a fasta - like format . the folding decoy database is unique among ppt - db 's property databases as it actually consists of coordinate data rather than pure sequence data . among the 28 pure sequence databases in the ppt - db , each sequence is annotated with the name of the sequence , the swissprot accession number ( if it exists ) and a pdb accession number ( if it exists ) . global protein properties ( folding rate or contact order ) are always displayed on the fasta header line , while local properties are displayed on a line immediately below the sequence , under the residue(s ) with that property ( see figure 1 for examples ) . for certain properties ( b - factors , rmsf , order parameters ) the horizontal ( fasta ) format requires that the multi - digit numbers be scaled to a single digit integer value between 0 and 9 so that they can be displayed directly underneath the single - character sequence data . the vertical format displays both the sequence and the actual ( i.e. multi - digit , real ) numbers for the associated property in vertical columns . additional details about the format , content , scaling and labeling conventions in each database are given in the database details link located at the top of each database query page . ( a ) an example of a typical sub - database query page , with an example of the content found the database details. ( b ) and ( c ) illustrate the two kinds of blast search output , with ( b ) showing the standard horizontal or fasta format and ( c ) showing the vertical or column format for displaying residue - specific values / properties that are multi - digit numbers . ( a ) an example of a typical sub - database query page , with an example of the content found the database details. ( b ) and ( c ) illustrate the two kinds of blast search output , with ( b ) showing the standard horizontal or fasta format and ( c ) showing the vertical or column format for displaying residue - specific values / properties that are multi - digit numbers . as mentioned earlier , the ppt - db is a dual - purpose resource , serving as either a fully downloadable database for software developers and data miners or as a general property prediction service for protein chemists and structural biologists . access to both components of ppt - db is through a relatively simple web interface ( figure 1 ) . as seen in this figure , the left margin of the ppt - db home page consists of a hyperlinked list of all of its component databases . clicking on any one of these sub - database hyperlinks generates a database - specific query page ( figure 1a ) . at the top of each database query page is the name of the protein property database followed by a database details button . clicking on this button provides detailed information on the database format and how the database was constructed ( figure 1a ) . below this ( with the exception of the folding decoy database ) is a text search box . users may search their selected database using the protein name ( or part thereof ) , the swissprot i d ( or part thereof ) , pdb i d ( or part thereof ) or any other text within the sequence header . this text search will rapidly generate a 3-column hyperlinked table showing the name of the protein , the swissprot i d and the pdb i d . clicking on the protein name will display that proteins sequence along with its ppt - db property annotations . clicking on the swissprot i d will open the corresponding swissprot entry for that protein while clicking on the pdb i d will open the corresponding pdb web page for that protein . the ppt - db is also searchable via sequence queries using a local version of blast . however , the main purpose of ppt - db 's blast search is not necessarily to locate a given protein in the database , but rather to predict the properties of a query ( i.e. an unknown or uncharacterized ) protein through a technique known as homology - based property prediction or homology - based property mapping ( 2224 ) . in other words , the blast sequence search is part of ppt - db 's general protein property prediction service . details concerning the performance of these property predictions are described in the database details link at the top of each database query page , as well as in the section entitled ppt - db 's blast search accepts both fasta and raw sequence data as input and uses a default e ( expect ) value of 10 as a cutoff for selecting sequence matches . each blast query in the ppt - db has an example button which uploads a sample sequence , allowing new users to test ppt - db 's search utilities and investigate the output format for each kind of database query . figure 1b and c illustrate the type of output generated by ppt - db 's blast search , showing examples of both the horizontal and vertical output formats . the ppt - db also supports blast queries against all of ppt - db 's protein properties through the all properties database located at the top of the database list . predict all ppt - db properties ( 24 of 28 ) that can be displayed in a fasta ( horizontal ) format . at the bottom of each database query page this is a hyperlinked table providing information about the version number , the release date , the size and number of sequences in each version of a given ppt database . pressing on the click to download link allows users to retrieve or install a local copy of each ppt sub - database on their own computer . the structure and annotation details for each version of every ppt - db sub - database are contained in a header at the top of each database file . all of ppt - db 's databases are stored as simple , uncompressed text files . the download section of the ppt - db is primarily intended to support the activities of software developers interested in training , testing and validating their property prediction software or data miners interested in extracting statistics , trends or heuristics about certain protein properties . as described in the following section , every effort is made to ensure that these downloadable databases are as current , complete and correct as possible . table 1 briefly describes the sources , protocols or programs used to generate most of the databases in ppt - db . additional details concerning the preparation and maintenance of each database are provided in the database details link located in ppt - db 's menu bar or via the database details button located within each ppt - db sub - database . as seen from table 1 , many of the databases created for ppt - db were developed internally using a program called vadar ( 25 ) to help derive or extract the data from existing pdb files . other ppt databases were assembled from information contained explicitly in swissprot ( 26 ) or the pdb ( 27 ) . in these cases , database - specific programs were written to extract data from swissprot headers or pdb comment fields and to map this information on to the sequence extracted from the corresponding database . essentially , all databases that were primarily derived from the pdb ( or subsequently by vadar ) were processed by the pdb culling / filtering service called pisces ( 28 ) . structures were selected using a 95% identity sequence - redundancy cutoff and a requirement for better than 3 resolution ( for x - ray structures ) . these files were further filtered to remove electron microscopy models and protein chains having fewer than 30 residues . while the vast majority of the databases in ppt - db were derived using automatic methods , some databases ( such as the transmembrane helix , the transmembrane beta barrel database , the folding rate and folding decoy database ) were assembled manually . other databases , such as the beta hairpin and the beta edge / central strand were created using specialized programs that re - interpreted standard vadar output . still other databases such as eva ( 19 ) , spdb ( 21 ) , the coiled coil / paircoil2 database ( 15 ) and tmh - benchmark ( 20 ) were obtained from external sources but were re - formatted and manually edited or upgraded to make them compatible with the ppt - db annotation standards . each database and each update to a database is numbered and dated allowing a well - defined audit trail to be assembled . this is intended to allow external software developers and external data miners the opportunity to share and compare testing / training data . with the exception of the signal peptide databases , almost all sequences in the ppt - db were derived from the original pdb sequence file . as a consequence , the sequence for the corresponding swissprot entry may sometimes differ from the sequence listed in either the ppt - db or the pdb . all databases , with the exception of the folding decoys database and the folding rate database , have automated or semi - automated scripts to facilitate updating . depending on their size and ease of curation , ppt - db databases are updated as frequently as once per month ( i.e. secondary structure databases ) or as infrequently as once per year ( i.e. the folding rate database ) . in preparing and updating the ppt - db , every effort has been made to ensure that each database is as complete , correct and current as possible . certainly many of the programs used in the data generation process , such as vadar ( 25 ) and superpose ( 29 ) , have had more than a decade of testing and are considered very robust . ppt - db sanity checker has been written to ensure that impossible numeric values or disallowed characters are flagged in any existing database and any subsequent updates . these problem entries are then manually assessed and manually corrected as required . as a further quality control measure , spot checks are routinely performed on many entries by senior members of the curation group , including two phd - level biochemists . one of the most useful and important applications of ppt - db lies in its ability to help predict protein properties through homology - based property mapping . just as sequence searches through genbank and swissprot allow evolutionary relationships or functional annotations to be made for newly sequenced proteins , so too it is possible to use sequence searches through ppt - db to accurately predict both structural and dynamic properties of proteins . previous studies have shown that homology - based property prediction can significantly outperform the best ab initio ( neural net , svm or hmm ) prediction methods if the query is sufficiently similar to a protein in the database ( 2224 ) . these homology - based predictions are also very fast ( < 1 s ) compared to most other advanced property prediction methods ( most of which take minutes ) . however , one obvious limitation of homology - based property prediction is that it only works if some level of sequence homology exists . surprisingly , this requirement is not as onerous or as infrequent as one might think . to evaluate the performance of each of the ppt - db 's property predictors specifically , we randomly selected 10 sets of 100 proteins ( or fewer if the database had < 1000 sequences ) from each ppt database and used these as queries for the corresponding ppt - db blast search using an expect value cutoff of 10 . after the exact match was excluded , the second highest scoring hit ( if such a hit was found ) was used to predict the property of the query protein . the prediction was then scored using standard q2 or q3 methods ( i.e. % correct ) for categorical predictions , such as secondary structure , or correlation coefficients for numeric predictions , such as accessible surface area . for global properties , such as folding rate , contact order or secondary structure content , the prediction is only accepted if the sequence length of the matching protein is 100 20% of the query protein 's length . results for each of these 10 prediction sets were tabulated , both in terms of performance ( average and standard deviation ) and overall coverage . results for individual prediction sets were also plotted using scatter plots ( performance versus sequence identity ) along with the proportion of queries that exhibited significant hits ( i.e. the coverage ) . all of these scatter plots and performance statistics are available by clicking the database details button for each ppt sub - database . this is a fairly typical result with the performance generally dropping as the sequence identity drops below 3540% . as noted in this figure , ppt - db achieves an average q3 of 89.1% . this compares quite favorably to q3 's of 75% reported for most conventional secondary structure prediction methods ( 5 , 22 ) for instance , ppt - db 's accessible surface area ( asa ) prediction achieves a correlation coefficient of 84.5% , which is between 8 and 15% better than the best methods for asa ( 3-state only ) prediction ( 16 ) . likewise ppt - db obtains a q2 for beta hairpin prediction of 93.0% , while the best ab initio method attains a q2 of 77% ( 14 ) . ppt - db also performs quite well in identifying edge / central beta strands with a predictive accuracy of 90.1% compared to only 55% for the best ab initio method ( 13 ) . space limitations prevent a detailed comparison between all of ppt - db 's property predictions and those reported in the literature . however , interested readers can view the performance statistics by clicking the database details button for each ppt sub - database . as seen from the tables and scatter plots in these database details pages , predictions derived from ppt - db 's similarity searches are typically 8595% correct ( for categorical or global property predictions ) or exhibit correlations of > 0.80 ( for numeric predictions ) . this performance is typically 1020% better than what is obtained from the best ab initio predictions . our data also suggests that reliable ppt - db predictions are possible for 75% of all query sequences , as long as the database contains > 10 000 sequences . certainly as ppt - db 's databases increase in size , the property prediction performance and level of coverage would be expected to increase as well . figure 2.(a ) a scatter plot showing of the predictive performance ( q3 versus% sequence identity ) for secondary structure prediction for 1000 random query sequences that were submitted to ppt - db 's secondary structure database . ( b ) a moving average of the same data shown in a ( using 5% sequence identity intervals ) . using 10-fold cross - validation ( 10 sets of 100 random query proteins ) , the average coverage was 77.1 9.3% and the average q3 for all secondary structure predictions was 89.1 1.3% . ( a ) a scatter plot showing of the predictive performance ( q3 versus% sequence identity ) for secondary structure prediction for 1000 random query sequences that were submitted to ppt - db 's secondary structure database . ( b ) a moving average of the same data shown in a ( using 5% sequence identity intervals ) . using 10-fold cross - validation ( 10 sets of 100 random query proteins ) , the average coverage was 77.1 9.3% and the average q3 for all secondary structure predictions was 89.1 1.3% . in summary , the ppt - db is a comprehensive , open - access , continuously updated , protein property database . it was developed to fill a database void in the field of protein property prediction , which currently lacks both a uniformly formatted and a centralized repository of known or predicted protein properties . the ppt - db was also designed to appeal to two very different audiences : ( i ) programmers and ( ii ) biologists . software developers should find it helpful in developing , testing , comparing and improving their own protein property prediction programs while structural biologists and protein chemists should find it useful as a fast and accurate tool to help predict a wide range of important protein properties . while the protein properties covered by the ppt - db are comprehensive , they certainly are not complete . over the coming year site modification databases ( glycosylation , sulfation , phosphorylation sites ) , protein solubility databases , thermostability databases , subcellular location databases and amyloid propensity databases . likewise we are certainly open to suggestions for new kinds of databases or new database formats . submissions from external sources of new protein property databases to the ppt - db ( if appropriately formatted , described and validated ) are welcomed . overall it is hoped that the ppt - db will help improve the quality and reliability of protein property predictions as well as the frequency with which these kinds of predictions are made or reported in the literature .

the protein property prediction and testing database ( ppt - db ) is a database housing nearly 30 carefully curated databases , each of which contains commonly predicted protein property information . these properties include both structural ( i.e. secondary structure , contact order , disulfide pairing ) and dynamic ( i.e. order parameters , b - factors , folding rates ) features that have been measured , derived or tabulated from a variety of sources . ppt - db is designed to serve two purposes . first it is intended to serve as a centralized , up - to - date , freely downloadable and easily queried repository of predictable or derived protein property data . in this role , ppt - db can serve as a one - stop , fully standardized repository for developers to obtain the required training , testing and validation data needed for almost any kind of protein property prediction program they may wish to create . the second role that ppt - db can play is as a tool for homology - based protein property prediction . users may query ppt - db with a sequence of interest and have a specific property predicted using a sequence similarity search against ppt - db 's extensive collection of proteins with known properties . ppt - db exploits the well - known fact that protein structure and dynamic properties are highly conserved between homologous proteins . predictions derived from ppt - db 's similarity searches are typically 8595% correct ( for categorical predictions , such as secondary structure ) or exhibit correlations of > 0.80 ( for numeric predictions , such as accessible surface area ) . this performance is 1020% better than what is typically obtained from standard ab initio predictions . ppt - db , its prediction utilities and all of its contents are available at http://www.pptdb.ca

a variety of stress factors ( i.e. , exposure to microorganisms , hyperthermia , heavy metals , prolonged immobilization , swimming stress , and noise ) are known to inhibit male reproductive functions . noise is one of the major problem today that enhances the apoptosis of the testicular germ cells . this environmental stress may cause increase in the number of free radicals ( i.e. , hydrogen peroxide [ h2o2 ] and reactive oxygen species [ ros ] ) that can cause apoptosis in the spermatogenic cell lineage to decrease the rate of normal sperms . many of these may lack specificity , have low efficiency , or have potentially severe side effects on the treated men . it is generally believed that the antioxidants prevent infertility in men via scavenging of free radicals . honey is full of enzymatic and non - enzymatic antioxidants such as catalase , ascorbic acid , flavonoids , and alkaloids . it acts against oxidative stress and prevents oxygen contact with non - saturated fatty acids and also lipoprotein oxidation . in addition , honey is rich in fructose , glucose , minerals , magnesium , potassium , calcium , sodium chloride , sulfur , ferrous , phosphate , and vitamins c , b6 , b5 , b3 , b2 , and b1 . the efficacy of honey on the fertility of males of different species was also tested in some studies . generally , it was suggested that honey can affect the spermatogenesis process and sperm fertility in males . vitamin e is another antioxidant that presents in cell membranes , it neutralizes h2o2 and prevents cell membrane damage . indeed , -tocopherol is the most biologically active form of vitamin e and is the most abundant form in the body . although the absorption processes of all forms of vitamin e are same , the alpha form is found more in the organs and blood . food supplemental prescriptions that contain vitamin e may contribute to the functional improvement of spermatozoa . also , vitamin e decreases lipid peroxidation produced in oxidative stress and enhances the motility and fertilization power of sperms . to our knowledge , there has been no research regarding the effects of both honey and vitamin e on the testes of rat exposed to noise stress . therefore , this study suggests that treatment by antioxidants like honey and vitamin e may compensate the oxidative damages as a result of noise stress on the germinative and somatic cells of testis parenchyma . honey was obtained from a beekeeping center in uremia , iran and prepared as a 5% solution . mature wistar male rats ( n = 24 , 200 15 g ) were obtained from the laboratory animal reproduction and breeding center , ahvaz , iran . the cages of groups 1 - 3 were placed in a woody and acoustic room ( dimensions of 3 4 3 m ) and then , routinely , exposed to 90 - 130 db voice with 300 - 350 hz frequency of noise emitted by a white noise generator from 7:00 pm to 7:00 am for 50 days . it is worth mentioning that the timer was set in a way that the sound - generating ( speech ) device was turned on for 1 h and turned off for 15 - 60 min and then turned on again . group 1 was exposed to voice along with feeding of 5% honey solution by gavage method . group 2 was exposed to voice waves along with receiving of vitamin e ( 75 mg / ml ) by gavage method and group 3 was exposed to the voice without any complementary material . after 50 days that equals with the duration of one spermatogenesis cycle of rat , testes of rats of all groups were removed and then testicular cells of them were cultured in dulbecco 's modified eagle medium ( dmem ) medium with 10% fetal bovine serum ( fbs ) and penicillin / streptomycin . for this purpose , briefly after separating the capsule under stereo microscope , seminiferous tubules were digested in two steps . first , seminiferous tubules were placed in tubes contains 1 mg / ml collagenase type iv and 200 - 700 g / ml dnasei for 15 min in 37c incubator with gently pipetting action . next , the tubes were centrifuged ( 100g for 5 min ) , the supernatant was discarded , and the cells were resuspend in 1 ml trypsin - ethylenediamminetetraacetate ( edta ; sigma ) and 200 g / ml dnasei for 5 min in 37c incubator . the cells were washed in pbs solution and incubated in fixing buffer ( 1% paraformaldehyde solution ) . next , the cells were centrifuged ( 300g for 5 min ) , washed twice in pbs , resuspended in solution containing dna - labeling , and then incubated ( 37c ) for 1 hour . subsequently , the supernatant was discarded and the cells pre - coated with an antibody - anti brdu solution ( at room temperature for 5 min ) . the cells were then incubated in propidium iodide / rnse staining buffer according to the manufacturer 's instructions . finally , after removing of propidium iodide / rnse staining , the cells were washed again with pbs twice , and assayed for apoptosis by flow cytometry and tanell kit ( invitrogen , ca , usa ) according to the manufacturer 's directions . the significance of differences among different groups was assessed by one - way analysis of variance ( anova ) . honey was obtained from a beekeeping center in uremia , iran and prepared as a 5% solution . mature wistar male rats ( n = 24 , 200 15 g ) were obtained from the laboratory animal reproduction and breeding center , ahvaz , iran . the cages of groups 1 - 3 were placed in a woody and acoustic room ( dimensions of 3 4 3 m ) and then , routinely , exposed to 90 - 130 db voice with 300 - 350 hz frequency of noise emitted by a white noise generator from 7:00 pm to 7:00 am for 50 days . it is worth mentioning that the timer was set in a way that the sound - generating ( speech ) device was turned on for 1 h and turned off for 15 - 60 min and then turned on again . group 1 was exposed to voice along with feeding of 5% honey solution by gavage method . group 2 was exposed to voice waves along with receiving of vitamin e ( 75 mg / ml ) by gavage method and group 3 was exposed to the voice without any complementary material . after 50 days that equals with the duration of one spermatogenesis cycle of rat , testes of rats of all groups were removed and then testicular cells of them were cultured in dulbecco 's modified eagle medium ( dmem ) medium with 10% fetal bovine serum ( fbs ) and penicillin / streptomycin . for this purpose , briefly after separating the capsule under stereo microscope , seminiferous tubules were digested in two steps . first , seminiferous tubules were placed in tubes contains 1 mg / ml collagenase type iv and 200 - 700 g / ml dnasei for 15 min in 37c incubator with gently pipetting action . next , the tubes were centrifuged ( 100g for 5 min ) , the supernatant was discarded , and the cells were resuspend in 1 ml trypsin - ethylenediamminetetraacetate ( edta ; sigma ) and 200 g / ml dnasei for 5 min in 37c incubator . the cells were washed in pbs solution and incubated in fixing buffer ( 1% paraformaldehyde solution ) . next , the cells were centrifuged ( 300g for 5 min ) , washed twice in pbs , resuspended in solution containing dna - labeling , and then incubated ( 37c ) for 1 hour . subsequently , the supernatant was discarded and the cells pre - coated with an antibody - anti brdu solution ( at room temperature for 5 min ) . the cells were then incubated in propidium iodide / rnse staining buffer according to the manufacturer 's instructions . finally , after removing of propidium iodide / rnse staining , the cells were washed again with pbs twice , and assayed for apoptosis by flow cytometry and tanell kit ( invitrogen , ca , usa ) according to the manufacturer 's directions . the significance of differences among different groups was assessed by one - way analysis of variance ( anova ) . in the control group , the mean percentage of apoptotic , necrotic , and live cells were 0.9 0.1 , 0.24 0.01 , and 0.98.41 0.06 , respectively . the level percentage of apoptotic and necrotic cells in the noise group ( 89.31 0.5 and 9.88 0.3 , respectively ) was increased as compared to the control group ( p = 0.003 and p = 0.001 , respectively ) . also , the mean percentage of viability of cells was decreased when compared with that in the control group ( p = 0.003 ) . however , in the case of group pre - treated with honey , the mean percentage of apoptotic and necrotic cells ( 1.89 0.7 and 0.26 0.01 , respectively ) decreased when compared with that in the noise group ( p = 0.002 and p = 0.002 , respectively ) . the level of apoptotic and necrotic cells in groups pre - treated with vitamin e ( 3.42 0.6 and 0.43 0.08 , respectively ) was decreased when compared with that in the noise group ( p = 0.01 and p = 0.01 , respectively ) . it is noteworthy that honey increased the live cells percentage more than did vitamin e ( 97.1% 0.03 vs. 95.56 0.07 , respectively , p < 0.05 ) , but the necrotic cells level was similar between both honey and vitamin e treated groups ( p < 0.05 ) [ figure 1 ] . cells were analyzed for green fluorescence ( fitc ) and for red fluorescence ( pi ) by flow cytometry . percentages of viable , apoptotic and necrotic testicular parenchyma cells were determined by the dot plot of fl1 ( in the x axis ) to fl3 ( in the y axis ) . lower left indicates viability percent and lower right and upper right indicate percentage of apoptosis and necrosis respectively . the comparisons between cell cycle / apoptotic responses of honey , vitamin , noise stress and control groups are showed . in the present study , it was suggested that the noise stress may have negative influences on germinative and somatic cells of testes parenchyma by increasing apoptotic and necrotic cells . considering the current results , some studies confirmed that , in generally , stress can injure testicular cells . yazawa et al . , reported the effect of immobilization stress on the apoptotic rate of the germ cells as well . in normal situation , apoptosis process occurs permanently for maintaining the tissue homeostasis during spermatogenesis . however , high occurrence of apoptosis can cause negative effects in male genital system . apoptotic activity is dependent on the expression of some apoptosis - related genes and proteins ( i.e. , caspase , apaf-1 , nf - kb , p53 , and death receptors ) and also some anti - apoptosis - related genes and proteins ( i.e. , bcl-2 ) . furthermore , some studies have shown that there is a relationship between apoptosis - related proteins and genes and male infertility . in addition , some studies determined whether there is a relationship between steroid hormones or testicular cells and the expression patterns of apoptosis - related proteins and genes . the results of abovementioned studies showed that reduction or inhibition of gonadotropin as well as steroid hormones secretion can lead to apoptosis of testicular cells . therefore , it seems that every factor that can cause , reduce , or even inhibit gonadotropin and sex hormones secretion , normally can lead to induction of apoptosis in germinative and somatic cells of testes parenchyma . therefore , noise stress may disrupt steroid hormones concentration and neuroendocrine gonadal axis in turn is result in increased rate of cellular damage by expressing , as mentioned for testicular tissue , apoptosis - related proteins and genes in testicular cells . in accordance with the above suggestion , chandralekha et al . , reported that the testosterone serum level in rats under noise stress ( 100 db ) was reduced . also , more structural changes in the testis tissue was observed following the noise stress . in addition , it was reported that apoptosis ratio is negatively correlated with normal morphology and motility of sperm and conversely positively correlated with sperm tail defects . the results in this study also show that the necrosis in cells increased following noise stress . consequently , it seems that noise stress can directly lead to induce necrosis in cells population and destruct germ cells of the testicular tissue . alternatively , this study reported that honey and vitamin e decreased apoptosis and necrosis in cells by noise stress and thereby increased cell viability . in fact , focusing on the above suggestions , the only thing that can be concluded from this finding is that vitamin e and especially honey , being rich in enzymatic and nonenzymatic antioxidants , work by neutralizing the expression patterns of apoptosis - related proteins and genes ( may be due to destructive effects of noise stress ) or by regulation of anti - apoptotic patterns . abdul - ghani et al . , claimed that honey enhances the spermatogenesis process without any disruption in sexual hormone concentrations . however , asiyah et al . , reported that honey has no more positive influence on the sex hormones concentration in male . in general , each factor that affects the function of interstitial space cells ( leydig and myoid cells ) as well as epithelium germinal cells ( sertoli and germ cells ) , may consequently lead to suffer a harmful effect on the neuroendocrine gonadal axis and , in turn , on the spermatogenic cell lineage . the above information suggests that honey and vitamin e , because of their effect on both neuroendocrine gonadal axis and testicular cells , may create conditions that modifies or decreases the process of apoptosis and necrosis in the cells . the second assumption is that honey and vitamin e , because of enhanced steroid hormones level to sertoli cells , can have positive effect on the nutrition of germ cells . mahanem et al . , as well as syazana reported that if appropriate dose of honey is applied to rats , the spermatogenic cells lineage and sperm rate increases . therefore , considering the above results and the results of this study , it is suggested that honey and vitamin e can be useful for enhancing the longevity of cells that suffer from deleterious factors such as noise pollution . the findings of this study may specify a novel natural therapeutic approach based on modulation of the apoptotic process induced by pathogenesis stress , i.e. , noise and enhancing spermatogenesis through honey and vitamin e as possible future antioxidant in fertile men .

aims : a variety of stress factors are known to inhibit male reproductive functions . so this study was conducted in order to investigate the effects of honey and vitamin e on the germinative and somatic cells of testes of rats exposed to noise stress.materials and methods : mature male wistar rats ( n = 24 ) were randomly grouped as follows : group 1 ( honey + noise stress ) , 2 ( vitamin e + noise stress ) , 3 ( noise stress , ) and 4 as the control group . in groups 1 , 2 , and 3 , rats were exposed to noise stress . in groups 1 and 2 , rats also were given honey and vitamin e , respectively , orally for 50 days . after that , the germinative and somatic cells of testes parenchyma were isolated by digesting the whole testes by a standard method . next , viability , apoptosis , and necrosis of the cells were evaluated by tunel kit and flow cytometry.results:the rates of apoptosis and necrosis of the testicular cells were increased ( p = 0.003 and p = 0.001 , respectively ) , but viability of these cells decreased in testes of rats exposed to noise stress ( p = 0.003 ) . however , administration of honey and vitamin e were significantly helpful in keeping the cells of testis parenchyma alive , which suffers from noise pollution ( p < 0.05 and p < 0.05 , respectively).conclusions : noise stress has negative influences on the cells of testicular tissue by increasing apoptotic and necrotic cells . however , the associated enhancement in healthy cells suggests that honey and vitamin e have positive influences on the testis parenchyma .

we tested the drug sensitivity of neuraminidases ( nas ) ( 2 ) from influenza ( h5n1 ) from chickens , ducks , geese , and quail from 2004 from vietnam and malaysia ( provided by n. long , regional animal health centre , ho chi minh city , vietnam ; and s. hassan , veterinary research institute , ipoh , malaysia ) , from 200405 from cambodia ( provided by s. san , national animal health production and investigation center , phnom penh , cambodia ) , and from 2005 from indonesia ( provided by t. usman ) . in the absence of a validated cell culture assay , the 50% inhibitory concentration measured in the na enzyme inhibition assay is used as the benchmark for measuring drug sensitivity ( 3 ) . despite their origins in different countries and different avian species , all clade 1 and clade 2 viruses had a similar sensitivity to zanamivir as the reference influenza ( h1n1 ) na , ( table 1 , figure , panel a ) . however , sensitivities of the nas to oseltamivir ( oseltamivir carboxylate ) fell into 3 groups when compared to the na of a reference human influenza ( h1n1 ) strain ( table 1 , figure , panel b ) . the clade 1 isolates from 2004 were all more sensitive to oseltamivir than was the human influenza ( h1n1 ) control , consistent with recent findings of rameix - welti et al . however , the nas of our 2005 cambodian viruses showed a 6- to 7-fold decrease specifically in oseltamivir sensitivity in comparison to our 2004 cambodian isolates . these 2005 isolates came from the same area as 1 of the more sensitive 2004 isolates ( kandal ) , which suggested that at least regional evolution had occurred . * sigma , saint louis , mo , usa . ic50 , drug concentration for 50% inhibition of enzyme activity ; na , neuraminidase ; wt , wild type . drugs provided by glaxosmithkline ( stevenage , hertfordshire , uk ) . nos . in brackets indicate the nos . of isolates tested against zanamivir and oseltamivir in the same assay . nineteen influenza ( h5n1 ) isolates were retested in an independent assay against oseltamivir and 4-amino - neu5ac2en . ic50s for each isolate were calculated by using the sigmaplot nonlinear curve - fitting function ( systat software inc . , values in parentheses are standard deviations for the means of the individual ic50s for the isolates in each group . several plaques were tested from the a / mississippi/3/2001 against zanamivir and oseltamivir , but only 1 plaque of each was tested against 4-amino - neu5ac2en . sensitivity of clade 1 and clade 2 influenza a ( h5n1 ) viruses to zanamivir , oseltamivir , and 4-amino - neu5ac2en in a munana - based enzyme inhibition assay ( sigma , saint louis , mo , usa ) . plots are the mean values for inhibition of enzyme activity for each drug concentration of all isolates from that country and year ; bars represent standard deviations of values for all isolates from that group . a ) sensitivity to zanamivir ; b ) sensitivity to oseltamivir ; c ) sensitivity to 4-amino - neu5ac2en . of more concern was the third group . the nas from all the clade 2 2005 indonesian viruses demonstrated a 15- to 30-fold decrease in sensitivity specifically to oseltamivir compared with clade 1 viruses ( table 1 , figure , panel b ) . ( 5 ) also recently showed that the a / turkey/15/2006 clade 2 virus was almost 60-fold less sensitive to oseltamivir in a plaque reduction assay than was the clade 1 a / vietnam/1203/2004 virus . ( 4 ) contrast to those recently published by hurt et al . , who found no difference between the sensitivities of clade 1 and clade 2 isolates to oseltamivir in the enzyme assay ( 6 ) , although many of the isolates were the same as those tested here . the decrease in sensitivity is comparable to that conferred by the n294s recently detected in egypt ( 7 ) , known to be selected for by oseltamivir treatment ( 8) . although both drugs are based on the transition state analog of sialic acid , zanamivir has a single substitution of a guanidinium group at the 4 position on the sugar ring , whereas oseltamivir has an amino group at the 4 position and , more importantly , a bulky hydrophobic pentyl ether group replacing the glycerol side chain at the 6 position . reorientation of e276 in the active site is required to create a hydrophobic pocket necessary to accommodate this pentyl ether group . mutations that prevent this reorientation from occurring lead to high levels of specific oseltamivir resistance ( h274y , r292k ) . decreased binding to oseltamivir can also be due to altered interactions with its 4-amino group ( e119v ) ( 9 ) . we therefore tested several viruses from each group for inhibition by a drug that shares the 4-amino substitution . the clade 1 and 2 viruses had similar sensitivity to 4-amino - neu5ac2en ( table 1 , figure , panel c ) , indicating the decreased binding to oseltamivir was specifically due to altered interactions around the 6-pentyl ether group , thus explaining why no altered binding to zanamivir was seen . because of the potentially important implications of our findings for public health and stockpiling strategies , and because of the apparent discrepancy with the results of hurt et al . ( 6 ) , we retested 2 viruses from each group against an independent source of oseltamivir carboxylate ( provided by biota , notting hill , victoria , australia ) . we chose a / indonesia / wates/77/2005 , which had a further slight decrease in sensitivity compared to other indonesian isolates , as well as one that was in the same range as the remaining indonesian isolates . results shown in table 2 confirm this decreased sensitivity of the clade 2 indonesian isolates and a small further decrease in sensitivity of a / indonesia / wates/77/2005 . * ic50 , drug concentration for 50% inhibition of enzyme activity ; wt , wild type . ic50s and standard errors ( in parentheses ) were calculated by using the nonlinear curve - fitting function in sigmaplot ( systat software inc . , since we only had access to clade 2 isolates from indonesia , we do not know whether this decreased sensitivity occurs only in the indonesian clade 2 isolates or globally with all clade 2 isolates . comparisons of the sequences in the public databases show several mutations in the stalk region , which vary between clade 1 and clade 2 nas , but 1 mutation in the globular head , h252y , varies between all clade 1 and clade 2 isolates . a further 3-aa variation occurs in most clade 1 and clade 2 nas , s343p , e387 g , and g459s ( n2 numbering ) , including the indonesian isolates studied here . an additional v338 m variation is found in most indonesian isolates , including the ones studied here ( sequences submitted by n. komadina ) . the a / indonesia / wates/77/2005 na has an additional unique i117v variation , which is adjacent to one of the catalytic arginines , r118 ( which would be consistent with a further effect on drug sensitivity to oseltamivir ) , and a slight decrease in sensitivity to zanamivir ( 2.3 nmol / l vs mean of 1.4 we have shown here that , compared with clade 1 isolates from 2004 , some clade 1 cambodian isolates and clade 2 indonesian isolates from 2005 demonstrate reduced sensitivity to oseltamivir . because none of the sequence variations in the public databases correlates with any mutation known to confer oseltamivir resistance , and none of the variations are in the active site ( 10 ) , this suggests that the decrease in sensitivities may be due to drift mutations rather than from exposure to oseltamivir . recent results show that human isolates can also demonstrate decreased sensitivity to oseltamivir and zanamivir with drift mutations in the na remote from the active site ( 11 ) . the difference in the amino acid at position 252 may partially account for the altered binding between the clade 1 and clade 2 nas as other researchers have suggested that the amino acid at position 252 can affect reorientation of the e276 ( 4,10 ) . in clade 1 nas , this is normally subtype h252 , but all clade 2 nas have subtype y252 . in the recently published n1 structure , although it is from a clade1 subtype h5n1 nas , the authors had mutated the na to subtype y252 ( 10 ) , which is also found in human nas . the e276 in this structure does not appear to have undergone full reorientation , which could contribute to reduced oseltamivir binding found in the indonesian clade 2 isolates here . testing the sensitivities of both nas would provide valuable information on the role of subtype h252 . however , mutational analysis of the indonesian and cambodian isolates and structure determinations of the different nas are necessary to fully understand the mechanisms of altered binding . the specific decrease in sensitivity to oseltamivir of both 2005 cambodian clade 1 and especially the indonesian clade 2 influenza ( h5n1 ) isolates is disturbing , especially since they maintain their pathogenicity and transmissibility in birds and are clearly pathogenic in humans , since indonesia has the highest death rate from influenza ( h5n1 ) infections of any country . this finding is in contrast to recent observations that mutations conferring zanamivir resistance in human strains have poor viability and are not genetically stable ( 12 ) . such a decrease in oseltamivir sensitivity could lead to suboptimal drug dosing in treating persons infected with these isolates , which is thought to facilitate selection of viruses with a high level of resistance ( 13 ) . several groups have reported the emergence of resistant viruses in clade 1infected influenza ( h5n1 ) patients treated with oseltamivir and suggested that higher doses of oseltamivir may be needed ( 1,8 ) . because the clade 2 viruses studied here have a 15- to 30-fold decrease in sensitivity compared to the clade 1 viruses , this suggests the standard dosing of oseltamivir may be even less effective in treating clade 2 influenza ( h5n1)infected patients . many laboratories are developing rapid pcr sequencing methods for detecting the known mutation ( h274y ) that confers high - level resistance in influenza ( h5n1 ) viruses . however , we have shown here the importance of phenotypic testing of isolates in an enzyme assay rather than just genotypic screening ( 14 ) . because the clade 2 virus is now spread through parts of europe and africa , continued global collaboration and phenotypic testing of drug sensitivity of circulating highly pathogenic avian isolates for na inhibitor sensitivity are critical . no altered sensitivity to zanamivir occurred , which further supports the hypothesis of minimalist drug design ( 15 ) and of maintaining the inhibitor as close as possible to the natural substrate to minimize the emergence of resistance .

we tested the neuraminidase drug sensitivity of clade 1 and clade 2 influenza a virus ( h5n1 ) . all viruses demonstrated similar sensitivity to zanamivir , but compared to the 2004 clade 1 viruses , the cambodian 2005 viruses were 6-fold less sensitive and the indonesian clade 2 viruses were up to 30-fold less sensitive to oseltamivir .

the 1980s were a seminal period for the study of primary headache , as they were characterized not only by a wealth of investigational activity and significant progress in the knowledge of the disease , but above all by an increasingly rigorous approach from the scientific point of view . the decade opened with the results of the danish studies [ 1 , 2 ] on regional cerebral blood flow ( rcbf ) in migraine which was still named these studies paved the way for the trigeminovascular theory , which is still considered a good explanation for the underlying pathogenetic mechanisms of the disease . then , at the close of the decade came a number of accurate controlled trials that demonstrated the efficacy of sumatriptan in the symptomatic treatment of migraine , leading to the introduction of a new class of drugs that are still considered the therapy of choice for migraine attacks . in between , there were two all - important steps : the joint commitment from researchers of several countries to draw up the first version of the international headache society ( ihs ) classification , which was published in 1988 and represented a true cultural and practical turning point ; and the first demonstrations of investigators interest for those migraine patients who over time experience a progressive increase in the frequency of attacks until they develop a so - called chronic daily headache ( cdh ) [ 5 , 6 ] . among headache experts , the debate on cdh has been heated and controversial since the very beginning and is not yet over . to sum up the question in a few words on the one hand , there were those who thought that , in the wide - ranging headache chapter , cdh was a group that naturally included different subtypes some of which still undefined but was nonetheless autonomous and therefore should be considered , assessed and classified independently of the other primary headache forms [ 68 ] . on the other hand , there were those who thought that cdh was nothing but an entirely generic definition , a hotchpotch containing a non - homogeneous group of disparate headache forms that had already found their place in the current classification systems : in their opinion , this was a non diagnosis , or a diagnosis still waiting to be defined , if not a diagnosis made by unskilled people [ 9 , 10 ] . both views were based on elements and remarks that were worthy of the utmost respect . the former view was more closely related to clinical practice , the latter view paid more attention to formal aspects ; the former view was more critical of the current nosographic identification and classification systems ; the latter view reflected the conviction that the ihs classification has always been thoroughly exhaustive since its first 1988 edition . in the next 20 years , the authors who originally embraced either view continued to investigate the issue of cdh in general and of its subtypes in particular ; while clinging to their initial beliefs , they were nonetheless willing to pay attention to their counterpart s opinions and advances . this intelligent , non - dogmatic approach led to very important , albeit not yet conclusive , achievements . we will first deal with the milestones that led to the current state of knowledge on cdh in general and chronic migraine in particular . the current official view of the issue will be expounded with all relevant critical points and a proposal will be advanced for changes and adjustments to the classification of the main cdh subchapter : the one about migraine that evolves unfavourably over time . were the first to call attention to the possibility that migraine may be transformed into daily headache over time . five years later , the same group of researchers reported a case series of 630 cdh patients and attempted to subdivide them into three different groups depending on their headache type : type i starts as daily or near - daily headache with no change in the severity and lacks migrainous features ; type ii starts as daily or near - daily headaches with occasional more severe headache with some migrainous features ; type iii ( transformed or evolutive migraine ) starts as a clear - cut occasional episodic migraine . with increasing frequency over the next many years ( 77.7% ) belonged to type iii , while only 57 ( 9.0% ) and 84 ( 13.3% ) belonged to type ii and type i , respectively . these authors in 1987 were not yet talking about chronic tension - type headache ( ctth ) , nor about new daily persistent headache ( ndph ) , a form of primary headache first described just 1 year earlier by vanast . both were still unknown definitions , even though the two first cdh types could seemingly be identified with these two headache forms . however , back then they already suggested the term transformed or evolutive migraine for the third type . ( tm ) , which he included in the first true cdh classification , alongside ctth and ndph ( table 1).table 1classifications of chronic daily headache introduced in the mid-1990smathew 1 . transformed migraine 2 . new daily persistent headachesilberstein et al . 1 . transformed migraine 1.1 with medication overuse 1.2 without medication overuse 2 . chronic tension - type headache 2.1 with medication overuse 2.2 without medication overuse 3 . new daily persistent headache 3.1 with medication overuse 3.2 without medication overuse 4 . hemicrania continua 4.1 with medication overuse 4.2 without medication overusemanzoni et al . 1 . evolution of migraine 1.1 migraine with interparoxysmal headache 1.1.1 migraine with interparoxysmal headache fulfilling the criteria for chronic tension - type headache 1.1.2 migraine with interparoxysmal headache not fulfilling the criteria for chronic tension - type headache 1.2 chronic migraine 2 . chronic tension - type headache classifications of chronic daily headache introduced in the mid-1990s the following year , silberstein , together with lipton , solomon and mathew again , proposed that hemicrania continua ( hc ) should be added to these three forms and that each of the four forms thus identified should be distinguished depending on the presence or absence of medication overuse ( table 1 ) . tm criteria are reported in table 2.table 2diagnostic criteria for transformed migraine by silberstein et al . a. daily or almost daily ( > 15 days / month ) head pain for > 1 monthb . history of increasing headache frequency with decreasing severity of migrainous features over at least 3 months 3 . . such a disorder is suggested , but it is ruled out by appropriate investigations 3 . such a disorder is present , but first migraine attacks do not occur in close temporal relation to the disorder diagnostic criteria for transformed migraine by silberstein et al . in 1995 , following the description of a broad case series of cdh patients seen at the parma and pavia headache centres in italy , manzoni et al . first introduced the name chronic migraine ( cm ) , which they included , alongside migraine with interparoxysmal headache ( mih ) , within the migraine forms that evolve unfavourably over time until they lose the typical symptom - free interval between an attack and the next ( table 1 ) . according to the italian authors , cm and mih differentiate from each other for the type of headache that sets in the originally free intervals between attacks : while retaining the clinical features of migraine in cm , in mih this interval headache loses its similarities to migraine : in some cases it has the same features of tension - type headache , but in other cases it has undefined features and this prevents it from being definitely included in the migraine or the tension - type headache group . the proposals introduced in the mid-1990s by the us authors and the italian authors differ from each other in several respects . first , they use different approaches to identify cdh entities : while silberstein et al . adopt a more detailed and exhaustive classification that includes also ndph and hc , manzoni et al . prefer a classification that takes into account only the forms most frequently encountered in clinical practice . secondly , and more importantly , the differences between them concern : ( a ) the terminology used to describe migraine forms that evolve unfavourably over time ( tm for silberstein and cm for manzoni ) ; ( b ) the temporal requirements for these headache forms ( presence of headache at least 15 days per month for at least 1 month for silberstein and at least 6 days per week for at least 1 year for manzoni ) ; and ( c ) manzoni et al.s attempt to identify possible clinical subtypes in the group of migraine forms evolving unfavourably ( cm and mih with interval headache with or without tension - type headache features ) . if we browse the scientific cdh literature produced from the mid-1990s to as late as 2006 , we can see how predominant silberstein et al.s systematization became . almost all studies conducted over this long period of time and aimed at defining the epidemiological , pathogenetic and therapeutic aspects of cdh basically followed silberstein et al.s classification , both in terms of headache forms included in the classification , and in terms of their respective diagnostic criteria and the terminology proposed to give an official name to each form . in this connection , it is worth noting that there was a general and widespread acceptance of the tm name . on the other hand , we are sorry to say that the 2004 edition of the international classification of headache disorders classification ( ichd-2 ) only partially and not always appropriately integrated those considerations in its final changes over the 1988 first edition of the ihs classification . in the first place , the ichd-2 editors did not deem it advisable to devote a separate chapter to cdh within their classification which , all things considered , is actually a decision we can agree on . ( table 1 ) , only one , ctth , was already included in the 1988 first edition of the ihs classification and then again in the 2004 ichd-2 classification . two other forms , hc and ndph , did not appear in the ihs classification but were included in the ichd-2 classification , where they were coded to group 4 other primary headaches . the last of silberstein et al.s cdh forms , tm , was not recognized as such in the ichd-2 , which however included for the first time cm and its diagnostic criteria , coded to 1.5.1 as a complication of migraine ( table 3 ) . thus , with respect to the most important and certainly most frequent of all cdh forms , i.e. migraine evolving unfavourably over time , the ichd-2 eventually retained the cm name , originally proposed by manzoni et al . , but with diagnostic criteria ( > 15 days per month for at least 3 months ) that are very different from those suggested by the italian authors ( at least 6 day per week for at least 1 year ) . the result is that only for some of the patients affected by this clinical entity precisely those with an interval headache resembling tension - type headache can the ichd-2 provide a diagnosis , but even so it would be a dual diagnosis , of migraine without aura and of tension - type headache . mih patients with an interval headache not resembling either migraine or tension - type headache remain unclassified.table 3diagnostic criteria for chronic migraine by the ichd-2 ( 2004 ) and by the ichd-2r ( 2006)ichd-2 ( 2004 ) a. headache fulfilling criteria c and d for migraine without aura on 15 days per month for > 3 months b. not attributed to another disorderichd-2r ( 2006 ) a. headache ( tension - type and/or migraine ) on 15 days per month for 3 months b. occurring in a patient who has had at least five attacks fulfilling criteria for 1.1 migraine without aura c. on 8 days per month for 3 months headache has fulfilled c.1 and/or c.2 below ; that is , has fulfilled criteria for pain and associated symptoms of migraine without aura : 1 . has at least two of a d : a. unilateral location b. pulsating quality c. moderate or severe pain intensity d. aggravation by or causing avoidance of routine physical activity ( e.g. , walking or climbing stairs ) and at least one of a or b below : a. nausea and/or vomiting b. photophobia and phonophobia 2 . treated and relieved by triptan(s ) or ergot before the expected development of c.1 above d. no medication overuse and not attributed to another causative disorder diagnostic criteria for chronic migraine by the ichd-2 ( 2004 ) and by the ichd-2r ( 2006 ) with respect to silberstein et al.s classification , the ichd-2 classification neither recognizes the tm definition nor agrees with its diagnostic criteria ( tables 2 , 3 ) . additionally , the ichd-2 does not envisage , either for cm or even for ctth , hc and ndph , a differentiation based on the presence or absence of medication overuse , because it prefers to retain medication - overuse headache as an autonomous clinical entity ( coded to 8.2 of the 2004 classification ) , much as the 1988 ihs classification had already done . as defined by the diagnostic criteria of the ichd-2 classification , cm seems : ( a ) to resemble more a high - frequency migraine than a migraine evolving over time to a daily or near - daily form and eventually losing some , and in certain cases , many of its typical migraine features , such as unilateral and/or throbbing pain and/or nausea and vomiting as accompanying symptoms ; ( b ) to be scarcely relevant to actual clinical practice , because a patient with more than 15 days of headache per month is highly unlikely to use symptomatic drugs for less than 1015 days per month . since its inclusion in the ichd-2 classification , then , cm has always appeared as an ambiguous clinical entity and one that would not be of much use either for clinical practice or research . bigal et al . in the us tried to re - classify 638 cdh patients seen over a period of 20 years at the new england medical center for headache in stamford , connecticut . by strictly applying the diagnostic criteria of the ichd-2 in contrast , using the diagnostic criteria proposed by silberstein et al . , the number of patients that in the same case series could be classified as suffering from tm without medication overuse would be as high as 158 . therefore , the authors concluded that cm diagnosis , as defined and formally applied in the ichd-2 , does not offer any considerable benefit to the cdh diagnostic issue . another major shortcoming of the ichd-2 classification is that , as was clearly demonstrated in the same analysis by bigal et al . , most cdh patients receive multiple diagnoses , in several cases as many as four or five , including some that are only probable . since the very introduction of the ichd-2 in 2004 , then , it has been clear that the diagnostic criteria of cm needed to be changed . even the committee that worked them out was aware of this problem and new criteria were formulated as a result ( ichd-2r ) . today , these criteria ( table 3 ) are considered standard reference and , according to olesen , all of them could be integrated in the future ichd-3 classification [ 18 , 19 ] . the new criteria are much more relevant to actual clinical practice than the previous ones of the ichd-2 classification and may provide a good starting point to get to a better understanding of this complex and all - important chapter of headaches [ 2022 ] . nevertheless , a few questions remain to be solved and these basically concern two aspects that the headache clinician knows well and the headache researcher can not underestimate . the first major question is that a severity gradient exists in cm , as defined in the ichd-2r classification diagnostic criteria , and it is so wide - ranging as to carry the risk of including exceedingly different cases under the single group of cm . patients who for 3 months have had 8 days of migraine and 7 days of tension - type headache per month have almost nothing to do with patients who have suffered from migraine - like headache every day for years . the former have a good chance of improving , if adequately treated ; the latter generally do not respond well to standard treatments and require a special , customized therapeutic approach . in other words , the minimum time limits set in the ichd-2r for diagnosis of cm ( 15 days per month for 3 months ) appear to be too short to allow inclusion of cm itself among complications of migraine . these might actually be cases of migraine with a transiently high frequency or of a transient association of medium - frequency migraine and medium - frequency tension - type headache , as can often be seen in the natural history of migraine . however , none of these cases would represent a true complication of migraine . creating a single loose - net subchapter of headaches , as would be the case with cm according to the ichd-2r , will likely be of no use either to the clinician or to the researcher or even the drug trial investigator . indeed , it could ultimately jeopardize the key goal of all those who are concerned with the issue , i.e. providing both basic and clinical research with a reliable instrument that can be effectively used to collect homogeneous and well - defined case series . if the tool we are using does not allow an adequate selection of the case series under study , the results obtained whether from epidemiological surveys , pathogenetic investigations or drug trials will be misleading and the effort made to achieve them useless [ 2429 ] . the second major question is the problem of symptomatic medication overuse , which affects most patients with cm [ 3035 ] . as the ihs classification before it , the ichd-2 classification also includes medication - overuse headache in group 8 ( headache attributed to a substance or its withdrawal ) . the ichd-2 diagnostic criteria were revised twice in the last few years and those that are currently recognized as valid ( ichd-2r ) ( table 4 ) for diagnosis of medication - overuse headache no longer require that headache resolve or revert to its previous patterns within 2 months after discontinuation of the overused medication . thus , according to the ichd-2r classification , a patient with a form of migraine that has worsened over the years or has become complicated evolving into daily or near daily , with daily or near - daily use of symptomatic drugs , should have a dual diagnosis : medication - overuse headache and probable cm . apart from the drawbacks of multiple diagnoses , it is difficult to see a patient with cm who will not use drugs to block , or at least relieve pain , and if the headache recurs at least 15 days per month as must happen for the diagnosis of cm to be established this patient will likely report medication overuse as a consequence , if not necessarily as a cause . instead of a dual diagnosis of medication - overuse headache and probable cm , would not a single diagnosis of cm with medication overuse be preferable in such a patient until he / she is freed from the overused drugs?table 4diagnostic criteria for medication - overuse headache by the ichd-2r ( 2006)a . regular overuse for > 3 months of one or more acute / symptomatic treatment drugs as defined under subforms 1 . ergotamine , triptans , opioids , or combination analgesic medications on 10 days / month on a regular basis for > 3 months 2 . simple analgesics or any combination of ergotamine , triptans , analgesic opioids on 15 days / month on a regular basis for > 3 months without overuse of any single class alonec . headache has developed or markedly worsened during medication overuse diagnostic criteria for medication - overuse headache by the ichd-2r ( 2006 ) the uncertainties about medication - overuse headache are also related to the lack of specific scientific data on the role that the individual symptomatic drugs taken by migraineurs might play on the underlying course of the disease , if overused . as things stand now , we can not but agree with what was recently stated by bigal et al . : the ultimate question that needs to be discussed by the scientific community is not how to better classify migraine overuse headache , but if migraine overuse headache should exist as a single entity or is more appropriately viewed as a risk factor . the next edition of the ichd classification ( ichd-3 ) , due out by the end of 2012 , will certainly introduce some changes to cm as defined in the ichd-2 classification ( table 3 ) . it may be that for cm the ichd-3 will merely replace the much criticized diagnostic criteria of the ichd-2 with the more acceptable ones proposed by the ichd-2r ( table 3 ) . this would certainly be a significant step forward over the ichd-2 , but a few questions remain unsolved . olesen himself criticized the use of the adjective chronic , which , as was reported in a recent article by seshia et al . the term transformed migraine is to be preferred , because it is less ambiguous and more indicative of the type of patients we are referring to [ 12 , 13 , 3941 ] . even though we avoid use of the adjective chronic , in order to establish a diagnosis of tm we nonetheless have to set a minimum period of time for the duration of this daily or near - daily headache pattern . the 3 month period generally taken as reference until now seems too short and carries the inherent risk of considering tm as a form of migraine that merely undergoes an entirely transient worsening . the same applies to the other temporal parameter , which must define and therefore better specify the vague expression daily or near - daily originally used by mathew et al . [ 5 , 6 ] . quantifying this daily or near - daily parameter as 15 days appears an oversimplification . in order to avoid too loose a categorization , a more accurate statement would be 20 days / month , adding also that there are never > 5 headache - free consecutive days . then , there is the big question of medication - overuse headache . from the ihs classification to the ichd-2 and ichd-2r classifications , the addition of triptans , the addition of overuse from other symptomatic drugs , the addition of the limits of 10 days / month for overuse from certain drugs and 15 days / month for overuse from other drugs , the addition and then the removal of the criterion that required improvement within 2 months ( ! ) from withdrawal of medication , the addition and then the removal of a description of certain headache features for some types of overuse all these tentative changes clearly indicate that we are still a long way from knowing this topic well , because literature reports are still much too scarce . as is currently categorized in the ichd-2 or proposed in the ichd-2r , medication - overuse headache today is a largely arbitrary entity and its very existence appears questionable for certain subtypes . the only sure data we have are those about ergotamine , caffeine and combined medications containing barbiturates or codeine and , partly , triptans . waiting for future specific studies to yield more reliable results than are available today and help clarify whether medication - overuse headache should be considered less an autonomous entity than a complication of migraine , at the current state of knowledge perhaps we should better take a more cautious approach to this chapter . a basic reason that could explain why , in spite of the efforts made by so many authors for so many years , we have not yet come to share a common systematization for the classification of migraine that evolves unfavourably , is that international classifications the ihs of 1988 and the ichd-2 of 2004 are typically classifications of headache attacks , providing a snapshot of a headache attack at a given moment . for primary headaches at least , they are not and do not purport to be classifications of disorders or of patients . this approach certainly has merits and advantages and such classifications have become indispensable tools for epidemiological , pathophysiological and therapeutic research . however , they are not suitable for headache forms such as cm or tm , in which what matters most is the patient s history , and not a snapshot of his / her headache , which can only be blurred and confused . hopefully , in the future we will be able and willing to engage in the preparation of a classification of headache syndromes that may combine all current international headache classifications . this is an ambitious and difficult project , which will require several years to complete , but could allow an adequate and correct categorization of patients with cdh . waiting for this goal to be achieved , based on the current state of knowledge and on what was previously discussed and expounded in this paper , we think it reasonable to formulate a simple and practical proposal , which can be broken down as follows : differentiation of migraine without aura based on frequency of attacks , with the addition of a third - digit level ( table 5);table 5proposed revision of the ichd-2 for migraine1.1 migraine without aura 1.1.1 infrequent migraine 1.1.2 frequent migraine 1.1.3 chronic migraine 1.1.3.1 with medication overuse 1.1.3.2 without medication overuse1.5 complications of migraine 1.5.1 transformed migraine 1.5.1.1 with medication overuse 1.5.1.2 without medication overuseshifting of cm from its current coding position in the ichd-2 and the ichd-2r among migraine complications to a different coding position as a high - frequency subtype of migraine without aura ; cm , coded to 1.1.3 would then represent an evolution stage in the natural chronicization course of migraine;introduction of precise temporal parameters among the diagnostic criteria for the three migraine without aura subtypes ( infrequent , frequent and chronic ) ( table 6);table 6proposed diagnostic criteria for the three migraine without aura subtypes , and for transformed migraine1.1.1 infrequent migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on 3 days / month for 3 months b. not attributed to another disorder1.1.2 frequent migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on > 3 but < 10 days / month for 3 months b. not attributed to another disorder1.1.3 chronic migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on 10 but 20 days / month for 3 months b. not attributed to another disorder1.5.1 transformed migraine a. headache ( tension - type and/or migraine ) on > 20 days / month for 1 year and never with more than 5 headache - free consecutive days b. occurring in a patient who has had at least five attacks fulfilling criteria for 1.1 migraine without aura c. on 10 days per month for 1 year headache has fulfilled criteria for pain and associated symptoms of migraine without aura or patient has been successfully treated with an ergot or triptan d. not attributed to another disorderinclusion of tm among the complications of migraine : tm should be coded to 1.5.1 replacing cm ( table 5 ) and its diagnostic criteria should be different from those listed in the ichd-2r only in as far as frequency and duration are concerned ( > 20 days / month for 1 year and never with > 5 headache - free consecutive days ) ( table 6);differentiation of tm at the fourth - digit level depending on the presence or absence of symptomatic medication overuse ( table 5 ) ( i.e. use for > 20 days / month ) regardless of whether overuse played any role in the worsening of the headache;shifting of medication - overuse headache to the appendix with alternative diagnostic criteria to be defined . differentiation of migraine without aura based on frequency of attacks , with the addition of a third - digit level ( table 5);table 5proposed revision of the ichd-2 for migraine1.1 migraine without aura 1.1.1 infrequent migraine 1.1.2 frequent migraine 1.1.3 chronic migraine 1.1.3.1 with medication overuse 1.1.3.2 without medication overuse1.5 complications of migraine 1.5.1 transformed migraine 1.5.1.1 with medication overuse 1.5.1.2 without medication overuse proposed revision of the ichd-2 for migraine shifting of cm from its current coding position in the ichd-2 and the ichd-2r among migraine complications to a different coding position as a high - frequency subtype of migraine without aura ; cm , coded to 1.1.3 would then represent an evolution stage in the natural chronicization course of migraine ; introduction of precise temporal parameters among the diagnostic criteria for the three migraine without aura subtypes ( infrequent , frequent and chronic ) ( table 6);table 6proposed diagnostic criteria for the three migraine without aura subtypes , and for transformed migraine1.1.1 infrequent migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on 3 days / month for 3 months b. not attributed to another disorder1.1.2 frequent migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on > 3 but < 10 days / month for 3 months b. not attributed to another disorder1.1.3 chronic migraine a. headache fulfilling criteria c and d for 1.1 migraine without aura on 10 but 20 days / month for 3 months b. not attributed to another disorder1.5.1 transformed migraine a. headache ( tension - type and/or migraine ) on > 20 days / month for 1 year and never with more than 5 headache - free consecutive days b. occurring in a patient who has had at least five attacks fulfilling criteria for 1.1 migraine without aura c. on 10 days per month for 1 year headache has fulfilled criteria for pain and associated symptoms of migraine without aura or patient has been successfully treated with an ergot or triptan d. not attributed to another disorder proposed diagnostic criteria for the three migraine without aura subtypes , and for transformed migraine inclusion of tm among the complications of migraine : tm should be coded to 1.5.1 replacing cm ( table 5 ) and its diagnostic criteria should be different from those listed in the ichd-2r only in as far as frequency and duration are concerned ( > 20 days / month for 1 year and never with > 5 headache - free consecutive days ) ( table 6 ) ; differentiation of tm at the fourth - digit level depending on the presence or absence of symptomatic medication overuse ( table 5 ) ( i.e. use for > 20 days / month ) regardless of whether overuse played any role in the worsening of the headache ; shifting of medication - overuse headache to the appendix with alternative diagnostic criteria to be defined .

in the field of so - called chronic daily headache , it is not easy for migraine that worsens progressively until it becomes daily or almost daily to find a precise and universally recognized place within the current international headache classification systems . in line with the 2006 revision of the second edition of the international classification of headache disorders ( ichd-2r ) , the current prevailing opinion is that this headache type should be named chronic migraine ( cm ) and be characterized by the presence of at least 15 days of headache per month for at least 3 consecutive months , with headache having the same clinical features of migraine without aura for at least 8 of those 15 days . based on much evidence , though , a cm with the above characteristics appears to be a heterogeneous entity and the obvious risk is that its definition may be extended to include a variety of different clinical entities . a proposal is advanced to consider cm a subtype of migraine without aura that is characterized by a high frequency of attacks ( 1020 days of headache per month for at least 3 months ) and is distinct from transformed migraine ( tm ) , which in turn should be included in the classification as a complication of migraine . therefore , cm should be removed from its current coding position in the ichd-2 and be replaced by tm , which has more restrictive diagnostic criteria ( at least 20 days of headache per month for at least 1 year , with no more than 5 consecutive days free of symptoms ; same clinical features of migraine without aura for at least 10 of those 20 days ) .

among childhood brain tumors , choroid plexus carcinomas ( cpcs ) are rare and have a dismal prognosis . recently , nupponen et al . reported that cpc cells express platelet - derived growth factor receptor ( pdgfr ) alfa or beta , and koos et al . revealed that imatinib mesylate ( gleevec ) , a tyrosine kinase inhibitor , suppresses choroid plexus cell proliferation in a dose - dependent manner by blocking the pdgfr beta signaling pathway . herein , we report the case of a pdgfr alfa - positive cpc that showed no response to imatinib mesylate . brain computed tomography revealed a large tumor accompanied with bleeding in the right lateral ventricle . after twice operations , the tumor , which was pathologically diagnosed as cpc , was almost completely ( over 95% ) removed . she was subsequently administered chemotherapy with vincristine , cisplatin , cyclophosphamide , and etoposide ( the vcce regimen ) . following 5 courses of vcce , x - ray irradiation was performed ( 18 gy to the whole brain and spine ; boost of 32 gy to the scar of the resected tumor ) . despite all these treatments , the tumor recurred in the third ventricle and spine after 6 months . afterwards , salvage chemotherapy with adriamycin , cyclophosphamide , carboplatin , etoposide , and methotrexate ( the accem regimen ) was initiated . after completion of 6 courses of accem , high - dose chemotherapy with thiotepa and melpharan along with auto - peripheral blood stem cell transplantation ( autopbsct ) was administered . however , 4 months after autopbsct , the cerebrospinal fluid ( csf ) was positive for cpc cells , and the mass had recurred at the third ventricular profunda . therefore , imatinib mesylate ( gleevec , 400 mg/(mday ) perorally ) alone was administered as palliative treatment the patient finally died at the age of 62 months because of acute renal failure due to hemorrhagic shock caused by bleeding from the mass in the third ventricle . lower panels : immunohistochemical staining of platelet derived growth factor receptor ( pdgfr ) alfa ( lower left panel : tumor section at diagnosis ; lower right panel : cerebrospinal fluid at disease recurrence ) . comparison of the histological findings of the tumor samples obtained during the first operation and the cytological findings of the cerebrospinal fluid samples obtained at 4 months after auto - peripheral blood stem cell transplantation revealed that the choroid plexus carcinoma ( cpc ) cells had continued to express pdgfr alfa at the same rate ( nearly 30% of all cpc cells were positively stained ) , while both samples were negative for pdgfr beta expression . lower panels : immunohistochemical staining of platelet derived growth factor receptor ( pdgfr ) alfa ( lower left panel : tumor section at diagnosis ; lower right panel : cerebrospinal fluid at disease recurrence ) . comparison of the histological findings of the tumor samples obtained during the first operation and the cytological findings of the cerebrospinal fluid samples obtained at 4 months after auto - peripheral blood stem cell transplantation revealed that the choroid plexus carcinoma ( cpc ) cells had continued to express pdgfr alfa at the same rate ( nearly 30% of all cpc cells were positively stained ) , while both samples were negative for pdgfr beta expression . the pdgf and pdgfr system plays a role in cell growth and angiogenesis in some tumors . it is known that pdgfr signaling is blocked by imatinib ( gleevec ; a tyrosine kinase inhibitor with high specificity for pdgfr ) as well as c - kit , and c - abl . some trials are being conducted on the use of imatinib in the treatment of pediatric neoplasms such as solid tumors and malignant gliomas . furthermore , it was recently reported that cpc cells express pdgfr alfa and beta , and that pdgfr beta expression is attenuated by imatinib in z310 , which is one of immortalized choroid plexus epithelial cell lines expressing pdgfr beta . we consider the following assumptions as the causes of failure : i ) imatinib could not penetrate adequately into the csf because of the blood brain barrier ( baruchel et al . reported that csf levels of imatinib was less than 5% of those of plasma levels ) ; ii ) the cpc cells in our case did not respond to imatinib probably because the cpc cells lacked pdgfr beta expression . however , the expression of pdgfr alfa in our case is presumed to play an essential role in the tumor regrowth , since this phenotype have retained even after potent treatments including with anticancer drugs , irradiation , and the auto pbsct .

we herein report a female child with choroid plexus carcinoma treated with standard dose of imatinib at disease recurrence . this patient failed initial twice - surgical resections , central nervous system ( cns ) irradiation , and adjuvant chemotherapies and high - dose thiotepa and melphalan with auto peripheral blood stem cell rescue . finally , imatinib treatment was undergone as a palliative setting , however the tumor did not reduce and the patient died of tumor bleedings . we consider that the reasons for the failure are as follows : i ) adequate cns level of imatinib were not obtained because of the blood brain barrier , ii ) the lack of platelet - derived growth factor receptor beta expression in our case may have a crucial role .

micromotion of dental implants has been defined as minimal displacement of an implant body relative to the surrounding tissue which can not be recognized with the naked eye ( figure 1 ) . various authors have shown that excessive micromotion may interfere with the process of osseointegration of dental implants [ 2 , 3 ] . although exact data are missing , it has been postulated that micromotion between implant and bone must not surpass a threshold value of 150 micrometer ( m ) for successful implant healing [ 46 ] . in traditional loading protocols , where implants are allowed to heal undisturbed for periods of several months , the issue of implant micromotion is of limited importance . with the advent of modern treatment concepts including early and immediate loading of dental implants [ 7 , 8 ] , with implants being restored early in the healing phase , the issue of implant micromotion has gained significant importance [ 4 , 5 ] . numerous reports trying to relate clinical parameters to the phenomenon of implant micromotion can be found in the dental literature [ 811 ] . the nonuniform nomenclature , the varying experimental settings , and the partially contradicting results presented on the one hand indicate the complexity of the topic but on the other hand emphasize the need for clarifying basic engineering principles . from a biomechanical perspective , successful osseointegration of dental implants depends on the way mechanical stresses and strains are transferred to the surrounding bone and tissues . the multiple factors hereby affecting stress and strain transfer include the type of loading that occurs , the type of implant - bone interface being present , the length and diameter of the implant , implant geometry and its surface texture , and the quality and quantity of the surrounding bone [ 1319 ] . only by understanding the most critical of these variables , strategies for optimizing implant stabilization can be developed . for determining how implant mobility , often referred to as micromotion , relative motion , micromovement , and so forth , or implant loading affects bone response , a closer look at implant deformation , bone deformation , and stress or strain at the implant - bone interface is required . in this context , it was the purpose of this paper to mechanically describe the phenomenon of micromotion occurring between implant and alveolar bone using simple spring models , continuum mechanics models , and 3d - finite - element models simulating varying contact types between implant and bone [ 2123 ] . three basic scenarios reflecting different anchoring situations of dental implants were considered . in scenario 1 , the implant rests on an apically located fixed surface but neither has contact to cortical bone nor to trabecular bone at the vertical walls of the implant ( figure 2 ) . in this situation , maximum implant deformation under vertical loading occurs in the coronal part and diminishes gradually towards the apex . as a result , micromotion between the implant and the vertical walls of the socket also decreases towards the apical part of the implant . axial deformation of the implant as a consequence of vertical loading can be calculated according to ( 1)u = fea / l = fc with f standing for the vertical force applied , e being the young 's modulus of the implant , a being the cross section of the implant , l being the length of the implant , and c being the stiffness of the implant . maximum micromotion uc at the cortical area can then be calculated according to ( 2)uc=ulh with h reflecting the height of cortical and trabecular bone around the implant . for scenario 2 , the fixed apical rest of the implant was altered by adding a layer of elastic trabecular bone apically to the implant . here , an axial force acting on the implant predominantly causes compression of the elastic material the implant is resting on . due to the drastically smaller elastic modulus of trabecular bone as compared to titanium , the deformation of the implant can be neglected and the relative movement between implant and bone is independent from the region of the implant considered ( figure 3 ) . in this situation , implant displacement may be calculated according to ( 3)u = fesa / h[1+lhese]=fcs[1+ ] , where es is the young 's modulus of the trabecular bone and h the height of the bone underneath the implant . taking into account that the young 's modulus of the implant is much greater than the young 's modulus of the trabecular bone ( e es ) it is accepted 1 and furthermore the approximation for the micromotion ( 4)uc=ufcs. consequently , micromotion at the cortical area of the implant and the relative micromotion between implant and bone are identical , both being related to the stiffness cs- of the trabecular bone underneath the implant . further approximating the clinical situation of an osseointegrated implant , in scenario 3 the implant is elastically supported by surrounding cortical and trabecular bone . due to the fixed contact between implant and bone , micromotion at this interface neglecting the deformation of the implant ( e es ) the implant displacement may be calculated according to ( 5)u = fcs1(1+(cs / cs)+(cc / cs ) ) . under the circumstances of scenario 3 , no relative micromotion between implant and bone exists . the displacement of the implant equals the micromotion depending on the stiffness of both cortical and trabecular bone surrounding the implant . further approximating clinical reality , continuum mechanics models were considered revealing implant displacement due to elastic deformation of bone when no contact between implant and bone was modelled in a plane strain fe - model ( figure 5 ) . this model correlates with scenario 2 described above ( figure 3 ) . simulating contact between implant and bone , bone is also elastically deformed in the cervical portion of the implant when an axial load is exerted ; however , no relative micromotion between implant and bone occurs at the interface ( figure 6 ) , and the implant displacement ( micromotion ) is related to the elastic properties of the cortical and trabecular bone . for a more realistic representation of clinical conditions , three - dimensional fe models of dental implants with and without threads were generated ( figure 7(a ) ) which were subsequently embedded in a bony socket consisting of cortical and trabecular bone and an intermediate layer surrounding the implant ( figure 7(b ) ) . the geometry of the models was generated with a cad program ( solidworks 2011 , solidworks deutschland gmbh , haar , germany ) and imported in a fe program ( ansys workbench 12 , ansys inc . , canonsburg , pa , usa ) . combining both components , three - dimensional fe models ( figure 7(c ) ) were obtained for evaluating micromotion between implant and bone when an axial vertical force of 200 n was exerted which reflects an average biting force [ 24 , 25 ] . different stages of osseointegration were simulated by altering the elastic modulus of the intermediate bone layer [ 2123 ] . the contact type between implant and bone could be modified as friction free , only transferring compressive forces and allowing for sliding and gap formation , to friction ( friction coefficient 0.3 ) and force fit , respectively . in general , isotropic linear model parameters were applied , defining the contact type between the different layers of bone as bond . out of the large number of possible solutions for solving contact problems , the augmented lagrange method was chosen as accompanying optimization method . this method was applied for defining all contacts not allowing contacting components to penetrate each other . based on the results of previous investigations [ 22 , 23 ] indicating that the size of the models was sufficient for evaluating micromotion , model dimensions were reduced to a minimum and the borders of the models were fixed . depending on model type , 160000 hexaeder elements and 600000 to 650000 nodes were used to set up the models using the elastic modules given in table 1 . based on the fact that the elastic values and the strength limits of biologic materials in vivo such as the bone - implant interface are highly complex , only two states of osseointegration were considered ( starting point and end point of osseointegration ) . these different states were modelled by different elastic values in the areas ( 1 ) and ( 2 ) in figure 7(b ) . results of all simulations were recorded as von mises equivalent stress in addition to contour plots of global displacement . for calculating relative displacement between implant and bone ( relative micromotion ) , a total of six corresponding nodes at the implant bone interface were established as reference marks . as the displacement of a specific reference mark on the implant represents both displacement of bone and implant , the displacement of the corresponding reference mark on the bone ( figure 8) was subtracted . simulating 200 n axial force acting on an osseointegrated cylindrical implant with no friction between implant and bone caused a symmetric loading situation of the bone surrounding the implant with maximum loading and maximum displacement occurring at the apical part of the implant ( figures 9(a ) and 10(a ) ) . adding threads to the implant led to a decrease both in loading and displacement occurring at the apical part of the implant . simultaneously , greater distribution of loading and displacement was observed at the vertical walls of the implants ( figures 9(b ) and 10(b ) ) . introducing friction between implant and bone ( figures 9(c ) and 10(c ) ) further decreased global displacement and resulted in a more homogeneous distribution of loads as compared to the force fit situation ( figures 9(d ) and 10(d ) ) , where load transfer predominantly occurred in the cervical area of the implant , where cortical bone was modelled . for freshly inserted implants with a soft intermediate layer of bone modelled around the implants , the introduction of a friction coefficient led to a considerable reduction in micromotion between implant and bone as well as to reduced displacement of all reference marks on the implant . overall , comparable values for micromotion were recorded at all corresponding reference marks ( figure 11 ) . simulating an osseointegrated implant in general reduced all displacement values by about 50% compared to the situation of a freshly inserted implant . again the introduction of a friction coefficient led to a considerable reduction in micromotion between implant and bone as well as to reduced displacement of all reference marks on the implant . in contrast to a freshly inserted implant , the distribution of micromotion depended on the location of the reference mark . whereas in the cervical aspect some minor micromotion in the range of 0.75 m could be found , at the most apical reference almost no relative displacement between implant and bone occurred ( figure 12 ) . within the limitations of this investigation , the effect of friction phenomena and implant design ( cylindrical versus threaded ) on stress distribution and implant displacement could be demonstrated . both the introduction of friction between implant and bone as well as the addition of threads to a cylindrically shaped implant resulted in the reduction of implant displacement under an axial load of 200 n. simultaneously , a more homogeneously distributed loading situation at the implant bone interface could be observed . changing the contact type between implant and bone to force fit resulted in load transfer predominantly occurring in the cervical part of the implant surrounded by stiffer cortical bone . this is in strict contrast to a situation with no friction modelled resulting in maximum loading of bone surrounding the periapical region of the implant . from a clinical perspective , these findings indicate that screw - shaped implants are advantageous while bone quality probably plays the most important role in achieving sufficient primary implant stability for immediate loading . based on a comparison of freshly inserted and osseointegrated implants it could be shown that the healing status affects the occurrence of micromotion phenomena along the implant bone interface . for a soft implant bone interface , reflecting early stages of osseointegration , micromotion remained on a constant level regardless of the location considered . simulating mature bone reflecting an osseointegrated implant , the introduction of a friction coefficient between implant and bone dramatically changed the distribution of micromotion along the implant bone interface . in addition to generally reduced levels of micromotion as compared to a freshly inserted implant , a decrease in micromotion was noted . it may be seen as a limitation of this study that only one specific value for axial loading of the implants was chosen . based on studies by brunski and coworkers , axial components of biting forces can range from 100 to 2400 n , while the exact values depend on factors such as location in the mouth and nature of food . for patients having implant - supported dentures , axial closure forces ranging from 45 to 255 furthermore , besides the pure mechanical aspects addressed in this paper , also biologic factors play an important role in the process of osseointegration of dental implants . following implant placement , the healing period starts with the adherence of serum proteins , followed by the attachment and proliferation of mesenchymal cells . bone remodeling occurs as an adaptation to the implants environment . with these processes occurring simultaneously to mechanical loading in an immediate loading situation , the interaction of both mechanical and biologic factors seems to be critical to the integration of the implant . given the nonuniform distribution of micromotion between implant and bone , it appears questionable whether currently available methods for experimentally determining this phenomenon provide meaningful data . the only valid approach for evaluating micromotion phenomena at the implant - bone interface appears to be finite element analysis . however , care has to be taken to set proper materials and interface characteristics as these parameters may greatly influence the outcome .

micromotion of dental implants may interfere with the process of osseointegration . using three different types of virtual biomechanical models , varying contact types between implant and bone were simulated , and implant deformation , bone deformation , and stress at the implant - bone interface were recorded under an axial load of 200 n , which reflects a common biting force . without friction between implant and bone , a symmetric loading situation of the bone with maximum loading and displacement at the apex of the implant was recorded . the addition of threads led to a decrease in loading and displacement at the apical part , but loading and displacement were also observed at the vertical walls of the implants . introducing friction between implant and bone decreased global displacement . in a force fit situation , load transfer predominantly occurred in the cervical area of the implant . for freshly inserted implants , micromotion was constant along the vertical walls of the implant , whereas , for osseointegrated implants , the distribution of micromotion depended on the location . in the cervical aspect some minor micromotion in the range of 0.75 m could be found , while at the most apical part almost no relative displacement between implant and bone occurred .

cyp4fs are a family of cytochrome p450 ( cyp ) enzymes that were first identified for their ability to catalyze end - chain hydroxylation and inactivation of leukotriene b4 ( ltb4 ) . the potency of ltb4 as an inflammatory mediator in normal immune responses and pathologies is well established . ltb4 is generated by the 5-lipoxygenase pathway of arachidonic acid metabolism and is implicated in the progression of diverse immune disorders such as inflammatory bowel disease , ischemia - reperfusion injury ( iri ) , arthritis , and asthma [ 2 , 3 ] . therefore , cyp4fs are predicted to play a significant role in the regulation of inflammation and prevention of disease . there is growing evidence to support this possibility . cyp - dependent ltb4 hydroxylase activity limits neuroinflammation in mouse models and might contribute to the beneficial effects of retinoids in the treatment of inflammatory skin diseases [ 5 , 6 ] . neutrophils and colonic mucosa from patients with inflammatory bowel disease have reduced ltb4 hydroxylase activity [ 7 , 8 ] , and genetic association studies link variants of the cyp4f2 and cyp4f3 genes with celiac disease and crohn 's disease [ 9 , 10 ] . human neutrophils have been used for detailed studies of cyp - dependent ltb4 metabolism : hydroxylation at the terminal ( ) position generates 20-hydroxy ltb4 , which is subsequently converted to the inactive metabolite 20-carboxy ltb4 . this is the major pathway for the inactivation of ltb4 in human neutrophils [ 12 , 13 ] . the enzyme responsible for the initial -hydroxylation step was identified as cyp4f3 [ 14 , 15 ] , and subsequently we demonstrated that this enzyme is an alternative splice form of the cyp4f3 gene designated as cyp4f3a . a second splice form , cyp4f3b , has lower activity for ltb4 and is expressed in different locations such as liver and kidney . the unusual localization and high expression of cyp4f3a in human neutrophils , and its high activity for ltb4 as a substrate , suggest that inactivation of ltb4 is a specialized function of the enzyme . there is evidence for temporal expression of cyp4fs consistent with the resolution phase of inflammation in some experimental models , but expression of cyp4f3a in neutrophils does not fit this time frame . neutrophils are short - lived cells associated with the early stages of inflammation , and cyp4f3a is expressed at a high constitutive level both before and during inflammatory recruitment of the cells . it is possible that ltb4 inactivation functions to restrain neutrophil infiltration and prevent excessive inflammation . an alternative possibility is that ltb4 inactivation plays a role in neutrophil polarization , which is required to maintain normal chemotaxis . we developed mouse models to better understand the role of cyp4fs in neutrophil - dependent inflammation . we identified the cyp4f18 enzyme as the mouse homologue of cyp4f3a and generated targeted deletions in the cyp4f18 gene . neutrophils from cyp4f18 knockout mice exhibit a null phenotype for end - chain hydroxylation of ltb4 . however , there are significant differences between mice and humans . the cyp4f18 gene is not alternatively spliced and generates a single enzyme that is homologous to cyp4f3a in sequence , localization to neutrophils , and high activity for ltb4 . the products of end - chain hydroxylation by cyp4f18 are 19-hydroxy ltb4 , and to a lesser extent 18-hydroxy ltb4 , not 20-hydroxy ltb4 [ 21 , 22 ] . it is not known whether -1 and -2 hydroxylation of ltb4 represents an efficient inactivation pathway . furthermore , mouse neutrophils have an alternative pathway of ltb4 metabolism that involves a 12-hydroxydehydrogenase . knockout of cyp4f18 does not impact neutrophil infiltration into kidney tissue and disease pathology in a mouse model of renal iri , although inhibition of ltb4 synthesis does have observable effects in this model . it appears that cyp4f18 is redundant for ltb4 inactivation in mouse neutrophils , and we speculated that it might have an alternative function in these cells . since the discovery of cyp4fs , numerous in vitro substrates have been identified . there are 7 members of the human cyp4f family including the two splice forms of cyp4f3 ( 4f2 , 4f3a , 4f3b , 4f8 , 4f11 , 4f12 , and 4f22 ) and 9 members of the mouse family ( 4f13 , 4f14 , 4f15 , 4f16 , 4f17 , 4f18 , 4f37 , 4f39 , and 4f40 ) . cyps typically have broad and overlapping substrate specificity , and a single enzyme such as cyp4f3b might have the capacity to catalyze multiple reactions including inactivation of ltb4 , generation of 20-hydroxyeicosatetraenoic acid ( 20-hete ) , and modification of fatty acid epoxides [ 1 , 24 ] . this suggests potentially diverse and prominent roles for cyp4fs in immune regulation but creates a challenge for the identification of physiologically relevant substrates . it is possible that cyp4fs have different functions in different tissue locations , and new experimental systems will be required to determine the significance of particular reactions and disentangle the effects of multiple cyp4fs . cyp4f18 is the only cyp4f family member expressed at high levels in mouse neutrophils , so cyp4f18 knockout mice provide a novel system to dissect diversity of function . in this report we demonstrate that neutrophils from cyp4f18 knockout mice show increased chemotaxis to complement component c5a that is independent of ltb4 , an unexpected result that is not predicted by known cyp4f substrates . cyp4f18 knockout ( / ) mice were generated in a c57bl/6 background as previously reported and are available at the mutant mouse regional resource center ( mmrrc ) with the designation b6.129s4(cg)-cyp4f18 . cyp4f18 + / heterozygous mice were maintained at the massachusetts general hospital and mated to generate cyp4f18 / homozygous knockouts and cyp4f18 + /+ wild - type littermates for experiments . breeding and experimentation of mice were performed in accordance with the guidelines of the massachusetts general hospital / partners committee on research animal care . mouse bone marrow cells were isolated from femurs and tibias by perfusion with phosphate buffered saline ( pbs ) . the cells were filtered through a 40 m cell strainer , washed in pbs , and layered on top of a discontinuous two - layer gradient of histopaque 1077/1119 ( sigma ) . after centrifugation at 700 g for 30 min at rt , neutrophils were separated from other cells including erythrocytes and recovered at the interface of the 1077 and 1119 fractions . purity of the isolated polymorphonuclear cells was confirmed ( > 90% ) by staining nuclei with dapi and examination under a nikon eclipse ti microscope with confocal imaging . bone marrow neutrophils were isolated as described above and resuspended in migration medium ( rpmi + 0.5% fbs ) to a concentration of 3 10 cells / ml . chemotaxis was performed using a 12-well plate with transwell 12 mm polycarbonate membrane inserts of 3.0 m pore size ( corning 3402 ) . the cells were placed in the insert ( 0.5 ml , 1.5 10 cells per insert ) , and 1.5 ml of migration medium containing different concentrations of chemoattractant was placed in the lower well . migration medium containing vehicle but no chemoattractant was used as a control to measure background chemotaxis , and chemoattractant was added to both the insert and the lower well as a control for chemokinesis . the 12-well plates were incubated for 3 hours at 37c in a 5% co2 incubator . the medium in the lower well was transferred to a 12 75 mm sterile culture tube ( bd falcon ) , and the well was washed with pbs . the tubes were then centrifuged for 5 min at 200 g , and the cell pellet was resuspended in 0.1 ml migration medium . the total number of cells was determined using a hemocytometer . to calculate the chemotactic index , the number of cells migrated in response to chemoattractant was divided by the number of spontaneously migrated cells ( background ) . the chemoattractants tested were ltb4 ( cayman ) , mouse complement component c5a ( r&d systems ) , mouse cxcl1/kc ( r&d systems ) , and wkymvdm peptide ( sigma ) as an agonist for the mouse formyl peptide receptor . in some experiments , bone marrow neutrophils were incubated with or without inhibitors of eicosanoid synthesis or blt1 for 30 min at 37c prior to adding the cells to the 12-well plate inserts . this included incubations with 0.5 m of the flap inhibitor mk 886 ( cayman 10133 ) , 1 m of a cpla2 inhibitor ( calbiochem 525143 , pubchem cid 9833099 ) , and 10 m of the blt1 antagonist ly223982 ( cayman 10010024 ) . each experimental condition was performed in duplicate or triplicate on a single 12-well plate , and each experiment was performed at least 4 times ( n 4 ) . bone marrow cell suspensions in pbs + 0.5% bsa were preincubated with mouse fc block ( bd biosciences ) for 5 min at 4c ( 0.5 g/10 cells/100 l ) and then incubated with fluorophore - conjugated anti - mouse monoclonal antibodies for 30 min at 4c ( antibodies diluted to 1 g / ml ) . anti - cd45-percp ( clone 30-f11 ) and anti - ly6g - fitc ( clone 1a8 ) were from bd biosciences . the cells were fixed in bd stabilizing fixative ( bd biosciences ) . labeled cells were analyzed at the flow cytometry core facility , massachusetts general hospital , using a bd sorp 7 laser lsrii and flowjo software , as in previous studies [ 19 , 22 ] . the cells were gated on forward versus side scatter , then for cd45 expression , prior to double plot analysis of ly6 g and c5ar ( numbers were assigned to each quadrant to indicate percentage of total cd45 + cells ) . bone marrow samples from 5 wild - type mice and 5 cyp4f18 knockout mice were analyzed . total rna was isolated from cells and tissues using the rneasy plus mini kit with qiashredder ( qiagen ) . reverse transcription was performed with a high capacity cdna reverse transcription kit ( life technologies , applied biosystems ) . the cdna was analyzed for target gene expression using taqman primer sets and a steponeplus real time pcr machine from applied biosystems . a standard reaction protocol was followed ( 50c for 2 min , 95c for 10 min , 40 cycles of 95c for 15 sec and 60c for 1 min ) . relative quantification of gene expression in knockout samples compared to wild - type was performed by the ct method using mouse gapdh as endogenous control . each sample was run in triplicate to determine ct values , 2 values , or fold - differences in expression ( 2 ) . the taqman primer - probe sets were purchased from life technologies ( applied biosystems ) as listed in table 1 . dss ( mp biomedicals ) of average molecular weight 42 kda ( 3550,000 ) was administered to 8-week - old mice ad libitum at a concentration of 4% in drinking water for 9 days . control mice received the same drinking water without dss ( n = 10 mice in each group ) . changes in body weight were calculated every day . a disease activity index ( dai ) was determined by assigning a score of 14 for weight loss ( 1 : 15% , 2 : 510% , 3 : 1015% , and 4 : > 15% ) , rectal bleeding ( ranging from 1 : positive to 4 : gross bleeding ) , stool consistency ( ranging from 1 : loose stools to 4 : diarrhea with fecal material adherent to anal fur ) , and body posture / lethargy ( ranging from 1 : mild to 4 : severe ) . the total scores were averaged to give a value between 0 ( normal ) and 4 ( maximum ) . mice were sacrificed on day 9 , and colonic tissue samples were collected for further analysis . myeloperoxidase ( mpo ) was measured using an elisa kit from hycult biotech ( hk210 ) . the assay was performed on tissue samples that had been frozen and stored at 70c . the samples were thawed , weighed , and homogenized in a lysis buffer containing 200 mm nacl , 5 mm edta , 10 mm tris , 10% glycerin , 1 mm pmsf , 1 g / ml leupeptin , and 28 g / ml aprotinin , ph 7.4 ( 20 l lysis buffer per mg tissue ) . the homogenate was transferred to a 1.5 ml microfuge tube and centrifuged two times at 1500 g for 15 min at 4c . a t - test was used for comparisons of paired data , and multigroup data were analyzed by anova . ltb4-dependent chemotaxis of neutrophils from wild - type and cyp4f18 knockout c57bl/6 mice was compared using an in vitro assay . a dose - dependent increase in chemotaxis was observed ( figure 1 ) . for wild - type ( wt ) neutrophils , the chemotactic index increased from 1.27 0.12 at 1 nm ltb4 to 2.73 0.7 at 10 nm ltb4 and 8.53 0.64 at 100 nm ltb4 . for cyp4f18 knockout ( ko ) neutrophils , the chemotactic index increased from 1.4 0.1 at 1 nm ltb4 to 3.3 0.77 at 10 nm ltb4 and 9.27 0.77 at 100 nm ltb4 . at each concentration , there was no significant difference between the chemotactic index for wild - type and cyp4f18 knockout neutrophils ( n = 10 , p > 0.05 ) . the complement component c5a was originally used as a control in these experiments , because c5a - dependent chemotaxis was not expected to be affected by loss of cyp4f18 . surprisingly , significant differences in chemotaxis were observed when comparing wild - type and knockout neutrophils ( figure 2 ) . for wild - type neutrophils , the chemotactic index increased from 1.7 0.46 at 1 ng / ml c5a to 4.79 0.48 at 10 ng / ml c5a and 9.2 0.79 at 100 ng / ml c5a . for cyp4f18 knockout ( ko ) neutrophils , the chemotactic index increased from 2.2 0.38 at 1 ng / ml c5a to 11.2 1.1 at 10 ng / ml c5a and 17.3 1.7 at 100 ng / ml c5a . there was a significant difference between the chemotactic index for wild - type and cyp4f18 knockout neutrophils at 10 ng / ml c5a ( p < 0.01 ) and 100 ng / ml c5a ( p < 0.05 ) , but not at 1 ng / ml c5a ( figure 2(a ) ) . overall , the relative difference in chemotactic index for knockout compared to wild - type was 2.3-fold at 10 ng / ml c5a and 1.9-fold at 100 ng / ml c5a ( figure 2(b ) ) . no significant differences between cyp4f18 knockout and wild - type neutrophil chemotaxis were observed when cxcl1/kc or wkymvdm peptide was used as chemoattractant ( data not shown ) . previous studies demonstrated that ltb4 is a signal relay molecule during neutrophil chemotaxis : ltb4 synthesis and secretion are induced by primary chemoattractants such as c5a , and this amplifies neutrophil migration [ 20 , 26 ] . the amplification is reduced by mk 886 , a flap inhibitor that is known to block 5-lipoxygenase activation and leukotriene synthesis . as expected , we observed a decrease in c5a - dependent chemotaxis in neutrophils treated with mk 886 ( figure 2(a ) ) , but the decrease was equivalent in wild - type and cyp4f18 knockout neutrophils ( 0.450.56-fold ) , such that the relative difference in chemotactic index remained unchanged : 2.21-fold higher in cyp4f18 knockout neutrophils compared to wild - type at 10 ng / ml c5a and 1.92-fold higher at 100 ng / ml c5a ( figure 2(b ) ) . similar results were obtained when neutrophils were treated with a cpla2 inhibitor : there was an equivalent decrease in c5a - dependent chemotaxis in wild - type and cyp4f18 knockout neutrophils of 0.30.37-fold ( figure 2(a ) ) , and the relative difference in chemotactic index remained unchanged : 2.3-fold higher in cyp4f18 knockout neutrophils at 10 ng / ml c5a and 1.75-fold higher at 100 ng / ml c5a ( figure 2(b ) ) . the cpla2 inhibitor is less specific than the flap inhibitor : it blocks eicosanoid production by preventing release of arachidonic acid from membrane phospholipids and therefore inhibits synthesis of a wide range of lipid mediators in addition to leukotrienes . overall , the data show that loss of cyp4f18 results in increased neutrophil chemotaxis to c5a and suggest that this is independent of ltb4 and other eicosanoids . ly223982 is a synthetic blt1 ( ltb4 receptor ) antagonist that has been used to study neutrophil function . we incubated neutrophils with ly223982 prior to chemotaxis assays using 10 ng / ml c5a . this resulted in a 1.7-fold increase in c5a - dependent chemotaxis in wild - type neutrophils and a 2-fold increase in cyp4f18 knockout neutrophils , compared to cells that were not treated with ly223982 ( figure 2(a ) ) . there is previous evidence for cross - desensitization between neutrophil chemoattractant receptors , and this might account for our data : less cross - desensitization of the c5a receptor following inhibition of blt1 could result in an increased chemotactic response to c5a . however this does not account for the differences between cyp4f18 knockout and wild - type neutrophils , because the relative difference in chemotactic index remained undiminished following treatment with ly223982 : 2.8-fold higher in cyp4f18 knockout neutrophils compared to wild - type ( figure 2(b ) ) . we have previously used multicolor flow cytometry analysis of cells from mouse bone marrow and other tissues to investigate protein expression in different cell lineages [ 19 , 22 ] . ly6 g is a component of the myeloid marker gr-1 ( ly6 g + ly6c ) that is preferentially expressed in neutrophils , and fluorophore - conjugated monoclonal antibodies to ly6 g are useful to identify neutrophils in flow cytometry studies . using this approach , mouse bone marrow cells were stained with anti - ly6g - fitc and anti - c5ar - apc , and analysis of double plots shows an equivalent level of c5a receptor ( c5ar ) expression in neutrophils from cyp4f18 knockout and wild - type mice ( figure 3(a ) ) . real time pcr analysis of isolated bone marrow neutrophils ( figure 3(b ) ) determined that there is no significant difference in mrna expression of c5ar or blt1 in cyp4f18 knockout neutrophils compared to wild - type ( n = 4 , p > 0.05 ) . a plot of 2 values shows that c5ar is expressed at approximately 10-fold higher levels than blt1 in the cells ( figure 3(b ) ) . the values for relative expression ( 2 ) of c5ar in knockout neutrophils compared to wild - type ranged from 0.92 to 1.05 . a real time pcr assay designed to detect exons 8 and 9 of cyp4f18 was used to confirm loss of expression of these exons in knockout mice , as previously described . we routinely performed real time pcr analysis of isolated bone marrow neutrophils to determine if changes in expression of other cyp genes might account for differences in the cyp4f18 knockout . we have previously shown that cyp4f18 is the predominant cyp4f subfamily member in bone marrow neutrophils and that cyp4f13 and cyp4f16 are detected at lower levels . in this study , we extended the analysis to include all cyp4 family members in mouse ( figure 3(c ) ) . importantly , there were no changes in cyp4 expression in knockout neutrophils that might compensate for the loss of cyp4f18 or lead to differences in activity of the cells . a mouse model of dss colitis was used to investigate the consequences of increased c5a - dependent chemotaxis in vivo ( figure 4 ) . inhibition of c5a activity has been shown to reduce disease pathology in this model in c57bl/6 mice . continuous administration of 4% dss in drinking water to 8-week - old mice resulted in a rapid decline in body weight from 98.7 1.35% of initial weight on day 6 to 78.5 3.8% of initial weight on day 9 in wild - type mice . there was a corresponding increase in disease activity index ( maximum = 4 ) from 1.3 0.29 on day 6 to 3.2 0.24 on day 9 . there were no significant differences in the values for loss of weight or increase in disease activity in cyp4f18 knockout mice ( n = 10 , p > 0.05 ) . mpo , an enzyme produced mainly by neutrophils , was measured in colonic tissue to quantify inflammatory cell infiltration ( figure 5 ) . on day 9 , the mpo level was 57 9.0 ng / mg tissue in wild - type mice and 55 7.1 ng / mg tissue in cyp4f18 knockout mice ( n = 5 , p > 0.05 ) . based on the similarity of these indicators , we did not proceed with further histological analysis of colon tissue . we previously generated cyp4f18 knockout mice , and predicted that the mice would exhibit altered ltb4-dependent inflammation based on the ability of the cyp4f18 enzyme to metabolize ltb4 in neutrophils . however , there were no significant differences in inflammation and injury in a mouse model of renal iri compared to wild - type , although inhibition of ltb4 synthesis does ameliorate pathology in this model . the results of an in vitro chemotaxis assay are consistent with these previous observations in vivo . ltb4 stimulated chemotaxis of neutrophils in a dose - dependent manner , but there were no significant differences in the response of cyp4f18 knockout and wild - type cells ( figure 1 ) . in the renal iri model , we measured infiltration of neutrophils into kidney tissue by traditional histological approaches and flow cytometry and demonstrated a comparable time course and magnitude of infiltration in cyp4f18 knockout and wild - type mice . however , the loss of cyp4f18 might be compensated by other cyp4fs in the complex tissue physiology of inflammation . the in vitro assay reported here confirms that the similarity between wild - type and knockout is inherent to neutrophils . there are a number of possible explanations for the unaltered chemotactic response to ltb4 in cyp4f18 knockout neutrophils . cyp4f18 converts ltb4 to 19-hydroxy ltb4 , and to a lesser extent 18-hydroxy ltb4 , in mouse neutrophils , not 20-hydroxy ltb4 as seen in humans . these products were not detected in cyp4f18 knockout neutrophils , but it is not known whether -1 and -2 hydroxylation of ltb4 represents an efficient inactivation pathway . it is sometimes difficult to distinguish the , -1 , and -2 metabolites of cyp hydroxylases , but this can have physiological and pharmacological importance . for example , 19-hete is produced by a number of cyps and is an antagonist of 20-hete . we have provided experimental details for the identification of 18- and 19-hydroxy ltb4 [ 21 , 22 ] , but further studies are needed to clarify the roles of these metabolites . another possibility is that cyp4f18 is redundant for ltb4 metabolism , because mouse neutrophils have an alternative pathway for ltb4 inactivation involving a 12-hydroxydehydrogenase that is not affected in cyp4f18 knockouts . we speculated that cyp4f18 might have an alternative function in mouse neutrophils and that its homologue evolved to be the dominant ltb4-metabolizing enzyme in humans coincident with its ability to generate 20-hydroxy ltb4 . unexpectedly , a difference in response to complement component c5a was identified using the in vitro chemotaxis assay . compared to wild - type neutrophils , cyp4f18 knockout neutrophils show an increase in chemotaxis of 2.3-fold at 10 ng / ml c5a and 1.9-fold at 100 ng / ml c5a ( figure 2 ) . primary chemoattractants such as c5a stimulate ltb4 synthesis and secretion , which amplifies neutrophil chemotaxis [ 20 , 26 ] . therefore , it is possible that increased c5a - dependent chemotaxis in cyp4f18 knockout neutrophils might be caused by increased levels of ltb4 or other eicosanoids arising from endogenous synthesis . we used two different inhibitors to investigate this possibility : a flap inhibitor ( mk 886 ) that blocks activation of 5-lipoxygenase and a cpla2 inhibitor that blocks eicosanoid production by preventing release of arachidonic acid from membrane phospholipids . these inhibitors reduced c5a - dependent chemotaxis to an equivalent degree in both wild - type and cyp4f18 knockout neutrophils , such that the relative difference in knockout cells compared to wild - type remained unchanged ( figure 2 ) . a blt1 antagonist increased c5a - dependent chemotaxis in both wild - type and cyp4f18 knockout neutrophils ( figure 2 ) , possibly by blocking cross - desensitization between the chemoattractant receptors . the relative difference in chemotaxis was undiminished following treatment with the blt1 antagonist : 2.8-fold higher in cyp4f18 knockout neutrophils compared to wild - type ( figure 2 ) . overall , the data suggest that the increase in c5a - dependent chemotaxis in knockout neutrophils is unrelated to ltb4 , other eicosanoids , or receptor cross - desensitization . an alternative possibility is that cyp4f18 metabolizes a novel lipid substrate involved in the downstream signaling pathways and relay systems that mediate c5a activity ( c5a is a polypeptide and therefore not a cyp substrate ) . cyps typically have broad and overlapping substrate specificity , and redundancy of function is a consistent problem with cyp knockout studies . it is often difficult to observe a phenotype for deletion of one cyp in vivo , because of the effects of multiple related cyps in complex tissue environments . there are advantages to studying cyp4f18 knockout neutrophils in vitro , because cyp4f18 is the only cyp4 transcript expressed at high levels in these cells ( figure 3 ) . another problem with cyp knockout studies is that deletion of one cyp sometimes leads to up- or downregulation of a different cyp that generates an observed phenotype . for example , knockout of cyp4a14 led to male - specific hypertension that was caused by upregulation of cyp4a12 . we compared expression of all members of the cyp4 family in wild - type and cyp4f18 knockout neutrophils and detected no differences other than loss of cyp4f18 in the knockout ( figure 3 ) . we used a mouse model of dss colitis for a preliminary investigation of the consequences of altered c5a chemotaxis in vivo . a c5a receptor antagonist ameliorates pathology in this model , and genetic association studies link variants of the cyp4f2 and cyp4f3 genes with celiac disease in humans . mice were treated continuously with 4% dss for 9 days , but the profile of weight loss , disease activity , and colonic tissue mpo was not significantly different in wild - type and cyp4f18 knockout mice ( figures 4 and 5 ) . selection and design of future in vivo strategies will benefit from more in vitro information about cyp4f18 knockout neutrophils . a comparison of lipid metabolism in wild - type and knockout neutrophils will help to determine if novel cyp4f18 substrates are relevant to c5a chemotaxis and might point to other activities affected by loss of cyp4f18 . cyp4f enzymes have the ability to catalyze oxidation of a diverse range of lipid substrates related to inflammation . a significant challenge is to identify physiologically relevant substrates among multiple possibilities and to assign functions to individual cyps . knockout studies are problematic because of the ability of related cyps to compensate for function or to change expression and lead to phenotypes that are unrelated to the deleted cyp . neutrophils provide a useful tool for our studies of cyp4f18 knockout mice : cyp4f18 is the only cyp4 enzyme expressed at high levels in these cells , and other cyp4 enzymes do not change expression in cyp4f18 knockout neutrophils . in this report we demonstrate that there is no difference in ltb4-dependent chemotaxis of mouse neutrophils that lack cyp4f18 , despite the high activity of cyp4f18 for ltb4 as a substrate . this is significant , because many studies assume that cyp4fs regulate ltb4 function based on known activity as ltb4 hydroxylases . we identified an unexpected role for cyp4f18 in regulating c5a - dependent neutrophil chemotaxis , and this was independent of ltb4 . further studies of cyp4f18 knockout neutrophils in vitro will inform the design of in vivo strategies to investigate immune regulation .

cyp4fs were first identified as enzymes that catalyze hydroxylation of leukotriene b4 ( ltb4 ) . cyp4f18 has an unusual expression in neutrophils and was predicted to play a role in regulating ltb4-dependent inflammation . we compared chemotaxis of wild - type and cyp4f18 knockout neutrophils using an in vitro assay . there was no significant difference in the chemotactic response to ltb4 , but the response to complement component c5a increased 1.92.25-fold in knockout cells compared to wild - type ( p < 0.01 ) . this increase was still observed when neutrophils were treated with inhibitors of eicosanoid synthesis . there were no changes in expression of other cyp4 enzymes in knockout neutrophils that might compensate for loss of cyp4f18 or lead to differences in activity . a mouse model of dextran sodium sulfate colitis was used to investigate the consequences of increased c5a - dependent chemotaxis in vivo , but there was no significant difference in weight loss , disease activity , or colonic tissue myeloperoxidase between wild - type and cyp4f18 knockout mice . this study demonstrates the limitations of inferring cyp4f function based on an ability to use ltb4 as a substrate , points to expanding roles for cyp4f enzymes in immune regulation , and underscores the in vivo challenges of cyp knockout studies .

oral rehabilitation with dental implants requires sufficient vertical and horizontal dimensions of the residual alveolar ridge in order to accommodate and fully embed the endosteal implants in alveolar bone [ 1 , 2 ] . however , the dynamic hard tissue remodelling that occurs following tooth extraction typically results in significant resorption of the residual alveolar ridge , and the rate of this progressive postextraction bone loss has been shown to be fastest during the first three months [ 3 , 4 ] . since the loss of bone height and original ridge contour can lead to complications in implant placement , reducing the dimensional ridge alterations plays an important role in achieving mechanically stable and aesthetically pleasing clinical outcome in implant - retained prosthetic rehabilitation . several clinical and preclinical studies have described the morphological changes occurring in the alveolar process in both apical - coronal and buccal - lingual directions following extraction of teeth [ 2 , 3 , 5 , 6 ] . the healing sequence of an alveolar socket begins with the formation of a blood coagulum within the alveolus , which subsequently formed into a temporary matrix before osteoblastic reorganisation of the temporary matrix to woven bone . simultaneously , the socket walls undergo bone remodelling and osteoclastic resorption , and it has been shown that particularly the buccal aspect of the residual ridge is susceptible to reduction in bone volume . it has been suggested that immediate implant placement in fresh extraction sockets can help maintain the original ridge shape , but remodelling in the later stages of the healing sequence still results in marked reduction in the buccal bone volume as has been shown in experimental studies by arajo et al . this finding is in good agreement with recent clinical studies reporting that immediate implant placement fails to prevent the lateral resorption of the alveolar ridge following tooth loss [ 11 , 12 ] . an alternative approach in the attempt of dimensional ridge preservation has been the grafting of the fresh alveoli using porous bone graft substitutes , which ideally both stimulate the healing of the alveolar socket and inhibit the residual ridge resorption . several experimental and clinical studies have reported promising results in respect of dimensional ridge preservation for various synthetic and animal - derived bone graft substitutes [ 1418 ] . bioresorbable materials in granulated form have typically been employed as grafting material in extractions sockets where subsequent implant placement is expected . however , because of the low resorption rates of many currently used bone graft substitutes [ 19 , 20 ] , nonresorbable materials may also be considered a reasonable alternative for alveolar ridge preservation , particularly when highly porous materials are used as bone graft substitutes . due to its excellent biocompatibility and favourable osteogenic properties , ceramic titanium dioxide ( tio2 ) has been proposed as a promising material for nonresorbable synthetic bone grafts , or scaffolds , in the restoration of large bone defects [ 21 , 22 ] , and highly porous tio2 scaffolds are considered to be suitable for bone augmentation in dental applications . since the high surface - to - volume ratio and large accessible pore volume provide sufficient space for cell attachment , cell proliferation , and nutrient and metabolite transportation , the highly porous tio2 scaffold structure allows excellent conditions for initial osteoblast attachment and proliferation of human mesenchymal stem cells ( hmsc ) in vitro [ 23 , 24 ] . furthermore , the well - interconnected pore structure of the tio2 scaffolds has been shown to allow unobstructed bone ingrowth when placed in fresh , surgically modified extraction sockets in minipig mandibles , which together with the good osteoconductivity of the scaffold material resulted in good mechanical interlocking of the scaffold structure and the newly formed bone tissue within the alveoli . because of this mechanical stability of the scaffold structure in the alveoli , the porous tio2 scaffolds may also have the potential to inhibit the residual ridge resorption . hence , the aim of the present animal study was to assess the hard tissue remodelling in the alveolar ridge following tooth extraction in order to establish the effect of the highly porous tio2 scaffold structure on dimensional ridge preservation in minipig mandibles . ceramic tio2 scaffolds were fabricated by replication process . tio2 slurry was prepared by dispersing 65 g of tio2 powder ( kronos 1171 , kronos titan gmbh , leverkusen , germany ) in 25 ml sterilised h2o , and the ph of the dispersion was kept at 1.5 for the entire duration of the stirring with small additions of 1 m hcl . cylindrical polyurethane foam templates , 5 mm in diameter and 12.5 mm in height ( 60 ppi , bulbren s , eurofoam gmbh , wiesbaden , germany ) , were coated with the prepared slurry . prior to sintering at 1500c for 40 h , the polymer sponge was carefully burnt out of the green body at a lower temperature . after sintering , the scaffolds were recoated with slurry that was prepared using the previously described procedure , only this time mixing 40 g of tio2 powder with 25 ml of water . the recoated scaffolds were then sintered in 1500c for 24 h. for more details , see . the final dimensions of the scaffolds were 4 mm in diameter and 10 mm in height due to the shrinkage during the sintering phase . six female gttingen minipigs ( sus scrofa , ellegaard a / s , dalmose , denmark ) aged 18 to 24 months and weighing 3242 kg were acclimatised in the local animal facilities ( malm general hospital ( mas ) , malm , sweden ) for two weeks before the surgical procedure . preparation of animals as well as animal management and care followed routine protocols approved by the institutional review board of mas . ethical approval for the experiment had been obtained from the institutional review board at mas animal experimental ethical committee malm - lund m130 - 06 . the animals were maintained under general anaesthesia according to a standard procedure using ketalar 50 mg / ml ( pfizer , sollentuna , sweden ) and midazolam - hameln 5 mg / ml ( hameln pharma plus gmbh , hameln , germany ) . after visual inspection of the animals , the pigs were shaved around the mouth , and the skin was then rinsed with chlorhexidine solution ( chlorhexidine 5 mg / ml in 60% etoh , apoteksbolaget , stockholm , sweden ) . buccal and lingual incisions were made in the posterior premolar region in both quadrants of the mandibles . two premolars ( p3 and p4 ) were hemisected on both sides using fissure burs , and the distal roots were carefully removed using elevators and forceps . the extraction sockets were surgically treated to accommodate the cylindrical tio2 scaffold using burs 4 mm in diameter resulting in standardised cylindrical defects 10 mm deep and 4 mm in diameter . the resulting defects were either grafted with a porous tio2 scaffold or left untreated ( sham ) in alternate pattern so that each mandible contained equal number of scaffold and sham sites ( figures 1 and 2 , n = 12 ) . the surgical sites were closed with vicryl 4.0 resorbable sutures ( polyglactin 910 , ethicon inc . , somerville , nj , usa ) . particular care was taken to ensure that the flap completely covered the extraction sites and that passive stability of the flap had been obtained . antibiotic ( streptocillin vet 250 mg / ml + 200 mg / ml boehringer ingelheim gmbh , ingelheim , germany ) was administered for seven days after surgery , and 1 ml/10 kg analgesics ( temgesic 0.3 mg / ml schering - plough , brussels , belgium ) was administered once after surgery . six weeks after surgery , a lethal injection of 40 ml pentobarbital natrium 100 mg / ml in spiritus fortis 290 mg / ml was given intracardially to the animals . after 48 hours of fixation in formaldehyde , the biopsies were transferred to 70% alcohol , and the bone segments were later sequentially embedded in light - curing epoxy ( technovit 7200 vlc , heraeus kulzer gmbh , wehrheim , germany ) . were analysed using micro - ct imaging ( skyscan 1172 high - resolution micro - ct , skyscan n.v . , all samples were scanned at 17.1 m voxel resolution using a source voltage of 100 kv and a current of 100 a with 0.5 mm aluminium filter to optimise contrast . samples were rotated 360 around their long axes , and four absorption images were recorded every 0.4 of rotation . these raw images were then reconstructed with the standard skyscan reconstruction software ( nrecon ) to serial coronal - oriented tomograms using 3d cone beam reconstruction algorithm . for reconstruction , beam hardening the image analysis of the reconstructed axial bitmap images was performed using standard skyscan software ( ctan and ctvox ) . reconstructed cross - sectional images were recorded from the central area of each remaining root as well as their corresponding alveolus in both buccal - lingual and mesial - distal plane . three buccal - lingual micro - ct sections from each root and alveolus were recorded , and the following vertical and lateral aspects of the root / defect sites were evaluated : buccal and lingual total bone height ( bbh , lbh ) measured from the top of the buccal and lingual alveolar wall to the most apical point of the jaw ( figure 3(a ) ) , buccal and lingual alveolar wall bone width ( a , b , c ) , and total bone width ( a , b , c ) measured perpendicular to the long axis of the root / defect 1 , 3 , and 5 mm underneath the top of the alveolar crest ( figure 3(b ) ) . in addition , the vertical height difference between the most coronal positions of the buccal and lingual bone crest ( b - l ) was measured ( figure 3(b ) ) . each measurement for sites containing the remaining mesial root was subtracted from the equivalent measurement on the corresponding distal extraction site . the shape and dimensions of the alveolar process at the mesial and distal roots the vertical bone loss at the alveolar bone crest was measured in comparison with the original bone level marked by the top of the remaining mesial root ( figure 3(c ) ) . one mandible segment containing one scaffold and one sham site was excluded from the micro - ct analysis due to damage during sample preparation . histological sections were prepared according to the cutting - grinding technique described by donath and breuner and rohrer and schubert [ 26 , 27 ] . approximately 100 m thick sections were stained with haematoxylin and eosin for light microscopy and digital imaging . the bone tissue adjacent to the defect area ( scaffold and sham ) was examined using leitz dmrbe microscope ( leica , wetzlar , germany ) equipped with cell imaging system ( olympus soft imaging solutions gmbh , mnster , germany ) . all sections were evaluated qualitatively for the appearance of the alveolar ridge adjacent to the scaffold and sham sites as well as inflammation and/or immunological reactions in the extraction sockets . the normality and equal variance of the datasets were tested prior to further statistical analysis . when datasets were found normally distributed , statistical analyses between the datasets were performed using student 's t - test , while the datasets that failed the normality or equal variance test were analysed using nonparametric mann - whitney u test . all statistical analyses were performed using sigmastat software package ( sigmastat v. 3.5 , systat inc . , st . all defect sites demonstrated uneventful healing without clinical signs of inflammation at the time of harvest . after six weeks of healing , the morphological appearance of the scaffold , and sham sites did not differ markedly . both defect sites were sealed by newly formed hard tissue consisting of dense woven bone giving a dome - shaped outline for the bone crest of the defect sites in the buccal - lingual sections . typically , no distinct border that separated the newly formed bone from the old cortical bone of the buccal and lingual walls could be identified in the histological sections . for the majority of the scaffold sites , a bony bridge with the average height of 1.23 0.35 mm had formed above the scaffold material ( figures 2 and 4 ) as measured from the central mesial - distal cross - sections . in few isolated instances ( 2/11 ) which both were in the same individual , there was no bony bridge covering the entire scaffold and a small portion of the scaffold was exposed above the newly formed bone ( figure 4(e ) ) . the porous scaffold structures were well integrated in the newly formed bone tissue in the alveolar bone , and the tio2 scaffolds were not found to interfere with the normal healing sequence of the extraction socket ( figures 2 and 4 ) . both scaffold and sham sites showed a large bone volume within the alveoli , and only a small part of the alveolar ridge was occupied by loose connective tissue . the medians and interquartile ranges of the difference in buccal and lingual bone height ( bbh and lbh ) and height difference of buccal and lingual bone crest ( b - l ) in comparison to equivalent measurement on the corresponding mesial root of each defect site ( h = defect root ) as well as the vertical bone loss are shown in figure 5 . the difference in bbh was noticeably smaller for the scaffold group although no statistically significant difference was found between the scaffolds and shams . the results of the horizontal morphological measurements are presented in figure 6 as medians and interquartile ranges of the difference in each parameter in comparison to the equivalent measurement on the corresponding mesial root of each defect site ( w = defect root ) . no morphometric aspect apart from b ( buccal ) revealed statistically significant difference between the scaffold and sham sites ( p > 0.05 ) . both vertical and horizontal parameters were also compared without subtracting the equivalent measurement of the corresponding mesial root ( tables 1 and 2 ) . apart from the buccal bone height , no statistically significant difference in any of the measured parameters was detected ( p > 0.05 ) although the power of the performed comparisons was below the desired power of 80% for all aspects . this is the first study to investigate the effect of the highly porous tio2 scaffold construct on the dimensional ridge alterations occurring following tooth extraction . the highly porous tio2 scaffolds were well integrated in the alveolar bone with a dense bony bridge covering the entire scaffold at the most coronal point of the ridge ( figures 2 and 4 ) six weeks following their implantation in extraction sockets in the premolar region of minipig mandibles . the excellent osteoconductive capacity of tio2 scaffolds has previously been reported by the present authors . it was shown that the scaffold structure permits excellent bone tissue penetration to the entire pore volume when placed in fresh , surgically modified extraction sockets , and no significant delay in the healing of the socket was observed in comparison to empty sockets during the six - week healing period . the bone mineral density was also found higher for sockets containing tio2 scaffolds in comparison to the empty control sockets , thus indicating improved bone matrix mineralisation in the presence of the tio2 scaffold in mandibular extraction sockets . furthermore , it is likely that the porous tio2 scaffold can advance the closure of the marginal entrance of the alveolus with newly formed bone tissue , as indicated by the large volume of bone tissue observed above the scaffold structure for great majority of the grafted alveoli ( figure 4(a ) ) . early closure of the socket is desired in order to seal the defect site from soft tissue proliferation and invagination into the extraction socket as the ingrowth of endothelial tissue may result in reduced bone fill within the alveolus . for this purpose , bone grafts and bone graft substitutes are sometimes used in combination with a guided tissue regeneration ( gtr ) membrane in order to isolate the alveolus from the soft tissue , and thereby improve the maintenance of the ridge dimension . since the scaffold was completely embedded in newly formed bone tissue and enclosed by the marginal bone at great majority of the scaffold sites , there was no surface invagination of marginal bone , and the convex shape of the alveolar crest was preserved also in the absence of gtr membrane . however , it appears that the tio2 scaffold does not have the capacity to fully prevent the loss of bone height at the defect site in the present animal model . when the vertical bone loss was measured from the central mesial - distal sections , no marked difference in the lost bone height was observed between the grafted and nongrafted sites ( figure 5(b ) ) . this may be related to the cylindrical dimensions of the experimental tio2 scaffolds . while particulate and pastelike bone grafts can easily be made to fill the entire contour of the alveolus , with preformed rigid scaffold constructs ; this can only be achieved if the alveolus can be shaped to fit the scaffold dimensions or vice versa . since the scaffold structure had a predefined shape and dimensions that did not mimic the contours of the extraction alveoli , the tio2 scaffold did not always fill the entire defect volume close to the marginal entrance of the socket . if the graft material does not reach the original bone level or leaves a gap between the scaffold and alveolar walls , it can not provide sufficient mechanical support to the adjacent bone walls at the most marginal regions of the alveoli , which is likely to result in loss of bone height at the alveolar ridge as seen in the present study . various studies have suggested that bone graft substitutes can suppress the residual ridge resorption , while others have questioned the efficacy of bone graft substitutes in preserving the original contour of the alveolar ridge . several experimental studies in dogs have reported a favourable outcome in regard to dimensional ridge preservation when fresh extraction sockets were grafted with various bone graft substitute materials [ 17 , 2931 ] . the effect is commonly attributed to the capacity of the grafting material to provide a scaffold for new bone formation and to offer stability for the blood coagulum that forms in the initial stages of the healing process . it appears that good osteoconductive capacity alone can not serve to preserve the dimensions of the edentulous alveolar ridge , as grafting the sockets with highly osteogenic but fast resorbing autologous bone chips was not shown to prevent the ridge resorption following tooth extraction . moreover , other studies with similar experimental setups have concluded that various bone graft substitute materials fail to preserve the alveolar ridge height [ 3335 ] . reported that incorporation of deproteinised bovine bone xenografts ( dbbx ) into fresh extraction sockets has only a limited impact on the resorption of the buccal plate . similarly , a recent study by bashara et al . showed no statistically significant difference in the residual ridge resorption of empty extraction sockets and sockets grafted with either dbbx or porous titanium granules both with and without an additional gtr membrane after 6-month healing period . while the excellent osteoconductive capacity of the pore network of the tio2 scaffolds can provide a favourable environment for bone regeneration within the extraction socket , the mechanical stability of the tio2 scaffold block is expected to give good initial structural stability to the adjacent socket walls during the early phases of the healing sequence . however , it appears that the scaffold did not fill the entire defect volume as already mentioned above , and there was no conclusive evidence for the tio2 scaffold to prevent the reduction in the alveolar ridge dimensions . a statistical significance was observed in the buccal bone heights between scaffold and sham sites , while a more prominent reduction in buccal bone height in comparison to corresponding distal alveoli was also evident for the nongrafted extraction sockets as illustrated in figure 5(a ) . however , no significant difference in the lateral resorption was apparent between the grafted and no - grafted sockets apart from the statistical significance in the buccal total bone width 3 mm apical from the alveolar crest ( figure 6 ) . this difference was not found statistically significant when the actual horizontal bone widths at this level were compared , but all horizontal aspects at 3 and 5 mm apical from both buccal and lingual crest were found noticeably larger for the nongrafted group ( table 2 ) . this was also observed in figure 6 depicting the difference in bone width , particularly for the lingual bone wall . as the horizontal measurements were performed in respect to the buccal and lingual bone crests , the measured lateral dimensions are strongly affected by the vertical resorption with increasing vertical bone loss resulting in larger measured bone width values . therefore , the larger apparent increase in the alveolar bone width in the sham group can be seen as an indication of the resorption of alveolar crest and loss of ridge height . still , the lack of marked difference in the lingual height difference for scaffold and sham sites ( figure 5(a ) ) seems to be in disagreement with this notion . nevertheless , the observed tendency towards more pronounced vertical ridge resorption in the buccal bone wall of the nongrafted alveoli may indicate that the tio2 scaffold can suppress the postextraction resorption of the buccal bone wall . in addition , it must be noted that the dimensional ridge alterations were recorded after a relatively short healing period of six weeks , even if the bone resorption has been shown to occur at a fast rate during the first three months . longer observation periods may result in more discernible difference in the ridge of the grafted and nongrafted sites , and therefore further experimental studies with multiple healing periods are recommended to fully characterise the effect of the tio2 scaffolds in preserving the alveolar ridge dimensions . inaccuracies and technical complications in the evaluation of the dimensional ridge alterations may also have influenced the outcome of the comparisons between grafted and nongrafted sites . in the present study , all dimensional aspects of the alveolar ridge were measured from the cross - sectional micro - ct images rather than the histological sections as the nondestructive digital sectioning allowed more precise alignment of the cross - sectional images to the centre of each defect than what could be achieved by the cutting and grinding method used for the production of histological sections . for scaffold sites , the radiopaque tio2 structure clearly defined the lateral boundaries of the socket , and thus , the lateral endpoints of the measurements were easily distinguished . the same applies for root sites in which the remaining roots distinctly defined the boundaries of the alveolar socket . however , this was not the case for the sham sites as the boundaries of the original alveolus were not always clearly visible in either micro - ct cross - sections or histological slide as can be seen in figure 4 . therefore , defining the end - points for the horizontal measurements was not always straightforward for the nongrafted extraction sockets , and may have resulted in slightly erroneous width measurements . in addition , the measurements applied here are further restricted by the two - dimensional nature of the measurements which may partially omit the changes in the true three - dimensional ridge contour . moreover , the comparison of the different experimental animal studies performed to evaluate the effect of a given bone graft material in preserving the original ridge dimensions following tooth extraction is somewhat restricted due to the variation in the measured parameters and measurement techniques used in each study . using the distal alveolus with the remaining root as a reference for each experimental site , as described by arajo et al . , allows fairly good evaluation of the amount of ridge contraction in comparison to the original ridge dimensions . however , this method relies on the presumption that the mesial and distal alveoli , as well as the adjacent bone walls , have the same shape . in addition , it must be assumed that only minimal resorption occurs at alveolar walls adjacent to the root sites . to adjust to these assumptions , the results of dimensional measurements of the sham and scaffold sites were also compared to each other without the subtraction of the ridge dimensions measured for the mesial root sites . these results were mostly in line with the results obtained by calculating the dimensional differences , thus reinforcing the conclusions of the present study . however , it is important to keep these limitations in mind when interpreting the results of this study . the tio2 scaffolds were completely embedded in newly formed bone tissue , and the convex shape of the alveolar crest was preserved . however , it appears that the tio2 scaffold does not have the capacity to fully prevent the loss of alveolar bone height following tooth extraction in the present animal model . nevertheless , the observed tendency towards more pronounced vertical ridge resorption particularly in the buccal bone wall of the nongrafted alveoli indicates that the tio2 scaffold may suppress the postextraction resorption of the buccal bone wall . due to the short healing period of the present study , further experimental studies with longer healing periods are recommended to fully characterise the effect of the tio2 scaffolds in preserving the alveolar ridge dimensions .

despite being considered noncritical size defects , extraction sockets often require the use of bone grafts or bone graft substitutes in order to facilitate a stable implant site with an aesthetically pleasing mucosal architecture and prosthetic reconstruction . in the present study , the effect of novel tio2 scaffolds on dimensional ridge preservation was evaluated following their placement into surgically modified extraction sockets in the premolar region of minipig mandibles . after six weeks of healing , the scaffolds were wellintegrated in the alveolar bone , and the convex shape of the alveolar crest was preserved . the scaffolds were found to partially preserve the dimensions of the native buccal and lingual bone walls adjacent to the defect site . a tendency towards more pronounced vertical ridge resorption , particularly in the buccal bone wall of the nongrafted alveoli , indicates that the tio2 scaffold may be used for suppressing the loss of bone that normally follows tooth extraction .

pellagra is a nutritional disorder caused by niacin ( vitamin b3 ) deficiency , which leads to systemic disease with clinical manifestations from the skin , gastrointestinal tract and the nervous system . niacin forms into nicotinic acid and nicotinamide , which serve as precursors of two important coenzymes of cellular metabolism , namely nicotinamide adenine dinucleotide ( nad ) and nad - phosphate . the term pellagra - from pelle agra , italian for rough skin - was first used by frappoli in 1771 due to its dermatologic manifestations . however , since goldberger implicated vitamin b deficiency as the cause of pellagra in 1926 , the pellagra syndrome has historically been characterized by the 4 d s : dermatitis , diarrhea , and dementia leading to death . pellagra was a major widespread cause of death until the early 20 century , but fortification of flour with niacin has led practically to its eradication in developed countries . a 50-year old man was brought to the emergency department due to disturbed mental status . the patient was reported to be homeless , but no other information about his medical status or living conditions was available . the patient was unreliable in providing history of alcohol abuse or describing symptoms like fever in his recent past . clinical examination revealed normal vital signs , cachexia and dehydration , accompanied by extensive pigmentation and scaling eruptions on a red basis along the dorsa of his hands and the sun - exposed surface of his arms ( figure 1 ) . the patient was confused and disorientated but responded sufficiently to simple commands . laboratory investigation showed mild elevation of total white blood cell count , slightly elevated creatinine and low serum albumin . the remaining of the routine laboratory tests , including glucose , electrolytes and aminotransferases , were found within normal range . clinical follow - up did not reveal fever or other symptoms or signs indicative of an underlying infection . the patient was subjected to computed tomography imaging which excluded any brain or other pathology . following neurological assessment and the negative imaging studies , furthermore , as the characteristic skin lesions in an obviously malnourished homeless man were compatible with pellagra , pellagrous encephalopathy was the preferable diagnosis . although serologic and urinary assays confirming niacin deficiency were unavailable in our hospital , treatment with nicotinamide 300 mg orally in a daily basis was started . eighteen days later the patient died due to hospital - acquired pneumonia , without showing any remarkable improvement in his mental status . patients with pellagra have a brown discoloration of the skin , especially in sun - exposed areas . in advanced stages , the characteristic skin rash has a typical photosensitive distribution with welldefined borders , and is mostly observed on the face , the neck - forming the so - called casal s necklace - , the dorsa of the hands and the extensor surface of the forearms . in addition to skin changes , gastrointestinal involvement may lead to intractable diarrhea , stomatitis and glossitis , while the neurocognitive impairment - pellagrous encephalopathy - may present as apathy , memory loss , disorientation , depression or delirium . niacin deficiency should especially be suspected in the following conditions : i ) malnutrition ( homelessness , anorexia nervosa or severe comorbid conditions like end - stage malignancy or hiv ) ; ii ) malabsorption ( e.g. crohn s or hartnup disease ) ; iii ) chronic alcoholism ; iv ) hemodialysis or peritoneal dialysis ; v ) drugs like isoniazid , ethionamide , 6-mercaptopurine and estrogens ; vi ) carcinoid syndrome ( due to excess turnover of tryptophan , precursor of niacin , to serotonin ) . the characteristic - if not pathognomonic - skin lesions support its diagnosis on solely clinical grounds . clinicians should keep a high index of suspicion , in certain patient groups , in order to early diagnose and cure this potentially fatal but treatable disease .

we report a case of a 50-year old homeless male who presented with pellagra and pellagrous encephalopathy . the characteristic - if not pathognomonic - skin lesions of pellagra support its diagnosis on solely clinical grounds . clinicians should keep a high index of suspicion , in certain patient groups , in order to early diagnose and cure this potentially fatal but treatable disease .

the cardia study , sponsored by the national heart , lung , and blood institute ( nhlbi ) , is a multicenter longitudinal study consisting of 5115 black and white women and men aged 18 to 30 years at the baseline examination ( y0 ) in 19851986 who were free of cvd . seven follow - up examinations took place at years 2 , 5 , 7 , 10 , 15 , 20 ( y20 ) , and 25 ( y25 ) . participants in the cardia cohort were excluded from the analysis if they did not attend the y25 examination ( n=1617 ) , were missing data on all y25 outcome measures ( n=3 ) , were missing y25 bp measurements ( n=6 ) , were missing data on y25 covariates ( n=231 ) , or developed cvd prior to y25 ( n=94 ) . an additional person was excluded from analyses involving left ventricular ( lv ) mass due to an unlikely , large lv mass ( 502 g ) . participants were permitted to be missing data on some but not all y25 outcomes , thus the sample sizes for different outcomes may be slightly < 3164 participants . for participants who attended the baseline and y25 examinations and at least 4 of 6 intermediate examinations , 25-year cumulative sbp was computed . in the analyses relating 25-year cumulative sbp to lv mass index ( lvmi ) in untreated cardia participants ( n=2362 ) , 368 were excluded due to missing lvmi ( n=214 ) or missing 25-year cumulative sbp ( n=154 ) or had cumulative sbp outside of the range 2200 to 3600 mm hg years ( n=4 ) , leaving 1990 participants for that analysis ( figure 2 ) . in the exploratory analysis in table 4 , 236 participants ( 137 missing lvmi and 99 missing cumulative sbp ) were excluded from the 1669 participants with bp < 120/<80 at y25 , leaving 1433 participants . because the cohort is relatively young and only 2 years of cvd event follow - up data were available after the y25 examination , there were too few cvd events for statistical analysis . the multi - ethnic study of atherosclerosis ( mesa ) is also an nhlbi - sponsored multicenter longitudinal study consisting of 6814 white ( 38% ) , black ( 28% ) , hispanic ( 23% ) , and chinese ( 12% ) women and men aged 45 to 84 years who were free of cvd at baseline ( 20002002 ) . participants were excluded from analysis if they had fasting time < 8 hours ( n=6 ) ; missing sbp , low - density lipoprotein cholesterol , diabetes status , or smoking status ( n=121 ) ; missing antihypertensive medication information ( n=2 ) ; or prebaseline events ( n=4 ) . in addition , because most younger people were in the normotensive group , we excluded 883 participants aged < 50 years at baseline to enhance the age comparability between the bp treatment groups . all participants in both studies signed informed consent , and the studies were approved by the institutional review board of each field center . details regarding these 2 studies have been published previously.18,19 all measurements were obtained by centrally trained and certified technicians . participants were asked to fast for 12 hours and to refrain from smoking and from heavy physical activity for 2 hours before each examination . seated bp was measured using a random zero sphygmomanometer for the examinations from y0 to year 15 and an omron model hem907xl at y20 and y25 examinations . after a 5-minute rest in a quiet room , bp was measured on the right arm at three 1-minute intervals ; the average of the second and third measurements was used . a calibration study was conducted ( n=800 ) to convert y20 and y25 values to their y0 equivalents . total cholesterol was measured enzymatically by the northwest lipid laboratory , and low - density lipoprotein cholesterol was calculated using the friedewald equation.20 glucose was assayed using hexokinase coupled to glucose-6 phosphate dehydrogenase . diabetes was defined by fasting glucose 126 mg / dl , 2-hour glucose 200 mg / dl , hba1c 6.5% , or medication use . the estimated glomerular filtration rate ( egfr ) was estimated using the chronic kidney disease epidemiology collaboration equation.21 for height and weight measurements , the participants wore light clothing without shoes . participants were asked to bring their medications with them to the clinic visits . at y25 , participants underwent 2-dimensionally guided m - mode echocardiography ; all studies were read centrally at the johns hopkins university echocardiography reading center . lvmi was calculated by dividing the lv mass ( in grams ) by height ( in m ) . coronary calcium ( cac ) levels were measured at y25 using multidetector computed tomography of the chest.22 images were read centrally by the computed tomography reading center at wake forest university . three bp measurements , 1 minute apart , were collected using a dinamap pro100 model bp monitor , and the average value of the second and third measurements was used . details regarding lipid measurements have been published.24 serum glucose was measured by the glucose - oxidase method . the egfr was based on cystatin c.25 chest computed tomography was performed using either cardiac gated electron - beam or multidetector computed tomography scanners.22 images were read centrally for cac by the los angeles biomedical research institute at harbor ucla medical center . the average agatston score was used in the analysis.23 lv mass was obtained by cardiac magnetic resonance imaging . images were acquired by 1.5 t magnetic resonance imaging scanners using a protocol described by natori et al.26 the readings were performed centrally by the mesa magnetic resonance imaging reading center at johns hopkins hospital . lvmi was calculated using the dubois formula for body surface area.27 in cardia , participants were contacted every year to inquire about interim hospitalizations . for each self - reported event , medical records were obtained and adjudicated by 2 members of the morbidity and mortality committee . cvd events comprised nonfatal myocardial infarction or stroke ; hospitalization for angina pectoris , congestive heart failure ( chf ) , or transient ischemic attack ; revascularization for or angiographically or ultrasound - demonstrated obstruction of carotid artery disease or peripheral arterial disease ; fatal atherosclerotic coronary heart disease , fatal stroke , fatal atherosclerotic disease other than coronary or stroke , and fatal nonatherosclerotic cardiac disease . in mesa , incident cvd events were recorded over a mean follow - up of 9.5 years ( sd 2.4 years ) . every 9 to 12 months , participants were contacted to inquire about interim hospital admissions , cardiovascular outpatient diagnoses and procedures , and deaths . again , for each self - reported event , the medical and hospital records were obtained and adjudicated by 2 members of the morbidity and mortality committee . for this report , all chd events were classified as myocardial infarction , resuscitated cardiac arrest , definite angina , probable angina ( if followed by revascularization ) , and chd death . all cvd events included all chd events plus stroke , stroke death , other atherosclerotic death , and other cvd death . definite or probable chf required heart failure symptoms such as shortness of breath or edema . in addition to symptoms , probable chf required chf diagnosed by a physician and medical treatment for chf . definite chf required 1 criterion or more , such as pulmonary edema or congestion by chest x - ray , ventricular dilation or poor lv function by echocardiography or ventriculography , or evidence of lv diastolic dysfunction . cardia participants were classified into 6 bp treatment groups defined by whether they were treated or untreated with antihypertensive medication at y25 and the severity level of bp at y25 as categorized by the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure , or jnc 7 : normal ( untreated bp < 120/<80 mm hg ) , treated and well controlled ( bp < 120/<80 mm hg ) , untreated prehypertension ( sbp 120 to 139 mm hg or dbp 80 to 89 mm hg , or sbp 120 to 129 with dbp < 80 mm hg for participants with diabetes ) , treated and controlled ( bp in prehypertension ranges ) , untreated hypertension ( bp 140/90 mm hg or bp 130/80 mm hg for participants with diabetes ) , and treated and uncontrolled ( bp in untreated hypertension ranges).9 similar categories were used to define bp strata for mesa participants at baseline . for cardia participants who attended the baseline and y25 examinations and at least 4 of 6 intermediate examinations at years 2 , 5 , 7 , 10 , 15 , and y20 , the cumulative sbp ( in mm hg years ) was calculated by summing the product of the average sbp and the time interval ( in years ) between 2 consecutive examinations over the 25 years . analyses were conducted using sas statistical software ( version 9.4 ; sas institute ) and r software ( r foundation for statistical computing ) . cox regression was used to obtain hazard ratios ( hrs ) for the 6 baseline bp groups for incident chd , cvd , heart failure , and stroke in mesa participants , adjusting for the baseline covariates age , sex , race , body mass index , smoking status , low - density lipoprotein cholesterol , and cholesterol medication . in addition , we reran the cox regression models using the eighth report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 8) criteria28 due to changes in the treatment criteria . under the jnc 8 criteria , the initiation of antihypertensive medication for those aged > 60 years is delayed until they have sbp > 150 or dbp > 90 mm hg . the proportional hazards assumption was examined using the time interaction test and the supremum test . all tests were insignificant except for the comparison of incident cvd between the groups of people with bp < 120/<80 mm hg without medication and prehypertension patients without medication ; this comparison was borderline significant ( p=0.07 and p=0.04 for the time interaction and supremum tests , respectively ) . this comparison is not the focus of the paper , and the hr was not statistically significant . for cardia , a generalized estimating equation model was used to obtain estimates of age- , sex- , and race - adjusted sbp and dbp at the 8 examination visits ( figure 1 ) . ancova was used to estimate the cardia y25 covariate - adjusted prevalence and mesa baseline covariate - adjusted prevalence of cac score > 100 and egfr < 60 ml / min per 1.73 m for the 6 groups , and multiple logistic regression was used to test significance . ancova was used to compare the y25 covariate - adjusted lvmi among 6 bp groups . age- , race- , and sex - adjusted bp over time by y25 bp level and antihypertensive treatment status . mean bp for cardia participants at y25 : ( a ) bp < 120/<80 mm hg . ( b ) sbp 120 to 139 mm hg or dbp 80 to 89 mm hg ( or sbp 120 to 129 with dbp < 80 mm hg for people with diabetes ) , ( c ) bp 140/90 mm hg ( or bp 130/80 mm hg for people with diabetes ) . bp indicates blood pressure ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; sbp , systolic blood pressure ; y25 , year 25 . in addition , in cardia , a large number of participants were excluded from the analytic sample due to missing y25 examinations , cumulative sbp , or lvmi . we compared baseline age , sex , race , body mass index , smoking status , bp , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol , fasting glucose , education , serum creatinine , alcohol intake , and total physical activity between those who were included in the sample and those who were excluded from the sample . the variables that were significantly different between the 2 groups were used to develop a propensity equation for inclusion based on the logistic regression model for each end - organ - damage variable ( ie , lvmi , cac , and egfr ) . the inverse propensity probability of inclusion was used to perform the weighted regression analysis as sensitivity analyses for findings in table 3 . a generalized additive model was used to model the nonlinear relation of lvmi with cumulative sbp , adjusting for the y25 covariates . in the analysis , the adjusted mean of lvmi is a smooth function of the cumulative sbp with additive structure . specifically , we fit a special piecewise linear model , which assumes that lvmi is linearly related to cumulative sbp , with the slope of the regression line changing at an unknown cumulative sbp level . in addition , the davies test29 was performed to test the existence of the changing point . the main statistical analysis was conducted using the packages segmented and mgcv in r.3.1.3 ( r foundation for statistical computing ) ( figure 2 ) . adjusted relationship by spline regression between lvmi and 25-year cumulative systolic blood pressure in untreated cardia participants ( n=1990 ) . spline ( solid line ) with 95% pointwise confidence band ( dotted lines ) is adjusted for year 25 age , sex , race , body mass index , smoking status , diabetes , low - density lipoprotein cholesterol , and cholesterol medication . the slopes at the break point of 2885 cumulative mm hg years were estimated to be 0.5 g / m per 100 mm hg years ( 95% ci 0.2 to 0.8 ) before 2885 mm hg years and after 2885 mm hg years is 1.4 g / m per 100 mm hg years ( 95% ci 0.7 to 2.1 ) . cardia indicates coronary artery risk development in young adults ; lvmi , left ventricular mass index . the cardia study , sponsored by the national heart , lung , and blood institute ( nhlbi ) , is a multicenter longitudinal study consisting of 5115 black and white women and men aged 18 to 30 years at the baseline examination ( y0 ) in 19851986 who were free of cvd . seven follow - up examinations took place at years 2 , 5 , 7 , 10 , 15 , 20 ( y20 ) , and 25 ( y25 ) . participants in the cardia cohort were excluded from the analysis if they did not attend the y25 examination ( n=1617 ) , were missing data on all y25 outcome measures ( n=3 ) , were missing y25 bp measurements ( n=6 ) , were missing data on y25 covariates ( n=231 ) , or developed cvd prior to y25 ( n=94 ) . an additional person was excluded from analyses involving left ventricular ( lv ) mass due to an unlikely , large lv mass ( 502 g ) . participants were permitted to be missing data on some but not all y25 outcomes , thus the sample sizes for different outcomes may be slightly < 3164 participants . for participants who attended the baseline and y25 examinations and at least 4 of 6 intermediate examinations , 25-year cumulative sbp was computed . in the analyses relating 25-year cumulative sbp to lv mass index ( lvmi ) in untreated cardia participants ( n=2362 ) , 368 were excluded due to missing lvmi ( n=214 ) or missing 25-year cumulative sbp ( n=154 ) or had cumulative sbp outside of the range 2200 to 3600 mm hg years ( n=4 ) , leaving 1990 participants for that analysis ( figure 2 ) . in the exploratory analysis in table 4 , 236 participants ( 137 missing lvmi and 99 missing cumulative sbp ) were excluded from the 1669 participants with bp < 120/<80 at y25 , leaving 1433 participants . because the cohort is relatively young and only 2 years of cvd event follow - up data were available after the y25 examination , there were too few cvd events for statistical analysis . the multi - ethnic study of atherosclerosis ( mesa ) is also an nhlbi - sponsored multicenter longitudinal study consisting of 6814 white ( 38% ) , black ( 28% ) , hispanic ( 23% ) , and chinese ( 12% ) women and men aged 45 to 84 years who were free of cvd at baseline ( 20002002 ) . participants were excluded from analysis if they had fasting time < 8 hours ( n=6 ) ; missing sbp , low - density lipoprotein cholesterol , diabetes status , or smoking status ( n=121 ) ; missing antihypertensive medication information ( n=2 ) ; or prebaseline events ( n=4 ) . in addition , because most younger people were in the normotensive group , we excluded 883 participants aged < 50 years at baseline to enhance the age comparability between the bp treatment groups . all participants in both studies signed informed consent , and the studies were approved by the institutional review board of each field center . participants were asked to fast for 12 hours and to refrain from smoking and from heavy physical activity for 2 hours before each examination . seated bp was measured using a random zero sphygmomanometer for the examinations from y0 to year 15 and an omron model hem907xl at y20 and y25 examinations . after a 5-minute rest in a quiet room , bp was measured on the right arm at three 1-minute intervals ; the average of the second and third measurements was used . a calibration study was conducted ( n=800 ) to convert y20 and y25 values to their y0 equivalents . total cholesterol was measured enzymatically by the northwest lipid laboratory , and low - density lipoprotein cholesterol was calculated using the friedewald equation.20 glucose was assayed using hexokinase coupled to glucose-6 phosphate dehydrogenase . diabetes was defined by fasting glucose 126 mg / dl , 2-hour glucose 200 mg / dl , hba1c 6.5% , or medication use . the estimated glomerular filtration rate ( egfr ) was estimated using the chronic kidney disease epidemiology collaboration equation.21 for height and weight measurements , the participants wore light clothing without shoes . participants were asked to bring their medications with them to the clinic visits . at y25 , participants underwent 2-dimensionally guided m - mode echocardiography ; all studies were read centrally at the johns hopkins university echocardiography reading center . lvmi was calculated by dividing the lv mass ( in grams ) by height ( in m ) . coronary calcium ( cac ) levels were measured at y25 using multidetector computed tomography of the chest.22 images were read centrally by the computed tomography reading center at wake forest university . three bp measurements , 1 minute apart , were collected using a dinamap pro100 model bp monitor , and the average value of the second and third measurements was used . details regarding lipid measurements have been published.24 serum glucose was measured by the glucose - oxidase method . the egfr was based on cystatin c.25 chest computed tomography was performed using either cardiac gated electron - beam or multidetector computed tomography scanners.22 images were read centrally for cac by the los angeles biomedical research institute at harbor ucla medical center . the average agatston score was used in the analysis.23 lv mass was obtained by cardiac magnetic resonance imaging . images were acquired by 1.5 t magnetic resonance imaging scanners using a protocol described by natori et al.26 the readings were performed centrally by the mesa magnetic resonance imaging reading center at johns hopkins hospital . in cardia , participants were contacted every year to inquire about interim hospitalizations . for each self - reported event , medical records were obtained and adjudicated by 2 members of the morbidity and mortality committee . cvd events comprised nonfatal myocardial infarction or stroke ; hospitalization for angina pectoris , congestive heart failure ( chf ) , or transient ischemic attack ; revascularization for or angiographically or ultrasound - demonstrated obstruction of carotid artery disease or peripheral arterial disease ; fatal atherosclerotic coronary heart disease , fatal stroke , fatal atherosclerotic disease other than coronary or stroke , and fatal nonatherosclerotic cardiac disease . in mesa , incident cvd events were recorded over a mean follow - up of 9.5 years ( sd 2.4 years ) . every 9 to 12 months , participants were contacted to inquire about interim hospital admissions , cardiovascular outpatient diagnoses and procedures , and deaths . again , for each self - reported event , the medical and hospital records were obtained and adjudicated by 2 members of the morbidity and mortality committee . for this report , all chd events were classified as myocardial infarction , resuscitated cardiac arrest , definite angina , probable angina ( if followed by revascularization ) , and chd death . all cvd events included all chd events plus stroke , stroke death , other atherosclerotic death , and other cvd death . definite or probable chf required heart failure symptoms such as shortness of breath or edema . in addition to symptoms , probable chf required chf diagnosed by a physician and medical treatment for chf . definite chf required 1 criterion or more , such as pulmonary edema or congestion by chest x - ray , ventricular dilation or poor lv function by echocardiography or ventriculography , or evidence of lv diastolic dysfunction . cardia participants were classified into 6 bp treatment groups defined by whether they were treated or untreated with antihypertensive medication at y25 and the severity level of bp at y25 as categorized by the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure , or jnc 7 : normal ( untreated bp < 120/<80 mm hg ) , treated and well controlled ( bp < 120/<80 mm hg ) , untreated prehypertension ( sbp 120 to 139 mm hg or dbp 80 to 89 mm hg , or sbp 120 to 129 with dbp < 80 mm hg for participants with diabetes ) , treated and controlled ( bp in prehypertension ranges ) , untreated hypertension ( bp 140/90 mm hg or bp 130/80 mm hg for participants with diabetes ) , and treated and uncontrolled ( bp in untreated hypertension ranges).9 similar categories were used to define bp strata for mesa participants at baseline . for cardia participants who attended the baseline and y25 examinations and at least 4 of 6 intermediate examinations at years 2 , 5 , 7 , 10 , 15 , and y20 , the cumulative sbp ( in mm hg years ) was calculated by summing the product of the average sbp and the time interval ( in years ) between 2 consecutive examinations over the 25 years . analyses were conducted using sas statistical software ( version 9.4 ; sas institute ) and r software ( r foundation for statistical computing ) . cox regression was used to obtain hazard ratios ( hrs ) for the 6 baseline bp groups for incident chd , cvd , heart failure , and stroke in mesa participants , adjusting for the baseline covariates age , sex , race , body mass index , smoking status , low - density lipoprotein cholesterol , and cholesterol medication . in addition , we reran the cox regression models using the eighth report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc 8) criteria28 due to changes in the treatment criteria . under the jnc 8 criteria , the initiation of antihypertensive medication for those aged > 60 years is delayed until they have sbp > 150 or dbp > 90 mm hg . the proportional hazards assumption was examined using the time interaction test and the supremum test . all tests were insignificant except for the comparison of incident cvd between the groups of people with bp < 120/<80 mm hg without medication and prehypertension patients without medication ; this comparison was borderline significant ( p=0.07 and p=0.04 for the time interaction and supremum tests , respectively ) . this comparison is not the focus of the paper , and the hr was not statistically significant . for cardia , a generalized estimating equation model was used to obtain estimates of age- , sex- , and race - adjusted sbp and dbp at the 8 examination visits ( figure 1 ) . ancova was used to estimate the cardia y25 covariate - adjusted prevalence and mesa baseline covariate - adjusted prevalence of cac score > 100 and egfr < 60 ml / min per 1.73 m for the 6 groups , and multiple logistic regression was used to test significance . ancova was used to compare the y25 covariate - adjusted lvmi among 6 bp groups . age- , race- , and sex - adjusted bp over time by y25 bp level and antihypertensive treatment status . mean bp for cardia participants at y25 : ( a ) bp < 120/<80 mm hg . ( b ) sbp 120 to 139 mm hg or dbp 80 to 89 mm hg ( or sbp 120 to 129 with dbp < 80 mm hg for people with diabetes ) , ( c ) bp 140/90 mm hg ( or bp 130/80 mm hg for people with diabetes ) . bp indicates blood pressure ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; sbp , systolic blood pressure ; y25 , year 25 . in addition , in cardia , a large number of participants were excluded from the analytic sample due to missing y25 examinations , cumulative sbp , or lvmi . we compared baseline age , sex , race , body mass index , smoking status , bp , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol , fasting glucose , education , serum creatinine , alcohol intake , and total physical activity between those who were included in the sample and those who were excluded from the sample . the variables that were significantly different between the 2 groups were used to develop a propensity equation for inclusion based on the logistic regression model for each end - organ - damage variable ( ie , lvmi , cac , and egfr ) . the inverse propensity probability of inclusion was used to perform the weighted regression analysis as sensitivity analyses for findings in table 3 . a generalized additive model was used to model the nonlinear relation of lvmi with cumulative sbp , adjusting for the y25 covariates . in the analysis , the adjusted mean of lvmi is a smooth function of the cumulative sbp with additive structure . specifically , we fit a special piecewise linear model , which assumes that lvmi is linearly related to cumulative sbp , with the slope of the regression line changing at an unknown cumulative sbp level . in addition , the davies test29 was performed to test the existence of the changing point . the main statistical analysis was conducted using the packages segmented and mgcv in r.3.1.3 ( r foundation for statistical computing ) ( figure 2 ) . adjusted relationship by spline regression between lvmi and 25-year cumulative systolic blood pressure in untreated cardia participants ( n=1990 ) . spline ( solid line ) with 95% pointwise confidence band ( dotted lines ) is adjusted for year 25 age , sex , race , body mass index , smoking status , diabetes , low - density lipoprotein cholesterol , and cholesterol medication . the slopes at the break point of 2885 cumulative mm hg years were estimated to be 0.5 g / m per 100 mm hg years ( 95% ci 0.2 to 0.8 ) before 2885 mm hg years and after 2885 mm hg years is 1.4 g / m per 100 mm hg years ( 95% ci 0.7 to 2.1 ) . cardia indicates coronary artery risk development in young adults ; lvmi , left ventricular mass index . baseline characteristics of mesa participants and baseline and y25 characteristics of cardia participants are presented in tables1 and 2 , respectively , stratified by bp and treatment groups ( at baseline for mesa and at y25 for cardia ) . in mesa , the group with untreated bp < 120/<80 mm hg tended to be younger and leaner than the other groups ( table 1 ) . in cardia , the group with untreated bp < 120/<80 mm hg at y25 had a higher proportion of women and white participants , a lower prevalence of current smoking , and lower average body mass index at baseline and at y25 than the other groups ( table 2 ) . baseline ( 20002002 ) characteristics of mesa participants aged 50 years stratified by baseline bp level and antihypertensive treatment status values shown are mean ( sd ) or n ( % ) . bmi indicates body mass index ; bp , blood pressure ; dbp , diastolic blood pressure ; mesa , multi - ethnic study of atherosclerosis ; sbp , systolic blood pressure . baseline ( 19851986 ) and y25 characteristics of cardia participants stratified by y25 bp level and antihypertensive treatment status values shown are mean ( sd ) or n ( % ) . bmi indicates body mass index ; bp , blood pressure ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; sbp , systolic blood pressure ; y25 , year 25 . risk factor adjusted hrs for 9.5-year incident cvd , chd , heart failure , and stroke are presented in table 3 for the 6 bp groups , using the original jnc 7 criteria and the jnc 8 criteria . under the jnc 7 criteria , mesa participants with treated and well - controlled bp had hrs of 2.19 ( p<0.01 ) , 2.02 ( p<0.01 ) , 1.70 ( p=0.09 ) , and 2.56 ( p=0.01 ) for cvd , chd , heart failure , and stroke , respectively , compared with those with untreated bp < 120/<80 mm hg . for the other 4 groups , except for chd and stroke in the untreated prehypertensive group , the hrs for incident cvd , chd , and stroke were all significantly higher than for the group with untreated bp < 120/<80 mm hg . multivariable - adjusted * hazard ratios for cvd , chd , heart failure , and stroke stratified by baseline bp stratum and antihypertensive treatment status , mesa participants aged 50 years bp indicates blood pressure ; chd , coronary heart disease ; ckd , chronic kidney disease ; cvd , cardiovascular disease ; dbp , diastolic blood pressure ; jnc 7 , seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ; jnc 8 , eighth report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ; mesa , multi - ethnic study of atherosclerosis ; ref , reference ; sbp , systolic blood pressure . adjusted for age , sex , race , body mass index , current smoking , former smoking , diabetes , low - density lipoprotein cholesterol , and cholesterol medication measured at baseline . cvd : chd plus stroke , stroke death , other atherosclerotic death , and other cvd death . chd : myocardial infarction , resuscitated cardiac arrest , definite angina , probable angina ( if followed by revascularization ) , chd death . similarly , participants with treated and controlled hypertension had significantly higher hazards for incident cvd , chd , and heart failure compared with untreated prehypertensive participants . moreover , hrs for incident cvd , chd , heart failure , and stroke were similar between participants with treated but uncontrolled hypertension and untreated hypertension . when this analysis was rerun using jnc 8 criteria , the results were similar , except that in the prehypertension group , the hrs for chd and stroke were also significantly higher than for the group with untreated bp < 120/<80 mm hg . in addition , for the prehypertension group , based on the jnc 8 criteria , the hrs for cvd , chd , heart failure , and stroke were 1.65 , 1.45 , 1.45 , and 2.22 , respectively ; these hrs are all higher than the corresponding hrs1.42 , 1.29 , 1.41 , and 1.76 for cvd , chd , heart failure , and stroke , respectively for the prehypertension group based on the jnc 7 criteria ( table 3 ) . figure 1a shows the average bp at y0 and at years 2 , 5 , 7 , 10 , 15 , y20 , and y25 for normotensive participants in cardia at y25 ( closed circles ) and hypertensive participants with treated and well - controlled bp at y25 ( open circles ) . at y0 , the average bps in all 6 groups were much lower than 120/80 mm hg . the average sbps and dbps of participants who became hypertensive but had well - controlled bp with treatment at y25 were already much higher at y0 than the average sbps and dbps of normotensive participants . participants who became hypertensive over time had greater elevations in bp , as expected , and then returned to ideal bp levels with treatment by y25 . similar bp patterns were also observed between the y25 prehypertension group and the treated and controlled group ( figure 1b ) and between the treated but uncontrolled group and the untreated hypertension group ( figure 1c ) , although differences between the latter 2 groups were not as large . these data suggest that even participants on antihypertensive medication with well - controlled bp ( < 120/<80 mm hg at y25 ) have generally experienced a longer period ( in our study , 25 years ) of bp at much higher levels than those of the normotensive group . in both cardia participants at y25 and mesa participants at baseline with bp ( treated or untreated ) either < 120/<80 mm hg or from 120/80 to 139/89 mm hg , those on antihypertensive treatment had significantly higher lvmi , a significantly higher prevalence of egfr < 60 ml / min per 1.73 m , and a significantly higher prevalence of cac score > 100 than the corresponding group without treatment ( table 4 ) . adjusted * measures of end - organ damage stratified by y25 bp level ( cardia ) or baseline bp level ( mesa ) and antihypertensive treatment status bp indicates blood pressure ; cac , coronary calcium ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; egfr , estimated glomerular filtration rate ; lvmi , left ventricular mass index ; mesa , multi - ethnic study of atherosclerosis ; mri , magnetic resonance imaging ; sbp , systolic blood pressure ; y25 , year 25 . adjusted for age , sex , race , body mass index , smoking status , diabetes , low - density lipoprotein cholesterol , and cholesterol medication . ancova was used to estimate the prevalence rates in the 6 groups , adjusting for the covariates measured at y25 for cardia and at baseline for mesa ; multiple logistic regression was used to test for the significant differences between groups . in cardia , those who were excluded from the analyses tended to be younger , men , black , and current smokers , with higher sbp , dbp , low - density lipoprotein cholesterol , fasting glucose , alcohol intake , and creatinine and lower education at baseline . these baseline characteristics were used to develop a propensity equation for inclusion for each variable described for end - organ damage . a sensitivity analysis was performed using the inverse propensity probability as the weights in the regression analyses . the results were similar to those in table 4 , with no material change ( data not shown ) . figure 2 demonstrates the relationship between cumulative sbp over 25 years and lvmi in cardia . in general , lvmi was higher with greater exposure to cumulative sbp , and when cumulative sbp exposure was high , the increase in lvmi accelerated . in the changing point analysis , the davies test was significant for the existence of a change point , confirming that the association between cumulative sbp exposure and lvmi is not constant ( p=0.04 ) . the estimator of the change point in the piecewise linear regression was 2885 mm hg years . the slopes estimated before and after the break point of 2885 mm hg years were 0.5 and 1.4 g / m per 100 mm hg years , respectively . because stroke and transient ischemic attack are strongly associated with high bp and are unlikely to affect lv mass directly , the results may be overly conservative with the exclusion of these diseases . in a sensitivity analysis , we reran the change point analysis excluding cvd , except for stroke and transient ischemic attack , prior to y25 . the change point in this case was estimated at 2982 mm hg years with slopes before and after this point of 0.5 and 2.1 g / m per 100 mm hg years , respectively . the cardia data also allowed an exploratory examination of whether early detection of hypertension and controlling bp to < 120/<80 mm hg could prevent increasing end - organ damage . the group of cardia participants with treated bp < 120/<80 mm hg at y25 was further classified into 2 subgroups . group 2 included those who initiated treatment by year 15 , y20 , or y25 with cumulative sbp < 3000 mm hg years and bp < 120/<80 mm hg in subsequent years through y25 ; group 3 included those who initiated treatment by year 15 or y20 but were not always well controlled until y25 or those who had a cumulative sbp 3000 mm hg years . the adjusted average lvmis , prevalence of cac score > 100 , and prevalence of egfr < 60 ml / min per 1.73 m of group 2 were all lower ( significantly or borderline significantly ) than those of group 3 even though they were higher ( not statistically significant ) than those of group 1 ( table 5 ) . bp treatment status , cumulative sbp , and adjusted * measures of end - organ damage in people with bp < 120/80 mm hg at y25 , cardia bp , blood pressure ; cac , coronary calcium ; cardia , coronary artery risk development in young adults ; egfr , estimated glomerular filtration rate ; lvmi , left ventricular mass index ; sbp , systolic blood pressure ; y , year . adjusted for age , race , sex , diabetes , low - density lipoprotein cholesterol , cholesterol medication , body mass index , and smoking status measured at y25 . ancova was used to estimate the prevalence rates in the 3 groups , adjusting for the y25 covariates ; multiple logistic regression was used to test for the significant differences between groups . p<0.001 baseline characteristics of mesa participants and baseline and y25 characteristics of cardia participants are presented in tables1 and 2 , respectively , stratified by bp and treatment groups ( at baseline for mesa and at y25 for cardia ) . in mesa , the group with untreated bp < 120/<80 mm hg tended to be younger and leaner than the other groups ( table 1 ) . in cardia , the group with untreated bp < 120/<80 mm hg at y25 had a higher proportion of women and white participants , a lower prevalence of current smoking , and lower average body mass index at baseline and at y25 than the other groups ( table 2 ) . baseline ( 20002002 ) characteristics of mesa participants aged 50 years stratified by baseline bp level and antihypertensive treatment status values shown are mean ( sd ) or n ( % ) . bmi indicates body mass index ; bp , blood pressure ; dbp , diastolic blood pressure ; mesa , multi - ethnic study of atherosclerosis ; sbp , systolic blood pressure . baseline ( 19851986 ) and y25 characteristics of cardia participants stratified by y25 bp level and antihypertensive treatment status values shown are mean ( sd ) or n ( % ) . bmi indicates body mass index ; bp , blood pressure ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; sbp , systolic blood pressure ; y25 , year 25 . risk factor adjusted hrs for 9.5-year incident cvd , chd , heart failure , and stroke are presented in table 3 for the 6 bp groups , using the original jnc 7 criteria and the jnc 8 criteria . under the jnc 7 criteria , mesa participants with treated and well - controlled bp had hrs of 2.19 ( p<0.01 ) , 2.02 ( p<0.01 ) , 1.70 ( p=0.09 ) , and 2.56 ( p=0.01 ) for cvd , chd , heart failure , and stroke , respectively , compared with those with untreated bp < 120/<80 mm hg . for the other 4 groups , except for chd and stroke in the untreated prehypertensive group , the hrs for incident cvd , chd , and stroke were all significantly higher than for the group with untreated bp < 120/<80 mm hg . multivariable - adjusted * hazard ratios for cvd , chd , heart failure , and stroke stratified by baseline bp stratum and antihypertensive treatment status , mesa participants aged 50 years bp indicates blood pressure ; chd , coronary heart disease ; ckd , chronic kidney disease ; cvd , cardiovascular disease ; dbp , diastolic blood pressure ; jnc 7 , seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ; jnc 8 , eighth report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ; mesa , multi - ethnic study of atherosclerosis ; ref , reference ; sbp , systolic blood pressure . adjusted for age , sex , race , body mass index , current smoking , former smoking , diabetes , low - density lipoprotein cholesterol , and cholesterol medication measured at baseline . cvd : chd plus stroke , stroke death , other atherosclerotic death , and other cvd death . chd : myocardial infarction , resuscitated cardiac arrest , definite angina , probable angina ( if followed by revascularization ) , chd death . similarly , participants with treated and controlled hypertension had significantly higher hazards for incident cvd , chd , and heart failure compared with untreated prehypertensive participants . moreover , hrs for incident cvd , chd , heart failure , and stroke were similar between participants with treated but uncontrolled hypertension and untreated hypertension . when this analysis was rerun using jnc 8 criteria , the results were similar , except that in the prehypertension group , the hrs for chd and stroke were also significantly higher than for the group with untreated bp < 120/<80 mm hg . in addition , for the prehypertension group , based on the jnc 8 criteria , the hrs for cvd , chd , heart failure , and stroke were 1.65 , 1.45 , 1.45 , and 2.22 , respectively ; these hrs are all higher than the corresponding hrs1.42 , 1.29 , 1.41 , and 1.76 for cvd , chd , heart failure , and stroke , respectively for the prehypertension group based on the jnc 7 criteria ( table 3 ) . figure 1a shows the average bp at y0 and at years 2 , 5 , 7 , 10 , 15 , y20 , and y25 for normotensive participants in cardia at y25 ( closed circles ) and hypertensive participants with treated and well - controlled bp at y25 ( open circles ) . at y0 , the average bps in all 6 groups were much lower than 120/80 mm hg . the average sbps and dbps of participants who became hypertensive but had well - controlled bp with treatment at y25 were already much higher at y0 than the average sbps and dbps of normotensive participants . participants who became hypertensive over time had greater elevations in bp , as expected , and then returned to ideal bp levels with treatment by y25 . similar bp patterns were also observed between the y25 prehypertension group and the treated and controlled group ( figure 1b ) and between the treated but uncontrolled group and the untreated hypertension group ( figure 1c ) , although differences between the latter 2 groups were not as large . these data suggest that even participants on antihypertensive medication with well - controlled bp ( < 120/<80 mm hg at y25 ) have generally experienced a longer period ( in our study , 25 years ) of bp at much higher levels than those of the normotensive group . in both cardia participants at y25 and mesa participants at baseline with bp ( treated or untreated ) either < 120/<80 mm hg or from 120/80 to 139/89 mm hg , those on antihypertensive treatment had significantly higher lvmi , a significantly higher prevalence of egfr < 60 ml / min per 1.73 m , and a significantly higher prevalence of cac score > 100 than the corresponding group without treatment ( table 4 ) . adjusted * measures of end - organ damage stratified by y25 bp level ( cardia ) or baseline bp level ( mesa ) and antihypertensive treatment status bp indicates blood pressure ; cac , coronary calcium ; cardia , coronary artery risk development in young adults ; dbp , diastolic blood pressure ; egfr , estimated glomerular filtration rate ; lvmi , left ventricular mass index ; mesa , multi - ethnic study of atherosclerosis ; mri , magnetic resonance imaging ; sbp , systolic blood pressure ; y25 , year 25 . adjusted for age , sex , race , body mass index , smoking status , diabetes , low - density lipoprotein cholesterol , and cholesterol medication . ancova was used to estimate the prevalence rates in the 6 groups , adjusting for the covariates measured at y25 for cardia and at baseline for mesa ; multiple logistic regression was used to test for the significant differences between groups . in cardia , those who were excluded from the analyses tended to be younger , men , black , and current smokers , with higher sbp , dbp , low - density lipoprotein cholesterol , fasting glucose , alcohol intake , and creatinine and lower education at baseline . these baseline characteristics were used to develop a propensity equation for inclusion for each variable described for end - organ damage . a sensitivity analysis was performed using the inverse propensity probability as the weights in the regression analyses . the results were similar to those in table 4 , with no material change ( data not shown ) . figure 2 demonstrates the relationship between cumulative sbp over 25 years and lvmi in cardia . in general , lvmi was higher with greater exposure to cumulative sbp , and when cumulative sbp exposure was high , the increase in lvmi accelerated . in the changing point analysis , the davies test was significant for the existence of a change point , confirming that the association between cumulative sbp exposure and lvmi is not constant ( p=0.04 ) . the estimator of the change point in the piecewise linear regression was 2885 mm hg years . the slopes estimated before and after the break point of 2885 mm hg years were 0.5 and 1.4 g / m per 100 mm hg years , respectively . because stroke and transient ischemic attack are strongly associated with high bp and are unlikely to affect lv mass directly , the results may be overly conservative with the exclusion of these diseases . in a sensitivity analysis , we reran the change point analysis excluding cvd , except for stroke and transient ischemic attack , prior to y25 . the change point in this case was estimated at 2982 mm hg years with slopes before and after this point of 0.5 and 2.1 g / m per 100 mm hg years , respectively . the cardia data also allowed an exploratory examination of whether early detection of hypertension and controlling bp to < 120/<80 mm hg could prevent increasing end - organ damage . the group of cardia participants with treated bp < 120/<80 mm hg at y25 was further classified into 2 subgroups . group 2 included those who initiated treatment by year 15 , y20 , or y25 with cumulative sbp < 3000 mm hg years and bp < 120/<80 mm hg in subsequent years through y25 ; group 3 included those who initiated treatment by year 15 or y20 but were not always well controlled until y25 or those who had a cumulative sbp 3000 mm hg years . the adjusted average lvmis , prevalence of cac score > 100 , and prevalence of egfr < 60 ml / min per 1.73 m of group 2 were all lower ( significantly or borderline significantly ) than those of group 3 even though they were higher ( not statistically significant ) than those of group 1 ( table 5 ) . bp treatment status , cumulative sbp , and adjusted * measures of end - organ damage in people with bp < 120/80 mm hg at y25 , cardia bp , blood pressure ; cac , coronary calcium ; cardia , coronary artery risk development in young adults ; egfr , estimated glomerular filtration rate ; lvmi , left ventricular mass index ; sbp , systolic blood pressure ; y , year . adjusted for age , race , sex , diabetes , low - density lipoprotein cholesterol , cholesterol medication , body mass index , and smoking status measured at y25 . ancova was used to estimate the prevalence rates in the 3 groups , adjusting for the y25 covariates ; multiple logistic regression was used to test for the significant differences between groups . p<0.001 the results of this study demonstrate that in mesa participants aged 50 years at baseline , those with well - controlled hypertension ( < 120/<80 mm hg ) on antihypertensive medication still had twice the risk of incident cvd events in the next 9.5 years than participants with ideal bp levels without treatment . the cardia results indicate that middle - aged adults with well - controlled bp on medication had longer exposure to higher bp levels than adults with ideal bp without medication and had significantly higher risk of end - organ damage , as measured by lvmi , renal function , and subclinical atherosclerosis . we observed these findings even in cardia participants who had mean bp levels below the typical threshold for diagnosing hypertension . in addition , our exploratory analysis suggested empirically that when cumulative sbp was high , the increase in lvmi accelerated . previous studies have shown that higher bp levels , even those not considered clinically high , at younger ages are strongly associated with clinical and subclinical cvd.13,6,7 results from the framingham heart study showed bp levels in the remote past as more predictive of incident cvd than more recent bp levels.4 loria et al reported that bp levels in young adulthood ( ages 18 to 30 years ) were significantly associated with coronary calcium 15 years later and that this association was much stronger than the association with concurrent bp levels.6 pletcher et al reported that prehypertension during young adulthood was strongly associated with coronary calcium many years later.7 in addition , the framingham heart study , atherosclerosis risk in communities ( aric ) , and the cardiovascular health study ( chs ) all demonstrated that for the same bp levels , those on antihypertensive medication had higher risk of incident cvd and mortality.1417 the results are echoed in more recent broad - based risk prediction equations that include antihypertensive therapy as a covariate with a positive coefficient.30,31 in this study , we further demonstrated that even people with well - controlled bp ( to < 120/<80 mm hg ) on antihypertensive medication although bp was assessed at 1 point in time may have a risk of incident cvd double that of people who have never had high bp . an immense array of clinical trial data has established unequivocally that lowering bp with antihypertensive medications lowers the risk of incident cvd in middle - aged and older adults , particularly heart failure and stroke in patients with hypertension.1013,32 results from the losartan intervention for endpoint reduction trial also suggest the regression of the electrocardiographic evidence of hypertrophy on treatment.33 our data , however , indicate that based on the current approach for treating hypertension , the restoration of bp levels 120/80 mm hg with antihypertensive medication may not fully restore the low risk of persons maintaining these ideal bp levels consistently without medication . it is unclear whether earlier treatment to maintain similar cumulative exposure to bp would abolish this excess cvd risk . this study indicates that for participants with similar bp levels , those on antihypertensive treatment have much higher cumulative bp exposure over time than those not treated . consequently , many have developed end - organ damage that treatment may not completely reverse . several large - scale epidemiological studies indicate that cvd risk increases as bp increases , even within the normal bp range,8,32 thus the assumption that long - term exposure to higher bp levels may lead to end - organ damage is reasonable . to our knowledge , this study is the first that provides direct evidence to support this theory and to explain why , in observational studies and at the same bp levels , people on antihypertensive treatment have a higher risk of incident cvd than people not on treatment . in addition , the exploratory analysis of the relationship between cumulative sbp and lvmi suggests that when cumulative sbp is high ( eg , > 3000 mm hg years ) , despite good control of bp with antihypertensive medication , lvmi remains significantly higher than it does in people with normal bp without treatment . the cumulative sbp of 3000 mm hg years is not very high . if , for example , a person s sbp increases evenly from 110 to 140 mm hg over 25 years , that person s cumulative sbp is 3125 mm hg . this finding suggests that there may be a point of no return for bp , that is , after reaching high levels , damage to an end organ may be difficult to reverse to the levels of those who never had hypertension . the results of this study also raise a question about recent bp treatment recommendations to delay the initiation of antihypertensive medication for those aged > 60 years until they exceed sbp of 150 or dbp of 90 mm hg.28 the higher hrs for cvd , chd , heart failure , and stroke in the prehypertension group based on jnc 8 criteria compared with the corresponding hrs in prehypertension groups based on jnc 7 criteria suggest that postponing treatment from 140 to 150 mm hg in people aged 60 this delay in treatment will also increase cumulative bp exposure and thus may increase end - organ damage and the risk of reaching the point of no return , if it exists . results from the trial of preventing hypertension ( trophy ) study suggest that reductions in incident hypertension and lower overall cumulative exposure to bp levels can be achieved with pharmacological therapy in those with prehypertensive bp levels.34 an even more desirable approach would be effective implementation of social , public health , and medical care policies to promote maintenance of ideal bp levels from youth into older age and to help restore ideal bp in people on antihypertensive medication through healthier diets and lifestyle modifications . in the cardia study , a large number of participants were excluded due to nonparticipation in the y25 examination and missing lvmi or cumulative sbp . we compared those who were included in the analysis and those who were excluded from the analysis with respect to the baseline characteristics . we then developed a propensity equation for inclusion based on the characteristics that were significantly different between the 2 groups . for each end - organ - damage variable , we used the inverse propensity probability for inclusion to perform the weighed regression analyses . the results are very similar , thus the cardia findings are unlikely to be biased . these data from the cardia and mesa studies provide a unique opportunity to examine whether bp treatment can lower cvd risk to ideal levels ; however , the findings are from observational studies , not randomized controlled clinical trials , and take into account neither the potential adverse effects of antihypertensive medications nor their financial costs . in addition , because the bp measurements were taken at a single visit , there may be some misclassification due to day - to - day variability . nonetheless , among all 6 bp strata in both studies , the normotensive group showed the lowest risk of end - organ damage and clinical cvd . these results clearly indicate that , from a public health standpoint , health care providers should place more emphasis on primordial prevention of bp elevation to further reduce cvd morbidity and mortality . the coronary artery risk development in young adults study ( cardia ) is supported by contracts hhsn268201300025c , hhsn268201300026c , hhsn268201300027c , hhsn268201300028c , hhsn268201300029c , and hhsn268200900041c from the national heart , lung , and blood institute ( nhlbi ) , the intramural research program of the national institute on aging ( nia ) , and an intra - agency agreement between nia and nhlbi ( ag0005 ) . the multi - ethnic study of atherosclerosis ( mesa ) is supported by contracts n01-hc-95159 , n01-hc-95160 , n01-hc-95161 , n01-hc-95162 , n01-hc-95163 , n01-hc-95164 , n01-hc-95165 , n01-hc-95166 , n01-hc-95167 , n01-hc-95168 and n01-hc-95169 from the nhlbi and by grants ul1-tr-000040 and ul1-rr-025005 from the national center for research resources ( ncrr ) .

backgroundit is unclear whether antihypertensive treatment can restore cardiovascular disease risk to the risk level of persons with ideal blood pressure ( bp ) levels.methods and resultsdata from the multi - ethnic study of atherosclerosis ( mesa ) and the coronary artery risk development in young adults ( cardia ) study were analyzed . outcomes were compared among participants without or with antihypertensive treatment at 3 bp levels : < 120/<80 mm hg , systolic bp 120 to 139 mm hg or diastolic bp 80 to 89 mm hg ( 120 to 129/80 mm hg for participants with diabetes ) , and systolic bp 140 or diastolic bp 90 mm hg ( systolic bp 130 or diastolic bp 80 mm hg for participants with diabetes ) . among mesa participants aged 50 years at baseline , those with bp < 120/<80 mm hg on treatment had higher left ventricular mass index , prevalence of estimated glomerular filtration rate < 60 ml / min per 1.73 m2 , prevalence of coronary calcium score > 100 , and twice the incident cardiovascular disease rate over 9.5 years of follow - up than those with bp < 120/<80 mm hg without treatment . in cardia at year 25 , persons with bp < 120/<80 mm hg with treatment had much longer exposure to higher bp and higher risk of end - organ damage and subclinical atherosclerosis than those with bp < 120/<80 mm hg without treatment . an exploratory analysis suggested that when cumulative systolic bp was high ( eg , > 3000 mm hg years in 25 years ) , the increase in left ventricular mass index accelerated.conclusionsthe data suggest that based on the current approach , antihypertensive treatment can not restore cardiovascular disease risk to ideal levels . emphasis should be placed on primordial prevention of bp increases to further reduce cardiovascular disease morbidity and mortality .

cone beam computerized tomography ( cbct ) uses a collimated and cone - shaped x - ray beam rather than a larger fan or cone beam , which allows for a scan range with a more restricted field of view ( fov ) . the beam s cylindrical fov may differ from small to large fields for dental imaging or other facial examinations ( 1 - 3 ) . some cbct units allow for the selection of different fovs to suit a specific purpose ( 3 , 4 ) . the advantages of cbct over ct scanning include lower costs , smaller size , and a lower dose of radiation ( 1 , 3 , 5 , 6 ) . recent technological improvements have made it more comfortable for patients to undergo a cbct examination as well as more convenient for dentists to acquire and analyze images ( 7 , 8) . since the technology is new and the scanners are provided by different manufacturers , little of the process is currently understood by most radiologists , and some concerns have arisen regarding the patient s radiation dose ( 9 - 14 ) . due to the hazards of x - ray irradiation , it is imperative to reduce the radiation dose given to the patient to the lowest possible amount ( 8 , 15 ) . the cbct radiation dose is lower than that of a helical ct scan ( 8 , 16 , 17 ) . however , the examination dose varies considerably when using different cbct scanners , or when using a single cbct unit that is configured to different settings ( 8 , 10 - 14 , 16 ) . if the dose can be changed considerably by selecting different exposure parameters , it is necessary to thoroughly understand the various options as well as their impact on radiation safety ( 8 , 18 ) . moreover , technological advancements are expected to improve the efficacy of each newly introduced device , while at the same time reducing the adverse effects . such a health precaution can be achieved via altering multiple variables such as different structures ( filtration , the source - to - object distances , etc . ) and different radiation protocols ( kvp , mas , and fovs ) ( 3 , 8 , 10 , 16 , 17 , 19 - 22 ) . hence , when marketing a new model of cbct scanner , it is necessary to document its radiation doses . various studies have evaluated human phantoms in order to assess the radiation doses received ( 3 , 10 , 16 , 17 , 19 - 22 ) . of course , it is difficult if not impossible to compare the results of these studies because of methodological differences such as the number of sensors used , their types , and their positioning ( 23 ) . however , to the best of our knowledge , there has been only one study involving the newtom vgi ( in 15 15 and 23 23 cm fovs ) ( 23 ) , while there has been no study concerning the radiation doses of the newtom 5 g . therefore , we conducted this study to document the radiation doses of the newtom vgi ( in 8 8 and 6 6 cm fovs , none of which were documented before ) and the newtom 5 g ( not assessed before at any configurations ) in comparison to the promax three dimensional ( 3d ) scanner . large fovs result in volumes adequate for covering the maxillofacial area , while medium and small fovs generate volumes more appropriate for dentoalveolar and localized imaging , respectively ( 1 - 3 , 23 ) . some organs have higher tissue weightings , which increases the damaging impact of radiation . in the head and neck area , the thyroid , salivary glands , calvarium , mandible ramus / trunk , and cervical vertebrae are examples of such organs . this study analyzed the absorbed and effective doses of these critical organs under irradiation from two different fovs of three cbct scanners . this in vitro experimental study was performed on the ten upper sections of an anthropometric phantom ( radiation analogue dosimetry system ( rando ) , nuclear associates , hicksville , ny , usa ) . this phantom is similar to an average - sized man in terms of tissue density and radiation absorption . the ethical protocols of this study ( in terms of the health of the operators ) were approved by the research committee of the university . all devices used in this study were properly calibrated and their quality was assessed and approved . a total of 20 white circular thermoluminescence dosimeters ( tld ) of 0.8 mm in height and 4.5 mm in diameter ( ( lif : mg , cu , p ) , tld gr 200a , conqueror electronics technology co. ltd , china ) were used in this study . the most common crystal is lif , which has a radiation absorption capacity similar to that of human soft tissue . each tld was sensitive to 1 gy - 10 gy doses , with a linear response curve . calibration was repeated prior to the examination of each of the cbct devices as detailed below . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . the energy used for the calibration was similar for all of the tlds and it was determined based on routine energy usage in cbct devices . these formulae would be used again later to convert the tld output into the absorbed dose . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the number of tlds placed in each bony area was based on the relative proportion of bone marrow distributed among the different bones in the human body ( table 1 ) ( 24 ) . the remaining three tlds were positioned in different areas of the radiology center in order to measure the background radiation . for each imaging procedure , underhill et al . ( 25 ) suggested embedding at least three dosimeters in the calvarium for the assessment of calvarial dosage . therefore , we used four sensors so as to improve the accuracy of our tests . the lower sections of the rando were not used , since the dose received by the lower phantom sections is not significant in oral radiography ( 22 ) . the phantom was transferred to three private radiology centers , each of which housed a cbct device . at each clinic , radiologists positioned the phantom in such a way to accurately resemble a human subject . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. , five times per fov ) . the exposed area was adjusted to cover the mandible in such a way that the inferior border of the fov was 1 cm lower than the mandibular inferior border . also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the device automatically configured mas based on tissue mass and density in a smart and undisclosed manner . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . this device automatically configured the resolution at high ( voxel size = 100 m ) when choosing the smaller fov . this could not be manually reserved . however , the resolution was optional ( high or normal ) for the larger fov . in order to increase the variables verona , italy ) , with a constant kvp of 110 and automatically configured mas . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . as with the above device , we manually configured the resolution at normal for the larger fov . 3 . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . the time between each irradiation and each measurement was constant for all measurements ( 24 hours ) . once at the laboratory , the dosimeters were first extracted using a vacuum device ( ett , fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . this light was read and converted to the absorbed dose using the formula obtained from the abovementioned six - phase tld calibration and ecc estimation procedure . finally , using an annealing device ( ett , fimel , france ) , the dosimeters were reset and calibrated for the next exposure . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . the equivalent dose of red bone marrow was calculated based on the mandibular ( trunk and ramus ) doses , cervical vertebral doses , and calvarial doses ( 16 ) . the doses pertaining to the devices and the organs were compared using the kruskal - wallis test and a dunn post hoc test . the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . a total of 20 white circular thermoluminescence dosimeters ( tld ) of 0.8 mm in height and 4.5 mm in diameter ( ( lif : mg , cu , p ) , tld gr 200a , conqueror electronics technology co. ltd , china ) were used in this study . the most common crystal is lif , which has a radiation absorption capacity similar to that of human soft tissue . each tld was sensitive to 1 gy - 10 gy doses , with a linear response curve . calibration was repeated prior to the examination of each of the cbct devices as detailed below . first , the dosimeters were shipped to the dosimetry laboratory of the atomic energy organization for calibration purposes . in order to reduce the error , the exposure to radiation for calibrating and ecc calculation was carried out in six separate phases at the secondary standard dosimetry laboratory ( ssdl ) of the atomic energy organization . the energy used for the calibration was similar for all of the tlds and it was determined based on routine energy usage in cbct devices . these formulae would be used again later to convert the tld output into the absorbed dose . seventeen calibrated and annealed tlds were simultaneously positioned within the phantom in positions representative of the thyroid gland , parotid gland , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus ( table 1 ) . the number of tlds placed in each bony area was based on the relative proportion of bone marrow distributed among the different bones in the human body ( table 1 ) ( 24 ) . the remaining three tlds were positioned in different areas of the radiology center in order to measure the background radiation . for each imaging procedure , underhill et al . ( 25 ) suggested embedding at least three dosimeters in the calvarium for the assessment of calvarial dosage . . the lower sections of the rando were not used , since the dose received by the lower phantom sections is not significant in oral radiography ( 22 ) . the phantom was transferred to three private radiology centers , each of which housed a cbct device . at each clinic , radiologists positioned the phantom in such a way to accurately resemble a human subject . the phantom was exposed at two different fields of view ( a large and a small fovs ) ten times ( i.e. , five times per fov ) . the exposed area was adjusted to cover the mandible in such a way that the inferior border of the fov was 1 cm lower than the mandibular inferior border . also , the anterior border of the fov was 5 mm anterior to the most prominent point of the chin . the device automatically configured mas based on tissue mass and density in a smart and undisclosed manner . the average mas was measured as 6.87 and 12.24 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . this device automatically configured the resolution at high ( voxel size = 100 m ) when choosing the smaller fov . however , the resolution was optional ( high or normal ) for the larger fov . in order to increase the variables , we chose the normal resolution for the larger fov . 2 . newtom 5 g ( newtom inc . , verona , italy ) , with a constant kvp of 110 and automatically configured mas . the average mas was measured as 6.76 and 11.52 for the large and small fovs , respectively . the large and small fovs were 8 8 cm and 6 6 cm , respectively . as with the above device , we manually configured the resolution at normal for the larger fov . 3 . promax 3d ( planmeca , helsinki , finland ) , configured at standard parameters for an average mature person ( kvp = 82 , ma = 10 , and time = 12 s ) . the large and small fovs were 8 8 cm and 4 5 cm , respectively . to render the conditions similar to those of the above two devices , the resolution of this device also , the resolution was manually configured at normal for the larger fov in order to foster similarity with the other two devices . after each of the 30 exposures , the phantom and dosimeters were shipped to the tld laboratory at the university for reading ( fimel , france ) . the time between each irradiation and each measurement was constant for all measurements ( 24 hours ) . once at the laboratory , the dosimeters were first extracted using a vacuum device ( ett , fimel , france ) . then , they were read ( tld reader , fimel , france ) , preheated , and heated , so that the absorbed x - ray dose could be emitted as visible light . this light was read and converted to the absorbed dose using the formula obtained from the abovementioned six - phase tld calibration and ecc estimation procedure . finally , using an annealing device ( ett , fimel , france ) , the dosimeters were reset and calibrated for the next exposure . since the effective dose is still considered to be the most appropriate radiation risk estimator for live tissues , it was calculated according to the formula e = ( wt ht ) , wherein wt is the tissue weighting factor , which according to icrp-2007 is 0.04 , 0.01 , and 0.12 for the thyroid , salivary glands , and red bone marrow , respectively . ht is the equivalent dose , calculated using the formula ht = ( wr dr ) , wherein dr is the absorbed dose and wr is the radiation weighting factor , which is defined as 1 for x - ray . the equivalent dose of red bone marrow was calculated based on the mandibular ( trunk and ramus ) doses , cervical vertebral doses , and calvarial doses ( 16 ) . descriptive statistics were calculated for the absorbed and effective doses . the doses pertaining to the devices and the organs the small and large fov doses were compared using the mann - whitney u test . the effective doses related to the large and small fovs were compared using the wilcoxon signed - rank test . the average absorbed doses , respectively , for the large and small fovs were 17.19 and 28.89 mgy in the promax 3d , 19.25 and 35.46 mgy in the newtom vgi , and 18.85 and 30.63 mgy in the newtom 5 g ( figure 1 , table 2 ) . the highest variation between doses received by the different organs was seen in the newtom 5 g and promax 3d ( mainly with the small fov ; table 3 ) . the kruskal - wallis test indicated a significant difference between the organ dosages measured at both fovs together ( p < 0.001 ; figure 2 , table 4 ) . the dunn post hoc test indicated significant differences between the thyroid and the submandibular gland , the mandibular trunk , and its ramus , between the parotid and the submandibular glands , and finally between the calvarium and the submandibular gland , mandibular ramus , mandibular trunk , and bone marrow ( table 5 ) . when comparing the radiation doses absorbed by organs at the smaller fov , the kruskal - wallis test showed a significant difference among the organs ( p = 0.0015 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium , while the other comparisons were not significant ( table 5 ) . when comparing organ doses at the larger fov , a significant difference among the organs was detected by the kruskal - wallis test ( p = 0.0027 ; table 4 ) . the dunn test only showed significant differences between the submandibular gland and the thyroid and calvarium ( table 5 ) . abbreviations : ci , confidence interval ; cv , coefficient of variation ; fov , field of view ; sd , standard deviation ; min , minimum ; max , maximum . abbreviations : ci , confidence interval ; cv , coefficient of variation ; fov , field of view ; sd , standard deviation ; min , minimum ; max , maximum . . the ratios of the absorbed doses of the newtom vgi and newtom 5 g as compared to the promax 3d scanner are presented in table 6 . the kruskal - wallis test did not detect a significant difference between the doses of the devices when both of their fovs were combined ( three variables ) ( p = 0.8944 ) . when each fov of each device was considered as a separate variable ( six variables ) , the kruskal - wallis test showed a non - significant difference between them ( p = 0.834 ) . the dunn post hoc test also failed to show any difference between the two fovs of each device ( all p values > 0.05 ) or between each pair of two sets of device - fov variables ( all p values > 0.05 ) . the mann - whitney u test showed insignificant differences between the fovs of each device : promax 3d ( p = 0.550 ) , newtom vgi ( p = 0.291 ) , and newtom 5 g ( p = 0.535 ) . the difference between the fov doses was insignificant according to the mann - whitney u test ( p value = 0.1930 ) . the effective doses were all greater when the device was set at the smaller fov compared to the larger fov . this difference was statistically significant according to the wilcoxon test ( p = 0.0039 ; table 7 ) . abbreviations : s / l , the ratio of the smaller - to - larger fov effective doses . a ratio greater than 1.0 indicates a higher effective dose caused by the smaller fov . in most cases , the promax 3d scanner showed lower doses compared to the newtom vgi ( and compared to the newtom 5 g in the case of a few organs ) . the radiation generated by all three devices ( with a few exceptions ) was slightly lower when absorbed by the parotid and sublingual glands and the cervical vertebrae . the absorbed dose of the calvarium was similar between the large and small fovs when generated by the newtom 5 g . it was slightly lower and greater , respectively , when the promax 3d and newtom vgi were tested . the rest of the organs had much higher absorbed doses ( 1.5 to 5 times greater ) when the device , regardless of its brand / model , was configured to use the small fov . the non - parametric evaluation of the fovs also showed no significant differences between the doses caused by the large and small fovs . however , the effective doses were considerably affected by the fovs , with the smaller fovs causing greater effective doses . this surprising finding is unlikely to be an artifact , since the experiment settings were controlled very carefully . moreover , the trends of dose increase / decrease consistently changed from organ to organ in the case of all three devices . among the evaluated organs , the calvarium and submandibular gland received the lowest and highest absorbed doses , respectively . based on these results , it can be suggested that reducing the fov for the sake of lowering the patient s received dose , if it accompanies an automatic resolution increase , is not a very effective method . this necessitates protecting this particular organ during cbct scanning . by reducing the fov and increasing the resolution , the parotid radiation dose was reduced by about 20% in the case of the promax 3d scanner , 3% in the case of the newtom vgi , and 30% in the case of the newtom 5 g . this might be attributed to the location of the parotid and the fact that the focus of imaging was on the anterior mandible region , which might lead to a reduction in the parotid dose in line with the reduction of the fov and not an increase together with the increase in resolution . this did not occur in the other salivary glands , and all salivary glands together showed about a 1.5 fold increase when the fov was reduced and the resolution was increased . decreasing the fov and increasing the projections led to small changes in the calvarium dose in the promax 3d scanner ( 3% ) and the newtom vgi ( 1% ) , although there were no changes in the newtom 5 g . the reason for this might be the distance of the calvarium from the region of interest , which reduced both its received dose and the changes in it . the reason for the smaller calvarium dose received in the newtom 5 g compared to the other two scanners might be the position of the patient , since the patient stands during imaging in the newtom vgi and promax 3d , but acquires a supine position in the newtom 5 g . the latter may place the calvarium at a farther distance from the region of interest and so expose it to less reflected radiation . unlike the calvarium , the mandible ramus and its body showed a considerable increase in received dose after decreasing the fov and increasing the resolution . the mandible ramus showed the highest increase in the case of the newtom vgi scanner ( about 2.2 fold ) when compared to the other two ( about 1.5 to 1.6 fold ) . similar increases was observed in the case of the mandible body : the newtom vgi scanner showed a 2.4 fold increase , while other two showed 1.8 to 1.9 fold increases . similar to the parotid , which showed a decrease when decreasing the fov , the vertebrae also received about 10% to 30% less radiation when the fov was reduced . to the best of our knowledge , no study to date has statistically compared the radiation doses of different fovs and different organs . all of the previous studies have been limited to cataloguing the doses . in a study performed by palomo et al . in 2008 ( 14 ) , the doses received to the head and neck organs ( esophagus , midline thyroid , mandible body , submandibular , center c spine , midbrain , and orbital surface ) from the cbct ( cb mercuray ) scanner in different fovs of 6 , 9 and 12 inches were examined . the average dose absorbed by the thyroid gland , mandible body on the right and left sides , submandibular gland on the right and left , and the vertebrae were 40.8 , 70.6 , 30.6 , 60.7 , 30.7 , and 93.5 mgy , respectively . the average absorbed dose received by the thyroid gland from all three cbct devices investigated in this study when in the normal mode was greater , with doses of 0.335 , 0.376 , and 0.379 mgy , respectively , in the case of the promax 3d , new tom vgi , and new tom 5 g at fovs of 8 8 . the difference between studies might be related to differences in exposure configurations ( kvp = 120 and ma = 15 ) and fov sizes , with the fov being 12 inches in the previous study . also , palomo et al . concluded that alongside decreasing the fov , the absorbed dose reduces . they stated that the reduction in dose is greater if the organ is farther from the direct radiation beam , which is similar to our findings ( 14 ) . in a study conducted in 2008 by hirsch et al . ( 3 ) , the doses absorbed by the head and neck organs and generated by two different cbct scanners at different fovs and protocols were examined for the anterior region of the jaws . the average dose absorbed by the parotid gland from the vera view 3d scanner in the case of fovs of 4 8 and 4 4 were 2.49 and 2.22 mgy , respectively , while for the accuitomo scanner in the case of fovs of 6 6 and 4 4 , the absorbed doses were 2.24 and 1.26 mgy , respectively . the radiation absorbed by the parotid gland was about 2 to 4 times greater in the present study compared to those results ( 3 ) . hirsch et al . also evaluated the absorbed dose of bone marrow , which was lower than that observed in this study . the reason for the higher doses observed in this study compared to the study of hirsch et al . ( 3 ) could be the larger fovs and higher resolutions adopted in this research , in addition to the differences in their scanner settings ( kvp = 80 and ma = 5 ) ( 3 ) . ( 26 ) evaluated the absorbed dose of the head and neck organs in terms of four cbct scanners and two mdct scanners . their results pertaining to the promax 3d scanner at a fov of 8 5 were lower in the case of the submandibular and sublingual glands , calvarium , vertebrae , and mandible ramus and body ( 26 ) . the reason for the lower doses observed in the submandibular and sublingual glands in their study ( 26 ) might be their smaller fov . however , it was interesting that despite their smaller fov , the parotid dose observed was higher than that in our study . a probable reason for this finding might be the differences in the positioning of the tlds and their levels . indeed , as pauwels et al . ( 23 ) concluded , a slight change in exposure settings , size of fov , the location of the dosimeters and patient , and a slight shift in the fov of a few centimeters can notably alter the radiation received by the dosimeters ( 23 ) . it should be noted that comparing the performance of devices based solely on dosimetric studies is not possible . the purpose of dosimetry comparisons is not to determine a better device , and diagnostic needs dictate the extent of necessary doses . due to the availability of various fov sizes in dental cbct , as well as various positions of fovs within the head and neck region , each point around the main beam can show high variability on the basis of its position relative to the isocenter ( 23 ) . using the same phantom this technique has proved reproducible , although some dosimeter locations might have some degree of variation , such as those placed close to the cranial and caudal ends of the x - ray beam , as well as those close to the skin , thyroid , and back of the neck . hence , patient position can also alter the dose of the head and neck organs such as the thyroid . in order to reduce this , the use of smaller fovs is suggested , which might significantly reduce the dose ( 10 , 23 ) . since the absorbed dose is an average value , the only way to improve the accuracy of dosimetry is to use as many tlds as possible . in order to ensure the precision of the measurements in the present study , numerous tlds were placed throughout the head and neck area to cover the head and neck organs still , these results should be cautiously compared with those of other studies , since the number of tlds and their positions differ in all studies , especially as many prior studies have used too few tlds ( 3 , 10 , 11 , 16 , 19 , 21 , 23 ) . utilizing too few tlds might result in either the overestimation or underestimation of such positioning alterations , with variations of up to 80% . this can become more vivid in the case of certain tissues such as the red bone marrow , thyroid , and salivary glands ( 23 ) . such excessive variations in the doses received by each organ , as caused by different cone beam collimations and exposure factors , imply that the average effective doses should not be used for comparisons between different radiographic techniques . still , it seems that the cbct dose is higher than that involved in plain dental radiographic techniques , while still being below that of multi - slice ct methods ( 16 , 17 , 23 ) . this can be increased by increasing the mas and kv and using a larger fov ( 23 ) . depending on the collimation features , maximum fov , and the quality of the diagnostic image , cbct units could be applied for different purposes ( 3 , 9 , 16 , 17 , 23 ) . hence , an important factor when optimizing the radiation dose is to ensure the proper quality of the produced image by employing appropriate protocols such as the proper size and position of the fov ( 9 , 23 ) . the alara principle dictates the usage of strategies to lower the radiation dose to that which is reasonably achievable ( 14 , 19 , 27 ) by choosing the most appropriate settings , fov , and adequate lead protection ( 14 ) . decreasing the fov as a collimation method is one of the approaches suggested to reduce the radiation dose . the choice of fov should be the smallest option that would capture a given region of interest ( 8 , 14 ) . it is observed that reducing the size of the fov can reduce the radiation dose ( 8) . therefore , it is recommended to reduce the fov when the lesions are limited to one jaw in order to reduce the absorbed dose . however , in this study , reducing the fov did not reduce the absorbed dose , although it did increase the effective dose . both newtom scanners automatically adjust the resolution to a higher level when a smaller fov is selected , and they do not allow for manual correction of the resolution modification . we also manually simulated this reverse resolution fov association of the newtom devices on the promax 3d device . a higher resolution increases the radiation dose ( 8) . it is possible that the devices are designed this way in order to improve the signal - to - noise ratio with a higher resolution . still , this strategy contradicts the philosophy of reducing the fov for the sake of reducing the dose . usually , offering many options for the operator to manually change the device s settings is not practical and so it might actually be desirable to have fewer ( but less confusing ) options . however , manufacturers are also suggested to let the operator have a minimum of control over the configurations . if a greater resolution is needed for a particular diagnostic task , it is important that the signal - to - noise ratio is adequate for the task . the worst form of excess exposure is a level too low to provide adequate image quality , which necessitates a repeat . however , there might be instances where a smaller fov with a lower resolution suffices for adequate image quality . as we wanted to make the groups uniform , the promax 3d scanner , which allowed manual resolution adjustment , was manually configured at its high resolution option for the smaller fov . the high and normal resolutions employ the same exposure parameters , while the low resolution might reduce the effective dose to about 10% of the normal dose resolution . generally , a low dose leads to an image with a low signal - to - noise ratio ( 8) . these resolution increases might be the reason for the increases observed in the promax 3d group when the smaller fov was used . however , it should be noted that the number of tlds used in this study was more than the number used in many previous examinations . additionally , due to the limited number of tlds available as well as other technical difficulties , we were limited to disregarding some areas and focusing on more critical organs ( 16 ) . as another limitation , it might be argued that the failure to match the devices configurations might confound the results . it should be taken into account that it was impossible to match the devices , since their settings are determined by their manufacturers ; therefore , all previous studies faced this issue as well . these prior studies were limited to comparing the same - name settings of different devices , without attempting to match the exposure parameters ( 3 , 16 ) . furthermore , as another constraint , a cbct study should also evaluate the quality of the image together with the extent of the absorbed doses . according to lorenzoni et al . ( 22 ) , the important factors in cbct imaging are the size and position of the fov and the quality of the image . the latter was not evaluated in this study , although other studies were similarly limited by this factor . other limitations centered on the numerous technical difficulties must be recognized , including the severe rarity of rando phantoms and tlds , very high sensitivity of tlds , difficulty of their transportation due to their very high fragility , lack of adequate laboratory experts , which all made conducting this study very difficult . however , we re - performed most of the steps involved in this study from the scratch ( with each step consisting of all exposures plus all calibration steps ) in order to ensure the validity of the results . while recognizing the limitations of this study , it seems that the devices did not differ considerably in terms of the generated dose . decreasing the fov but increasing the resolution did not reduce the absorbed dose and might actually increase the effective dose . when reducing the fov for the sake of x - ray safety , the resolution should be taken into consideration . the risk of absorbed doses is higher in the submandibular gland , mandible trunk and ramus , and red bone marrow . in terms of the three evaluated devices , it seems that resolution might play a more important role than fov in determining the absorbed dose of organs inside the fov or close to it such as the thyroid gland . however , the doses absorbed by organs farther away from the fov seem to be more affected by the size of the fov than the resolution . the structures that are distant from the fov ( such as the calvarium ) seem less likely to be affected by changes in the size of the fov or resolution . it should be noted that without statistical comparisons , these suggestions should be considered as theorems and not as evidence . future studies should hence conduct more experiments and perform further statistical analyses to assess these suggested theorems ( 28 , 29 ) .

backgroundmarketing new radiography devices necessitates documenting their absorbed x - ray doses . since the current literature lacks studies on new devices , we assessed the doses of two new devices that had not previously been assessed.objectivesthe new devices were compared to the promax three dimensional ( 3d ) scanner at two fields of view ( fov ) in nine critical head and neck tissues and organs.materials and methodsseventeen thermoluminescence dosimeters positioned in an average - sized male rando phantom were used to determine the dosimetry of the three cone beam computerized tomography devices ( newtom vgi , newtom 5 g , and promax 3d ) at two field of views ( fovs ) , one small and one large . the exposure by each device per fov was performed five times ( 30 exposures ) . the absorbed and effective doses were calculated for the thyroid , parotid , submandibular gland , sublingual gland , calvarium , cervical vertebra , trunk of the mandible , and mandibular ramus . the doses pertaining to the different devices , the fovs , and the tissues were compared using the kruskal - wallis , mann - whitney u , and wilcoxon tests.resultsthe average absorbed doses , respectively , for the large and small fovs were 17.19 and 28.89 mgy in the promax 3d , 19.25 and 35.46 mgy in the newtom vgi , and 18.85 and 30.63 mgy in the newtom 5 g . the absorbed doses related to the fovs were not significantly different ( p value = 0.1930 ) . however , the effective doses were significantly greater at the smaller fovs / higher resolutions ( p = 0.0039 ) . the doses of the three devices were not significantly different ( p = 0.8944 ) . the difference among the nine organs / tissues was significant ( kruskal - wallis p=0.0000).conclusionthe absorbed doses pertaining to the devices and the fovs were not significantly different , although the organs / tissues absorbed considerably different doses .

musculoskeletal disorders ( msds ) are a major problem for patients as well as for society and can lead to functional limitation and absence from work [ 1 , 2 ] . health care workers have physically and psychologically demanding work and are at high risk of developing long term msds and sickness absence [ 35 ] . research regarding work ability and prevention of sickness absence is a great challenge because of its complexity . the international classification of function ( icf ) provides a classification system for function and disability associated with health . the theoretical model of icf explains functioning as all body function , activity and participation as well as personal and environmental factors that interact with these concepts . hence , work ( dis)ability may be explained by physical , mental and social aspects of functioning , in addition to environmental and organizational demands of a person s work and personal factors that influence his or her capacity to meet these demands . socio - demographic factors such as age , gender and educational level are important predictors for work ability [ 711 ] . other factors associated with insufficient work ability are heavy physical work [ 9 , 10 , 12 , 13 ] , high pain intensity [ 12 , 14 ] , social and environmental workplace factors [ 15 , 16 ] , and psychological variables [ 8 , 11 , 17 ] . besides these factors , some studies have focused on the relation between deconditioning and poor work ability [ 11 , 1820 ] . deconditioning refers to a decrease of capacity over time expressed by weakened muscle strength , reduced aerobic fitness or altered coordination during activity . although it is argued that deconditioning may be a result of fear avoidance and altered behavioral performance , the evidence is inconclusive [ 19 , 2124 ] . there is also conflicting evidence concerning deconditioning among patients with chronic low back pain ( lbp ) [ 19 , 25 , 26 ] . although self - reported functioning and physical tests have been used to predict and evaluate work ability in several studies , only a few studies have compared the function of employees on sick leave and employees still working despite msds [ 17 , 2730 ] . it has been found that employees on sick leave have poorer health and more disability [ 17 , 28 ] , higher perceived workload , more fear - avoidance beliefs [ 27 , 29 ] , lower pain acceptance [ 17 , 27 , 28 ] and lower functional capacity compared to employees still working . more knowledge about the differences between employees on sick leave due to msds and employees staying at work despite msds can give us insight into what could be emphasized in work interventions and contribute to increase work participation . employment policies in scandinavian countries have focused on active approaches for employees with reduced work ability . partial sick leave has been used to give employees the possibility to combine work and sickness benefits . however , there is a lack of evidence regarding functional ability in workers on partial sick leave compared to those on full sick leave . the aim of this study was to describe self - reported and physically tested function in health care workers with msds and to examine how function was associated with work participation . by using the icf s model to understand the complexity of work ability , a wide range of bio - psycho - social and work - related factors were investigated . this study examines possible differences of functioning in ( a ) health care workers staying at work despite msds , ( b ) on partial sick leave , or ( c ) on full sick leave . this study was part of a larger study called function , activity and work of health care workers with msds in the municipality of bergen . the participants were recruited from the department of health and social service in the municipality of bergen , norway , from january 2012 to december 2013 . about 7,000 health care workers are employed in this department ; working in nursing homes , home care service and in special homes for disabled . through their managers and/or brochures we invited employees who were on sick leave or at risk of being sick - listed due to msds , to a functional evaluation . health care workers with msds took direct contact with the university of bergen and booked an appointment with a physiotherapist in the project . exclusion criteria were insufficient knowledge of the norwegian language and being on full sick leave for more than 4 months continuously . within 2 weeks after requesting an appointment , the participants met for an evaluation completed by a physiotherapist in the project . the evaluation ended with a verbal and written presentation of the self - reported and physical findings for all participants , except 56 who were recruited to an randomized controlled trial for participants with low back pain ( lbp ) . they practiced several times together before the start of the project and also examined the first 10 participants together . the participants performed the tests that required minimal effort first , in order to prevent fatigue and pain from having a significant impact on scores . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . test retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities . retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . chicago , il , 2011 ) and matlab ( version 7.10 ; mathwork , 2010 ) . differences between groups ( full sick leave , partial sick leave , not sick leave / working ) were analyzed by chi square exact for categorical data and kruskal wallis and mann whitney u tests for continuous variables . a personal mean was given for missing data if < 30 % of a sub - scale was missing . to examine which factors were associated with being on sick leave , a logistic regression analysis was performed using sick leave groups as the dependent variable and several independent variables ( gender , age , self - reported physical and mental function , perception of work environment , physical tests ) . we estimated both an unadjusted model for each independent variable and a fully adjusted model containing all independent variables . from those models and a correlation analysis we selected a final model based on statistical significance and clinical relevance . work demands were reported in both back - ground data and in the dcsq and reflect similar aspect . we chose the dcsq in the logistic regression model because this is a standardized measurement tool . taking into account multiple effects , a bonferroni adjustment was too conservative , therefore we used p 0.01 as marginal level . the participants were recruited from the department of health and social service in the municipality of bergen , norway , from january 2012 to december 2013 . about 7,000 health care workers are employed in this department ; working in nursing homes , home care service and in special homes for disabled . through their managers and/or brochures we invited employees who were on sick leave or at risk of being sick - listed due to msds , to a functional evaluation . health care workers with msds took direct contact with the university of bergen and booked an appointment with a physiotherapist in the project . exclusion criteria were insufficient knowledge of the norwegian language and being on full sick leave for more than 4 months continuously . within 2 weeks after requesting an appointment , the participants met for an evaluation completed by a physiotherapist in the project . the evaluation ended with a verbal and written presentation of the self - reported and physical findings for all participants , except 56 who were recruited to an randomized controlled trial for participants with low back pain ( lbp ) . they practiced several times together before the start of the project and also examined the first 10 participants together . the participants performed the tests that required minimal effort first , in order to prevent fatigue and pain from having a significant impact on scores . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . age , gender , marital status , number of children , education , exercise , smoking , and duration of sick leave were registered . in addition , different questionnaires regarding pain , function , psychosocial health and work environment were filled in . for the logistic regression analysis we dichotomized educational level into secondary school / vocational education and university degree , episodes of sick leave into 01 and 2 , exercises into < 1/week and 1/week , and smoking to yes ( yes , daily and yes , sometimes ) and no . the nprs has shown better reliability and responsiveness than the visual analogue scale [ 33 , 34 ] . the participants marked on a pain drawing the area or areas that had been painful the last 14 days . subjective health complaints inventory ( shc ) consists of 29 items regarding subjective somatic or psychological complaints experienced during the last month . hopkins symptoms checklist ( hscl-25 ) has 25 items with 10 items for anxiety symptoms and 15 for depression symptoms . the hscl has been shown to have a satisfactory validity and reliability in psychiatric outpatients and in a normal population [ 38 , 39 ] . the tampa scale of kinesiophobia ( tsk ) consists in short form of 13 items concerning fear of movement / re - injury . the tsk has been validated in numerous studies including patients with neck pain , acute and chronic lbp and fibromyalgia [ 4143 ] . the short form of the rebro musculoskeletal pain screening questionnaire has 10 items and is appropriate for clinical and research purposes since it is nearly as accurate as the longer version . norwegian function assessment scale ( nfas ) is an instrument for self - report of work related functioning with basis in the icf s classification system . retest reliability has been tested in a normal population and found acceptable . to measure social and psycho - social characteristics of jobs the demand - control - support questionnaire ( dcsq ) , based on a shortened and modified version of the job - demand- social support model ( jcq ) [ 46 , 47 ] . the psychometric properties of dcsq have been demonstrated to be satisfactory [ 46 , 47 ] . the short form-12 ( sf-12 ) , a 12-item version of the sf-36 , was used to measure physical and mental health - related quality of life . the sf-12 has shown good internal consistency , validity , and responsiveness in patients with lbp . bis can refer to high internal consistency , adequate reliability and good convergent and discriminative validity . body mass index was calculated by dividing weight ( kg ) by the square of height ( m ) . the physical tests were chosen to get a general impression of physical function according to body functions or activities in the icf s model . a more detailed description of the tests is given in table 1.table 1description of physical testsphysical testscontentscoreicf - dimensionsglobal body examination ( gbe ) ( 51 , 52)six tests : truncal flexibility and ability to relax during passive movements : elbow - drop flexibility , lumbar - sacral flexibility , head rotation resistance and resistance to hip circumduction , hip - knee flexion and arm / shoulder flexioneach test : 07 . healthy ( 34 individuals ) : median = 5.5 , mean = 7.2body functionback performance scale ( bps ) ( 53 , 54)five tests reflecting mobility - related activities for trunk and lower extremities ( sock - test , pick - up test , roll - up test , fingertip - to - floor and a lift test where a box weighing 4 kg ( women ) or 5 kg ( men ) is lifted from floor to waist for 1 min).each test : 03 . normative data for people without back pain ( n = 150 ) : median = 0 , mean = 0.8activity / participationhigh lift testa high lift test was a modified lift test included in bps . the participants lift a box of 2 kg ( for women ) or 3 kg ( for men ) from waist to shoulder height and back again . the lifting technique was optional.number of lifts performed in 1 min is counted.activity/participationbieringsrensen test ( 5558)static endurance of the back . participants are positioned prone with the upper body extending beyond the edge of the plinth and the lower body is fixed to the bench with three straps.the length of time holding the upper body straight is recorded . max time 240 s. healthy ( 31 individuals ) : median = 138body functionabdominal endurance / strength ( 59 , 60)three levels of dynamic sit - up test with increased demand for each level . the participants are supine with the knees flexed and with feet supported on the plinth by the tester.the number of completed repetitions is counted ( 015).body functiontender points ( 61)18 defined fibromyalgia tender points with four kilos pressure are tested.painful points are countedbody function description of physical tests the global body examination ( gbe ) is used to assess bodily function in patients with long - lasting musculoskeletal pain and/or with psychosomatic complaints . discriminating ability between healthy and different patients groups has shown to be very good to excellent . good inter - tester reliability has been demonstrated in a former version of the gbe . back performance scale ( bps ) consists of five tests reflecting mobility - related daily activities for trunk and lower extremities retest reliability and responsiveness to change have been demonstrated in patients with long - lasting lbp [ 53 , 54 ] . this is a modified lift test from the lifting test in the bps , but not described elsewhere . to assess static endurance of the back extensors we used the biering retest reliability has been reported as satisfactory , but variability has been high [ 5658 ] . for testing of abdominal endurance / strength we chose a three levels dynamic sit - up test with increasing demands for each level [ 59 , 60 ] . four kilos pressure of 18 defined fibromyalgia tender points were tested , and painful points counted . the study was accepted by the regional committee for medical and health research ethics , western - norway , and was performed according to the helsinki declaration . chicago , il , 2011 ) and matlab ( version 7.10 ; mathwork , 2010 ) . differences between groups ( full sick leave , partial sick leave , not sick leave / working ) were analyzed by chi square exact for categorical data and kruskal wallis and mann whitney u tests for continuous variables . a personal mean was given for missing data if < 30 % of a sub - scale was missing . to examine which factors were associated with being on sick leave , a logistic regression analysis was performed using sick leave groups as the dependent variable and several independent variables ( gender , age , self - reported physical and mental function , perception of work environment , physical tests ) . we estimated both an unadjusted model for each independent variable and a fully adjusted model containing all independent variables . from those models and a correlation analysis we selected a final model based on statistical significance and clinical relevance . work demands were reported in both back - ground data and in the dcsq and reflect similar aspect . we chose the dcsq in the logistic regression model because this is a standardized measurement tool . taking into account multiple effects , a bonferroni adjustment was too conservative , therefore we used p 0.01 as marginal level . a total of 250 participants ( 92.4 % women ) were consecutively recruited to the functional evaluation study . self - reports showed that 83 % of the participants had experienced their present complaints for more than 8 weeks . about 50 % reported previous contact with health personal for treatment of their msds . the group not on sick leave ( working group ) included 168 participants and the groups on partial and full sick leave each included 41 participants . in table 2 , workers on partial sick leave had statistically significant longer duration of sick leave compared to workers on full sick leave . the group on full sick leave reported more heavy physical work compared to the working group . the differences in function , health and work related variables between the three groups are presented in table 3 . major differences in self - reported and physically tested function were observed between the group on full sick leave and the working group . participants on full sick leave had statistically significant poorer function and higher ( worse ) score on rebro questionnaire compared to those working . when comparing those on partial and full sick leave , the group on partial sick leave had statistically significant ( p < 0.05 ) better scores on nfas , the physical dimension of sf-12 , nprs , rebro questionnaire , bsi , gbe and high lift test , compared to the group on full sick leave.table 2demographic variablesvariablesgr.1 working n = 168 n ( % ) gr.2 partial sick leave n = 41 n ( % ) gr.3 full sick leave n = 41 n ( % ) p value sosiodemographic factors age 49 ( 2164)47 ( 2662)49 ( 2167).414 gender , women155 ( 85.4)41 ( 100)35 ( 95.8).052 education.273 secondary school11 ( 6.6)2 ( 4.9)5 ( 12.2 ) vocational education82 ( 49.1)23 ( 56.1)24 ( 58.5 ) university degree74 ( 44.3)16 ( 39.0)12 ( 29.3 ) work status full - time work110 ( 65.5)23 ( 56.1)24 ( 58.5).436 sick leave ( weeks ) 0 ( 0)9 ( 262)3 ( 010 ) < .001 sick leave episodes ( number).214 027 ( 17.1)8 ( 19.5)10 ( 24.4 ) 132 ( 20.3)13 ( 31.7)10 ( 24.4 ) 299 ( 62.7)20 ( 48.8)21 ( 51.2)type of work ( mainly ) .034 sedentary work / sitting13 ( 7.8)1 ( 2.5)0 ( 0 ) standing / walking97 ( 58.1)23 ( 57.5)17 ( 42.5 ) heavy physical work57 ( 34.1)16 ( 40.0)23 ( 57.5 ) health related factors main disorder.067 neck- and shoulder pain53 ( 32.3)14 ( 34.1)7 ( 17.1 ) low back pain61 ( 36.3)19 ( 46.3)19 ( 46.3 ) widespread pain3 ( 25.6)3 ( 7.3)10 ( 24.4 ) other10 ( 6.0)5 ( 12.2)5 ( 12.2)smoking.403 yes , daily31 ( 18.8)9 ( 22.0)13 ( 32.5 ) yes , sometimes18 ( 10.9)4 ( 9.8)5 ( 12.5 ) no116 ( 70.3)28 ( 68.3)22 ( 55.0)exercise.752 < 1/week30 ( 17.9)7 ( 17.0)9 ( 21.9 ) 12/week73 ( 43.5)17 ( 41.5)13 ( 31.7 ) 35/week65 ( 38.7)17 ( 41.5)19 ( 46.4)body mass index 24.9 ( 18.842.1)25.2 ( 17.639.6)25.4 ( 17.236.4).904 median ( min max ) kruskal wallis test chi square , exact test bold = significant at p < 0.05table 3differences in health , work characteristics and function between three groups : those working , on partial sick leave , or on full sick leavevariablesngr wallis testmedian ( min max)median ( min max)median ( min max ) p values pain pain intensity2506 ( 210)5 ( 310)7 ( 210 ) 0.005 pain drawing area25010 ( 170)9 ( 237)10 ( 140)0.72 health factors and function rebro questionnaire25044 ( 1484)46 ( 1770)56 ( 3280 ) 0.001 sf-12 mental23250.1 ( 26.761.1)48.6 ( 30.563.2)48.4 ( 2.961.2)0.412sf-12 physical23245.5 ( 12.859.9)42.2 ( 24.33.2)38.7 ( 24.648.4 ) < 0.001 nfas2501.2 ( 1.002.10)1.23 ( 1 ( 1.00 1.84)1.42 ( 1.0 2.38 ) < 0.001 hscl2441.44 ( 1.002.87)1.42 ( 1.002.58)1.45 ( 1.043.08)0.665shc ( n)17910 ( 315)9 ( 313.0)10 ( 315)0.245tsk24721.7 ( 13.046.0)21.0 ( 13.035.8)21.0 ( 13.043.0)0.952bis24416.5 ( 042)17.0 ( 036)24.0 ( 241)0.065 work characteristics dcsq social2470.78 ( 0.221.00)0.78 ( 0.331.00)0.72 ( 0.331.00)0.108dcsq demand2460.67 ( 0.001.00)0.67 ( 0.270.93)0.67 ( 0.271.00)0.214dcsq control2400.67 ( 0.220.94)0.64 ( 0.390.83)0.67 ( 0.390.83)0.186 physical assessment acr - tender points ( n)2507 ( 018)6 ( 018)7 ( 018)0.616gbe flexibility25016 ( 235)16 ( 530)19 ( 535 ) 0.038 high lift test ( n)25016 ( 029)15 ( 824)13 ( 325 ) < 0.001 abdominal strength ( n)24812.5 ( 015)9 ( 015)5 ( 015 ) < 0.001 back strength ( s)24870 ( 0240)33 ( 0220)36 ( 0240 ) 0.002 bps2503 ( 015)4 ( 011)6 ( 013 ) < 0.001 sf-12 quality of life , short form-12 , nfas norwegian function assessment scale , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , bis bergen insomnia scale , bmi body mass index , dcsq demand - control - support questionnaire , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p < 0.05 demographic variables chi square , exact test bold = significant at p < 0.05 differences in health , work characteristics and function between three groups : those working , on partial sick leave , or on full sick leave sf-12 quality of life , short form-12 , nfas norwegian function assessment scale , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , bis bergen insomnia scale , bmi body mass index , dcsq demand - control - support questionnaire , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p < 0.05 the results of the logistic regression analysis are presented in table 4 . the group on full sick leave and the group on partial sick leave were compared with the working group . complete data were available in 210 participants ( 142 working , 30 on full sick leave , 38 on partial sick leave ) . reduced level of the physical dimension of sf-12 and on high lift test were significantly related to full sick leave ( or 0.86 , p < 0.001 ) ( or 0.79 , p = 0.002 ) . there was also a tendency ( p < 0.05 ) that being on full sick leave was associated with gender , the mental dimension of sf-12 , the hscl-25 , the demand dimension of the dcsq , and the abdominal strength test . the physical dimension of sf-12 ( or 0.91 , p = 0.005 ) was the only variable that was associated to partial sick leave ( table 4 ) . table 4logistic regression comparing group on full sick leave and on partial sick leave with the working groupvariablesnunadjusted model , full sick leaveadjusted model , full sick leave n = 30nunadjusted model , partial sick leaveadjusted model , partial sick leave n = 38or95 % ci p valueor95 % ci p valueor95 % ci p valueor95 % ci p valuegender410.49(0.171.38)0.1760.10(0.020.67)0.01841 age411.00(0.971.04)0.8840.96(0.921.01)0.126410.98(0.951.01)0.2410.97(0.941.01)0.164smoking400.67(0.461.00)0.0490.84(0.471.51)0.567410.92(0.611.41)0.7111.16(0.691.96)0.580sf-12 physical370.86(0.820.91 ) < 0.001 0.86(0.790.94 ) < 0.001 400.91(0.870.96 ) 0.001 0.91(0.850.97 ) 0.005 sf-12 mental370.96(0.911.01)0.1100.89(0.801.00)0.043400.98(0.931.04)0.5090.95(0.871.03)0.210hscl401.99(0.844.72)0.1180.10(0.010.90)0.040401.16(0.452.98)0.7540.25(0.041.43)0.119dcs support410.12(0.020.80)0.0290.67(0.0218.82)0.816411.20(0.168.77)0.8611.96(0.1526.35)0.611dcs demand403.63(0.324.86)0.18937.07(1.73792.84 ) . 021414.75(0.6932.65)0.1136.65(0.5876.37)0.128dcs control380.18(0.012.88)0.2234.43(0.03578.40)0.549400.21(0.013.34)0.2720.16(0.014.88)0.295gbe flexibility411.07(1.021.12 ) 0.009 1.02(0.941.10)0.676411.00(0.951.05)0.9570.98(0.921.04)0.559high lift test410.82(0.750.90 ) < 0.001 0.79(0.680.91 ) 0.002 400.97(0.891.06)0.4781.04(0.921.17)0.567abdominal strength400.84(0.780.91 ) < 0.001 0.84(0.730.97)0.014410.94(0.871.00)0.0650.95(0.851.06)0.335back strength410.99(0.981.00)0.0131.00(0.001.01)0.385410.99(0.991.00)0.0390.99(0.991.00)0.305bps411.29(1.151.45 ) < 0.001 0.85(0.661.09)0.206411.15(1.031.29)0.0161.06(0.891.28)0.513 sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , dcsq demand - control - support questionnaire , gbe global body examination , bps back performance scale . bold = significant at p 0.01 only womentable 5logistic regression - full model . comparing full sick leave and partial sick leave with the working groupn = 189nunadjusted model , full sick leaveadjusted model , full sick leavenunadjusted model , partial sick leaveadjusted model , partial sick leaveor(95 % ci ) p valueor(95 % ci ) p valueor(95 % ci ) p valueor(95 % ci ) p valuegender410.49(0.171.38)0.1760.06(0.000.70)0.02541age411.00(0.971.04)0.8840.96(0.911.02)0.160410.98(0.951.01)0.2410.98(0.931.02)0.284education410.52(0.251.09)0.0830.67(0.192.39)0.541410.80(0.401.62)0.5410.49(0.171.38)0.177working ( full / partly)411.37(0.682.75)0.3811.93(0.527.17)0.324411.51(0.753.03)0.2451.16(0.403.40)0.781sick leave episodes410.63(0.311.25)0.1840.42(0.131.38)0.153410.57(0.281.13)0.1090.53(0.201.38)0.190smoking400.67(0.461.00)0.0490.72(0.361.44)0.351410.92(0.611.41)0.7111.25(0.672.32)0.486exercise410.77(0.331.79)0.5470.94(0.194.70)0.937411.06(0.432.61)0.9061.49(0.346.45)0.594pain categories410.71(0.471.08)0.1070.83(0.332.09)0.692410.79(0.521.19)0.2660.89(0.461.73)0.738pain drawing area410.99(0.961.02)0.5820.98(0.931.04)0.572410.98(0.941.02)0.2930.97(0.921.02)0.280nprs411.33(1.101.60 ) 0.003 0.95(0.641.41)0.809410.95(0.791.13)0.5460.77(0.561.06)0.112rebro questionnaire411.05(1.021.08 ) < 0.001 1.04(0.981.11)0.211411.01(0.991.04)0.3261.02(0.961.08)0.470nfas4120.89(5.6277.70 ) < 0.001 2.47(0.1348.00)0.550412.99(0.7212.47)0.1320.56(0.039.48)0.685sf-12 physical370.86(0.820.91 ) < 0.001 0.88(0.790.98)0.015400.91(0.870.96 ) 0.001 0.89(0.810.97 ) 0.006 sf-12 mental370.96(0.911.01)0.1100.91(0.801.03)0.131400.98(0.931.04)0.5090.94(0.841.05)0.246hscl401.99(0.844.72)0.1180.08(0.011.15)0.063401.16(0.452.98)0.7540.66(0.085.35)0.699tsk411.00(0.951.06)0.8630.86(0.760.98)0.019410.99(0.941.05)0.7800.95(0.861.05)0.333dcs social410.12(0.020.80)0.0290.34(0.0116.50)0.583411.20(0.168.77)0.8613.13(0.1373.77)0.479dcs demand403.63(0.5324.86)0.189153.40(2.778,508.64)0.014414.75(0.6932.65)0.1135.53(0.27112.31)0.266dcs control380.18(0.012.88)0.2231.11(0.00363.95)0.973400.21(0.013.34)0.2720.14(0.0011.13)0.379bis401.04(1.001.07 ) . 2230.87(0.741.03)0.097410.94(0.871.00)0.0650.94(0.831.07)0.343back strength410.99(0.981.00)0.0131.00(0.991.02)0.597410.99(0.991.00)0.0390.99(0.981.01)0.314bps411.29(1.151.45 ) < 0.001 0.87(0.631.21)0.415411.15(1.031.29)0.0161.10(0.871.38)0.432 nprs numeric pain rating scale , nfas norwegian function assessment scale , sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , dcsq demand - control - support questionnaire , bis bergen insomnia scale , bmi body mass index , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . bold = significant at p 0.01 logistic regression comparing group on full sick leave and on partial sick leave with the working group sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , dcsq demand - control - support questionnaire , gbe global body examination , bps back performance scale . bold = significant at p 0.01 logistic regression - full model . comparing full sick leave and partial sick leave with the working group nprs numeric pain rating scale , nfas norwegian function assessment scale , sf-12 quality of life , short form-12 , hscl hopkins symptoms checklist , shc subjective health complaints , tsk tampa scale of kinesiophobia , dcsq demand - control - support questionnaire , bis bergen insomnia scale , bmi body mass index , acr - tender points american criteria of rheumatology , gbe global body examination , bps back performance scale . in this study we found that workers on full sick leave had reduced self - reported and physically tested function compared to workers still working despite msds , as well as compared to those on partial sick leave . lower physical function measured by the physical function score on sf-12 and the high lifting test were strongest associated with being on full sick leave . being female , lower mental health score ( worse ) on sf-12 , in addition to lower scores ( better ) on the hscl-25 , increased self - reported work demands ( dcsq ) and lower abdominal strength showed a tendency to be associated with being on full sick leave . for the group on partial sick leave , only the physical function scale of sf-12 was associated with being on sick leave , those on sick leave having lower ( worse ) scores . our findings are supported by several studies , but there are also new and interesting findings . low self - reported physical health and disability have been found to be associated with being on sick leave in patients with chronic lbp . in a systematic review of factors that promote staying at work with msds , an association was found between low perceived physical disability and staying at work . however , only a few studies have compared measures of physical tests / capacity between workers on sick leave and workers who continue working despite pain . compared functional capacity between workers staying at work despite msds , workers on sick leave due to msds and a group of healthy workers . in accordance with our findings , they found that the two groups with msds had significantly lower functional capacity than the healthy group , with the lowest capacity observed in the group on sick leave . other studies have shown that physical tests can predict return to work after being on sick leave . cardiovascular fitness was identified as one of the strongest predictors for return to work in a norwegian study . in a systematic review , better results on physical tests , and especially the lifting test , appeared to be predictive of work participation for patients with msds . as our study was cross - sectional , prediction of work participation could not be estimated . low lifting capacity was , however , strongly associated with being on full sick leave . an explanation may be that lifting captures several components such as gripping , holding , bending and lowering . several explanations were considered in order to explain why the participants on full sick leave in the present study had lower scores on the physical tests compared to workers not on sick leave . a possible explanation could have been different level of exercise between groups . however , the three groups in the present study reported quite similar level of regular exercising , in accordance with earlier research [ 12 , 27 ] . increased fear avoidance has been observed in workers on sick leave with msds [ 19 , 27 , 29 ] . our findings did not support this association , as scores on the tsk were similar for those on sick leave versus those working . even if a state of deconditioning is present , the functional capacity could still be sufficient to meet actual work demands , especially if they are not too excessive . however , health care workers usually have physically demanding work , including lifting , transferring patients and working in uncomfortable positions . in accordance with several studies showing that perceived workloads are associated with being on sick leave [ 12 , 19 , 27 ] , the workers on full sick leave in this present study reported higher perceived work demands than the other two groups . the reason might be more demanding work tasks for this group , but decreased physical capacity might also influence an individual s perception of work demands . this highlights the need for research that takes into account work demands and work environment for specific occupational groups . high pain intensity has also been associated with being on sick leave [ 8 , 11 , 17 ] . our study showed a statistically significant difference of pain intensity between the groups , with the highest level in the group on full sick leave and the lowest in the group on partial sick leave . however , there was only one point in difference on the nprs between those on full sick and the working group . it is therefore not likely that the pain level was of great importance for the result regarding physical functioning in the present study . in previous years , much attention has been given to the role of psycho - social factors related to work ability [ 17 , 64 ] . there were only small differences in measures of the psychological variables between the groups in our study . reduced physical function was more strongly associated with being sick - listed than pscyho - social factors , also reported in previous research [ 12 , 27 ] . there was only a tendency that being on full sick leave was associated with mental health , and the results were conflicting . the group on full sick leave showed worse function at the mental health component of sf-12 , but surprisingly , better score on hscl-25 . the hscl-25 has a higher number of items related to mental health and may therefore provide a more precise picture than the less detailed generic questionnaire sf-12 . being on short time sick leave , as in our study , may to a lesser degree influence psycho - social factors . the authorities in norway , sweden and denmark have strongly promoted the use of partial sick leave as the primary choice , if sick leave is needed . it is assumed that partial sick leave has positive effects on health and well - being , compared to full - time absence , and it is believed to facilitate return to full - time work . to our knowledge , the present study is the first study comparing self - reported and physical tested function in workers with those on full or partial sick leave , due to msds . the group on partial sick leave had statistically significant better function on some of the functional questionnaires and physical tests compared to those on full sick leave . more women than men have been on partial sick leave according to register data from norway . further research is needed to get insight into factors affecting workers on partial and full sick leave , and the decisions around sick leave . the high number of participants in our study ( n = 250 ) gave us enough power to detect differences between workers on full , partial or not on sick leave , and to identify variables related to work status . in accordance with the icf- model a variety of demographic variables , questionnaires and physical tests were used to cover the different dimensions in the model when evaluating the participants functioning and working ability . we used well - known standardized questionnaires measuring pain , physical- and mental functioning and conditions at work . this is in line with wand et al . who argued that both self - reported and physically tested functioning need to be assessed to get a better understanding of msds and how they could be managed . the tests demonstrate good levels of reliability and validity , but two of the tests ( abdominal and high lift tests ) are still under evaluation . the physical tests were able to discriminate functioning between workers on sick leave and not , although most of the workers were not on long - term sick leave . this indicates that the test battery could be a useful assessment of function at an early stage of sick leave and a tool when giving advice about rehabilitation and work adaption . different batteries of physical tests are designed to evaluate work ability and daily functioning [ 18 , 66 , 67 ] . most of them are costly and time - consuming and are mainly used as assessment tools in the return to work process . in contrast to this , our test battery is cheap , quick to apply and require little equipment and therefore could also be a useful clinical tool in private practice for physiotherapists . workers were provided with information about the project by their leaders and through pamphlets and took direct contact to participate . although several workers were pushed by their employer to participate , we can not be sure that the least motivated and the workers with more complex health problems actually contacted us . our target population was workers on sick leave or at risk of becoming sick - listed due to msds . only 20 % of all that were examined had never been on sick leave due to msds before ; indicating that we have included the target group . interviews with managers in the midst of the total project supported that we had managed to get a representative sample of participants ( not yet published ) . although self - reported sick leave data has been evaluated as being less reliable than register recorded data , other studies [ 69 , 70 ] have demonstrated good agreement between self - reports and register data in cross - sectional design . the length of the last sick leave and number of sick leave episodes the last years are reported , but not the length of all absences . workers on sick leave could recently have returned to work , and workers on partial sick leave could have changed to full sick leave . our study did not record this , and it is quite surprising that the differences between the groups still were so significant . over 90 % of the participants in the present study were women working in the health- and social sector . being male and/or having a less demanding work may not affect work ability in the same way . the present study was cross - sectional and therefore causality can not be inferred , and only associations are reported . it was conducted in a single country with a highly established social insurance system , thereby reducing generalizability of the study to countries that have similar social and security system . more specific knowledge about occupational sub - groups is needed to catch groups at risk for prolonged sick leave , and further research in this field should emphasize longitudinal studies . the high number of participants in our study ( n = 250 ) gave us enough power to detect differences between workers on full , partial or not on sick leave , and to identify variables related to work status . in accordance with the icf- model a variety of demographic variables , questionnaires and physical tests were used to cover the different dimensions in the model when evaluating the participants functioning and working ability . we used well - known standardized questionnaires measuring pain , physical- and mental functioning and conditions at work . this is in line with wand et al . who argued that both self - reported and physically tested functioning need to be assessed to get a better understanding of msds and how they could be managed . the tests demonstrate good levels of reliability and validity , but two of the tests ( abdominal and high lift tests ) are still under evaluation . the physical tests were able to discriminate functioning between workers on sick leave and not , although most of the workers were not on long - term sick leave . this indicates that the test battery could be a useful assessment of function at an early stage of sick leave and a tool when giving advice about rehabilitation and work adaption . different batteries of physical tests are designed to evaluate work ability and daily functioning [ 18 , 66 , 67 ] . most of them are costly and time - consuming and are mainly used as assessment tools in the return to work process . in contrast to this , our test battery is cheap , quick to apply and require little equipment and therefore could also be a useful clinical tool in private practice for physiotherapists . workers were provided with information about the project by their leaders and through pamphlets and took direct contact to participate . although several workers were pushed by their employer to participate , we can not be sure that the least motivated and the workers with more complex health problems actually contacted us . our target population was workers on sick leave or at risk of becoming sick - listed due to msds . only 20 % of all that were examined had never been on sick leave due to msds before ; indicating that we have included the target group . interviews with managers in the midst of the total project supported that we had managed to get a representative sample of participants ( not yet published ) . although self - reported sick leave data has been evaluated as being less reliable than register recorded data , other studies [ 69 , 70 ] have demonstrated good agreement between self - reports and register data in cross - sectional design . the length of the last sick leave and number of sick leave episodes the last years are reported , but not the length of all absences . workers on sick leave could recently have returned to work , and workers on partial sick leave could have changed to full sick leave . our study did not record this , and it is quite surprising that the differences between the groups still were so significant . over 90 % of the participants in the present study being male and/or having a less demanding work may not affect work ability in the same way . the present study was cross - sectional and therefore causality can not be inferred , and only associations are reported . it was conducted in a single country with a highly established social insurance system , thereby reducing generalizability of the study to countries that have similar social and security system . more specific knowledge about occupational sub - groups is needed to catch groups at risk for prolonged sick leave , and further research in this field should emphasize longitudinal studies . health care workers on full sick leave due to msds have reduced function on self - reported and physically tested function , compared to those working despite msds , as well as compared to those on partial sick leave . lower physical function measured by the physical dimension on sf-12 and the high lift test were strongest associated with being on full sick leave , and only the physical dimension on sf-12 was associated with being on partial sick leave . the authors , tove ask , jan sture skouen , jrg assmus , and alice kvle have no conflict of interest .

purpose the aim of this study was to describe self - reported and physically tested function in health care workers with musculoskeletal disorders ( msds ) and to examine how function was associated with work participation.methodsa cross - sectional study was conducted . 250 health care workers attended an evaluation where self - reported and physical function were measured . differences between groups ( full sick leave , partial sick leave , not on sick leave / working ) were analyzed for categorical data ( chi square exact test ) and continuous variables ( kruskal wallis and mann whitney u tests ) . logistic regression analysis was performed to examine which factors were associated with being on sick leave.results participants on full sick leave had statistically significant poorer function compared to those working and the group on partial sick leave . logistic regression showed that a reduced level of the physical dimension of sf-12 and a high lift test were significantly related to full sick leave ( or 0.86 , p < 0.001 ) ( or 0.79 , p = 0.002 ) . the physical dimension of sf-12 was the only variable that was associated to partial sick leave ( or 0.91 , p = 0.005).conclusionhealth care workers on full sick leave due to msds have reduced function on self - reported and physically tested function , compared to those working despite msds , as well as when compared to those on partial sick leave . more knowledge about work ability in occupational sub - groups is needed .

pheochromocytoma is a rare but critical neuroendocrine tumour arising from chromaffin cells of the adrenal medulla . similar tumours arising from extra - adrenal chromaffin cells are called paraganglioma or extra - adrenal pheochromocytoma . recurrence of pheochromocytoma is uncommon , with incidence of up to 3 - 10% cases . we hereby present a case report of a 20-year - old female who presented to us with recurrent pheochromocytoma , 2 years after laparoscopic left total adrenalectomy , which was then managed successfully using laparoscopy . a 20-year - old female underwent laparoscopic total left adrenalectomy for 3.8 3.2 cm left adrenal pheochromocytoma in our hospital 2 years ago . her postoperative course was uneventful , and the patient was discharged with a normal blood pressure on tablet amlodipine 2.5 mg once daily . the histopathology confirmed the features suggestive of pheochromocytoma . she was readmitted to the endocrinology department of our hospital with palpitations , headache and breathlessness , suggesting a recurrence of pheochromocytoma . her urinary noradrenaline and normetanephrines were raised . magnetic resonance imaging ( mri ) of abdomen showed 2 cm 2 cm mass in the left suprarenal fossa appearing hyperintense on t2-weighted image [ figure 1 ] . however , metaiodobenzylguanidine ( mibg ) scan did not show any uptake , but fluorodeoxyglucose positron emission tomography ( fdg pet ) showed a metabolically active lesion in the left para - aortic region . the biochemical parameters including serum cortisol , parathyroid hormone ( pth ) , thyroid - stimulating hormone ( tsh ) and thyroxine were within normal limits , making the diagnosis of familial syndrome less likely . the patient was taken up for laparoscopic excision of the mass . t2-weighted image of mri of abdomen showing hyperintense lesion in left suprarenal fossa ( arrow ) after laparoscopic total left adrenalectomy suggestive of recurrence of pheochromocytoma the patient was prepared for surgery . a well - defined tumour of about 2 2 cm was seen abutting the left renal vein [ figure 2 ] . the swelling was carefully mobilised and excised , taking care to prevent any injury to renal vessels . a small lymph node of 1 1 cm was seen which was also removed . laparoscopic view showing tumour ( green arrow ) lying over the kidney bed ( grey arrow ) abutting the renal vein ( blue arrow ) laparoscopic view showing the hem - o - lok ( arrow ) placed over the adrenal vein during previous surgery postoperatively , the patient did well . the drain was removed on the second postoperative day , and the patient was discharged on the fourth postoperative day without any anti - hypertensive medication . the histopathology revealed 3.2 cm 2.2 cm 1.2 cm mass consistent with features of pheochromocytoma . the separately sent lymph node showed features of a necrotising granulomatous inflammation that was negative for acid - fast bacilli ( afb ) staining . goldstein et al . , from the experience of 48 years of open surgical excision of pheochromocytoma , observed the recurrence in 5 out of 64 pheochromocytomas and 4 out of 26 extra - adrenal pheochromocytomas over a mean follow - up of 12.6 years and 11.5 years , respectively . amar et al . in their 25 years ' experience of surgical excision of pheochromocytoma observed a recurrence rate of about 10% , with the risk of recurrence being 3.4-fold higher in patients with familial disease than in those with sporadic tumours . the major familial diseases associated with recurrent pheochromocytoma include multiple endocrine neoplasia ( men ) type 2 due to ret ( rearranged during transfection ) oncogene mutation ; von - hippel - lindau ( vhl ) disease due to a mutation in the vhl gene ; neurofibromatosis ( nf ) due to nf type 1 gene mutation ; and familial pheochromocytoma / paraganglioma syndrome due to a mutation in succinate dehydrogenase ( sdh ) types b , c and d. in patients suffering from vhl , walther et al . reported a recurrence in 1 out of 13 patients , while neuman et al . observed recurrence in 1 out of 29 patients undergoing open partial adrenalectomy . in men2 syndrome , recurrence rates varied from 18.5% at a mean follow - up of 3.6 years to 33% during a median follow - up of 54 - 88 months . another retrospective study involving 552 patients with men2a syndrome reported a recurrence of around 1% in patients undergoing minimally invasive adrenalectomy as compared to a 3% recurrence rate after open procedure ( p = 0.24 ) over a follow - up of 6 - 13 years . in our case , the patient underwent laparoscopic total adrenalectomy without any significant perioperative complications . in spite of the complete surgical resection of the tumour with no histological or radiological remnant disease , the patient still presented with a recurrence . moreover , the patient did not show any feature suggestive of a familial syndrome , although a genetic study could not be done to confirm the same . recurrence could be probably due to formation of a new tumour due to genetic predisposition , or incomplete resection of tumour , or seeding of new tumour nodules secondary to intraoperative handling incomplete resection of the tumour or adrenal tissue inadvertently left after total adrenalectomy could be a possible reason . . however , li et al . proposed the theory of iatrogenic pheochromocytomatosis for recurrence . they showed laparoscopic - induced seeding of tumour due to local spillage of tumour as a possible mechanism of recurrence . nomura et al . reported a case of recurrent pheochromocytoma that occurred 5 years after laparoscopic total adrenalectomy , which was successfully managed using a laparoscopic approach . studied 61 chromaffin neoplasms including 52 pheochromocytomas and 9 paragangliomas managed successfully using minimally invasive surgery . only one patient with recurrent pheochromocytoma was managed using a laparoscopic approach , while the other three were successfully managed using the retroperitoneoscopic approach . they followed the policy of using the posterior retroperitoneoscopic technique for tumours cranial to the renal vessels as in three cases ( two pheochromocytomas and one paraganglioma ) and a laparoscopic approach for tumours placed anterior to the renal vein as in the remaining case of recurrent pheochromocytoma . in another study by nambirajan et al . four patients had recurrence after open transperitoneal partial adrenalectomy , and the remaining patient developed recurrence after laparoscopic partial adrenalectomy . they reported that the procedure is easier if the previous surgery was laparoscopic , due to less adhesion formation . at our centre , we commonly use a laparoscopic approach for excision of pheochromocytoma , and have used the same approach successfully for the management of its recurrence . a laparoscopic approach should be preferred in the management of recurrent pheochromocytoma because of less morbidity , less adhesion formation , better visualisation , better intraoperative haemodynamic control and easier reoperation for recurrences , but extensive experience in minimally invasive and endocrine surgery is indispensable .

recurrence of pheochromocytoma after a total adrenalectomy is uncommon . such recurrent tumours are mostly managed by the open technique , with very few studies reporting laparoscopic management . we hereby report a case of successful laparoscopic management of a recurrent pheochromocytoma after total adrenalectomy for left adrenal pheochromocytoma .

the population of over 60 years is estimated to be 605 million people in the world and it is predicted that this number would reach 2 billion by 2050 . statistical indicators have shown that the process of population aging has also started in iran and it is predicted that during the 20-year interval from 2006 to 2026 , the median age of iranian population would be increased by 10 years . as the elderly population increases , their problems also increase . the elderly , due to physical and motion changes caused by aging , including reduced vision , reduced hearing , poor balance , slow motions , disabling diseases , drug consumption , dizziness , decentralization , and inappropriate conditions , would be more prone to indoor and outdoor accidents . therefore , accidents among seniors have an essential importance in a way that falling is one of the top 10 causes of death among seniors . falling is defined as an unintentional accident where the individual would lie on the floor or any lower surfaces accidentally and their feet would not be able to tolerate their weight . regarding the statistics of falling among the elderly , in 2008 , oren et al . , in a study entitled falling - asleep - related injured falls in the elderly , stated that about 30% of the elderly experience falling once or twice a year . also , in 2007 , bayat , in a study entitled determine the risk factors for falls in the elderly in geriatric nursing tehran , demonstrated that about 40% of over 85-year - old people experience falling . in iran , 12% of every 8000 people who refer to hospitals due to trauma are seniors over 60 years old and 70% of them have suffered from falling . studies have shown that the reasons for falling of the elderly are divided into two groups of internal reasons ( including weakness of the lower extremity muscles , poor balance , and decreased mental ability ) and external reasons ( factors related to environmental conditions ) . duglas , in a study conducted on falling in female seniors in 2013 , revealed that cognitive disorders , aging , anemia , dizziness , history of stroke , and weakness are the risk factors of falling and accidents . also , salarvand and birjandi , in a study entitled factors related to falling down in older adults , believed that physiological changes like specific changes occurring in neuromuscular and musculoskeletal systems are important factors of falling . after falling , 2030% of people suffer from severe injuries like hip fracture or head trauma which consequently increase their dependence on others , decrease their self - esteem , and increase their risk of early death . in 2007 , bayat mentioned that fractures , soft tissue damage , bruising , ruptures , and subdural hematoma are common complications of falling in seniors . also , losing self - esteem , fear of falling again , inability to perform daily activities , and rejection by society and others are some of the concerns of seniors in this regard . falling in seniors is one of the common causes of hospitalization , decrease in physical activities , and decrease in quality of life . fathi believed that during old age , the risks of immobility and decreased physical activities would increase . physical activity would improve the self - esteem of seniors and distance them from inertia , uselessness , apathy , and unimportance . passing the older ages requires doing daily activities , which include activities that are required for having an independent life . in fact , functional status of the elderly in regard with their ability to perform daily activities , by determining their general health status , would reflect their independence and need for health care services . activities of daily living ( adl ) include eating , dressing and undressing , walking , performing activities related to the appearance , taking a bath or a shower , going to and getting off the bed , and going to the bathroom . disruption in adl is common among the elderly , so it is estimated that 20% of people aged from 65 to 74 years have limitations in performing these activities . some of the risk factors of disruption in adl are age , being female , cognitive disorders , chronic behaviors , depression , and physical problems . considering the above - mentioned facts , implementing necessary measures to prevent falling in the elderly is essential . the needs of today 's world have shown that primary prevention has a deeper impact and one of the methods to reduce falling among seniors is primary prevention that could be achieved by training prevention methods . a wide range of programs for prevention of falling exists that has been discussed in different studies . for example , in the study of karlsson et al . that was conducted in 2013 , training method was used to prevent falling . to promote knowledge about falling and to prevent it the first and most fundamental step in developing and implementing a training program is correct and reality - based execution of need assessment . in fact , need assessment is the foundation of training structure ; the more fundamental and strong the foundation , the more secure and invulnerable the structure . in the field of training , need assessment is considered as one of the basic and essential components of planning process , and whenever the matter of developing educational plans and adopting a series of educational measures is proposed , need assessment would repeatedly be mentioned and the logical base of every program is the existence of one or a series of needs . need assessment , in fact , is the process of gathering and analyzing the data , based on which the needs of individuals , groups , organizations , and societies would be determined . determining the educational needs is the first step of educational programming for staff and , in fact , the first factor of creating and ensuring the effectiveness of educational function and enhancement ; if performed correctly , a more objective basis for programming , as an effective plan , would be prepared . educational activities that would be designed and executed without considering these conditions are actually wasting valuable resources . therefore , in the present study , the researcher first evaluated the educational needs of seniors and trained them according to the need assessment . so , considering the importance of falling and its various complications and also lack of need assessment - based studies , the aim of this study was to evaluate the effect of need - based training about prevention of falling on seniors adl . this study was a before - after two - group ( case and control ) field trial . the case group received need - based training about prevention of falling and the control group received a brief educational card . after taking written permission from the health deputy of isfahan and the ethics committee of isfahan university of medical sciences , the researcher randomly selected health centers from 11 health centers of isfahan through multi - stage random sampling in which four centers were selected through drawing . then , from these four centers , two were randomly selected to be the case group and the other two became the control group . all the files of the elderlies of each center were studied and from them , those that satisfied the inclusion criteria ( willingness to participate in the study , being sufficiently aware and able to hear , knowing the persian language , being aged 60 or more , not being mentally or physically retarded , history of having at least one fall , and not using mobility aids ) were selected and coded . then , using the table of random numbers , 60 seniors were selected to participate in the study . it must be noted that participants were justified about the method of the study , the confidentiality of the data , and the aim of the study . since in this study trainings were based on needs , for preparing the educational package , some of the seniors were interviewed . through these interviews , educational needs of seniors ( including understanding the causes and risk factors of falling , its complications , strategies to prevent falling in common places of falling at home , strategies for fall - proofing the house , and muscle strengthening exercises ) were extracted , matched with related resources , necessary changes were made , and the final form of educational programs was developed . after selecting the participants , seniors were invited by phone calls to show up at the health center at the predetermined date . at the determined date , the aims and methods of the study were explained for both groups and written consent form was obtained from all the participants ; then , adl questionnaire was completed . in this questionnaire , questions on adl are answered with the options without help , with some help , and not able to perform it . without help is scored 2 , with some help is given 1 , and not able to perform it is scored 0 . after scoring the questions , the total score of the questionnaire was obtained by summing the scores of all questions and based on the obtained scores , seniors were divided into three groups of completely dependent , somewhat dependent , and independent ; a score of 06 indicated completely dependent , 710 was somewhat dependent , and a score of 1114 indicated independence . the validity of this questionnaire was evaluated through content validity method by habibi et al . in 2007 in their study that was conducted on 410 seniors of over 60 years old living in the west of tehran and for evaluating its scientific validity , test - retest method was used ( r. 9 ) . thus , it could be said that adl questionnaire is reliable and valid . after completing the questionnaire , the case group received six 45-min sessions for 1 day per week . during the sessions , lecturing , group discussion , and question and answer methods were used . educational content included teaching the nature , causes , and complications of falling , training strategies for fall - proofing the house , training methods for prevention of falling , and also , exercises for strengthening the muscles . at the end of the sixth session , the control group received a brief educational cart ( definition of falling and its complications ) at the first session after completing the questionnaire for the first time . one month after the intervention ( this time interval was selected based on literature review ) , participants of the case and control groups were invited again for completing the questionnaire again . data was analyzed using chi square , independent t - test , mann whitney , least significant difference ( lsd ) , and repeated measures analysis of variance ( anova ) . results showed that the mean age of the case and control groups was 69.4 ( 4.8 ) and 69.4 ( 5.8 ) years , respectively . the most frequent sex in both groups was female , and both groups had no significant difference regarding their demographic characteristics ( age , sex , marital status , educational level , and family combination ) before the intervention [ table 1 ] . demographic specifications of the case and control groups before the intervention based on the need assessment that was conducted on seniors , their needs were categorized into five groups including understanding the causes and risk factors of falling , complications of falling , strategies for prevention of falling in common places of falling at home ( like bathroom , kitchen , and stairs ) , strategies for fall - proofing the house , and exercises for strengthening the muscles . so , before the need - based educational intervention , no significant difference existed between the mean score of adl of both groups ( p = 0.38 ) . but this difference became significant right after the intervention ( p = 0.01 ) and 1 month after the intervention ( p = 0.01 ) , and the score of adl of the case group was significantly higher than the control group [ table 2 ] . also , in the control group , the mean score of adl showed no significant difference between the three different time intervals ( p = 0.907 ) . comparing the mean of activities of daily living in the case and control groups at three time intervals ( before the intervention , right after the intervention , 1 month after the intervention ) furthermore , the mean score of adl in the case group before the intervention had a significant difference with the mean scores obtained right after the intervention and 1 month after the intervention ; but the difference between the scores obtained right after the intervention and 1 month after the intervention was not significant [ table 3 ] . comparing the mean of activities of daily living between different time intervals in the case group the present study was conducted to evaluate the effect of need - based education about prevention of falling on adl in seniors . statistical results showed that the mean score of adl was increased in the case group after execution of educational program about prevention of falling . for example , in 2001 , rostami , in a study that evaluated the effect of training about prevention of falling on the level of awareness of seniors and caregivers and the performance of seniors living at kahrizak nursing home , reported that educational programs about prevention of falling had a significant effect on the level of awareness of the participants and performance of seniors . also , in 2014 , najafi et al . conducted a study evaluating the effect of multidimensional programs about prevention of falling on the incidence of falling and quality of life of seniors living at nursing homes and used multidimensional methods in their study , including training about prevention of falling , exercise , and environmental adjustment . results of the study showed that after 4 months of intervention , the number of falls decreased and the quality of life in seniors was increased . also , a study that was conducted in 2007 evaluating the relation between health improving behaviors with the level of adl and adl with mobility aids in seniors of western tehran revealed a significant relation between health improving behaviors and adl ( p < 0.05 ) . in the present study , to prevent falling , strategies for fall - proofing the house were trained , which is similar to previous studies . for example , in the study of karlsson et al . on prevention of falling , different methods have been mentioned and one of the most effective methods was training strategies for fall - proofing the house that caused a significant improvement in the quality of life of seniors . to confirm the results of the present study , studies of gillespie et al . , rajendran , shaw , and pereira et al . could be named that have used fall - proofing strategy for prevention of falling in seniors . another method that was used in the present study was training exercises for strengthening the muscles , which is also similar to the results of previous studies . in this conducted a study on the effect of selected yoga exercises on the balance of female seniors . the results of their study showed that exercising would increase the strength of muscles , increase the performance of motion and nervous system , and decrease the risk of falling in female seniors . in 2015 , tzn et al . conducted a study titled the effect of exercising programs on decreasing falling in seniors with dementia and their results showed that exercising interventions would decrease the number of falls in seniors . also , the results of studies of tozenof et al . , khaje nemat et al . , and bahgeri et al . confirmed this finding . in the present study the mean scores of adl in the case group before , right after , and 1 month after the intervention showed significant differences , but the difference between the scores right after and 1 month after the intervention was not significant . the researcher believes that in future studies , by considering factors like the time interval after the intervention and the interval between sessions and by increasing the sample size , a more desirable result could be achieved . one of the most important limitations of this study was the physical condition of the seniors that was an obstacle for the researcher . their impatience and tiredness was a barrier to better communication ; so , spending more time and conducting longer studies in future researches are necessary . also , lack of internal studies about falling in seniors was another limitation of this study that affected the strength of discussion about the results of this study . results of the present study showed that performing need - based educational programs about prevention of falling had a significant effect on adl in seniors . therefore , nurses and caregivers could help the elderly population through educational interventions to reach ideal seniority condition . currently , for various reasons , this logical need of seniors in less considered in provision of health cares and services , while this essential need could be provided through a developed and inexpensive program and nurses , who are a member of medical teams in health centers and have the most contact with seniors , could have more opportunities to deal with this matter .

background : falling has a great importance among the elderlies . even if no physical injury occurs , it can cause fear of falling down again and , consequently , reduce older adults activities . with regard to the prevalence of falling among older adults , its prevention is essential . therefore , the present study was aimed to define the effect of need - based education on prevention of older adults falling during their everyday life activities.materials and methods : this is a quasi - experimental study . study population comprised all the older adults of age 60 years and over referring to health care centers in isfahan . through multiple random sampling , 15 older adults were selected from four health care centers . data collection tool in the present study was daily activity questionnaire.results:results showed a significant difference between the mean of daily activity scores in the intervention group before , immediately after , and 1 month after the intervention ( 12 , 13.6 , and 13.5 , respectively ; p = 0.01 ) . meanwhile , there was no significant deference between the scores immediately after and 1 month after the intervention . there was no significant difference observed between the three time points in the control group ( mean = 12.3 ; p = 0.907).conclusion : implementation of education concerning prevention of older adults falling led to improvement of their daily activity in the intervention group .

malaria is the most prevalent parasitic disease in the world . each year 300 to 500 million cases occur worldwide , and approximately one to three million people die of malaria ( 1 , 2 ) . among the four species of human plasmodium , p. vivax , which causes malaria in temperate zones and also in large areas of tropical regions , is the second most common species . p. vivax malaria was endemic until the early 1970s in the korean peninsula ( 3 - 5 ) , and re - emerged in the republic of korea ( rok ) in 1993 ( 6 , 7 ) . based on epidemiological data , re - emergence of p. vivax malaria in rok was ascribed to infected mosquitoes originating from north korea . since the re - emergence , nearly a million cases of p. vivax malaria have occurred in rok and north korea ( 6 , 8 - 10 ) , and the malaria situation of rok and north korea provides a favorable environment to elucidate the parasitemia characteristics of p. vivax malaria . many parasitemia and clinical features of p. vivax malaria have been investigated so far , but most of the data have been acquired from tropical regions . transmission and symptom development of p. vivax malaria in temperate zones are largely influenced by seasonal change , whereas such seasonal interruption is less obvious in tropical zones . therefore , parasitemia characteristics of p. vivax malaria in temperate zones may be quite different from those in tropical zones . however , data from temperate regions have been scarce except those acquired from malaria therapy by artificially induced infection for neurosyphilis patients ( 11 - 13 ) . in that therapy , the number of inoculums was higher than in natural infections , which might have resulted in clinical and parasitemia differences from natural infections . the concentrated occurrence of p. vivax malaria near the demilitarized zone between rok and north korea provides a good opportunity to better understand the parasitemia characteristics of p. vivax malaria in a temperate zone . parasitemia characteristics including gametocytemia feature is the important factor related to clinical manifestation and transmission efficacy of plasmodium . in this study , we investigated the giemsa - stained peripheral blood smears and clinical laboratory data of p. vivax malaria patients in rok to clarify the parasitemia characteristics of the p. vivax strain in rok . subjects enrolled in this study were p. vivax malaria patients who were diagnosed during 2002 - 2006 at six private hospitals located in the northwestern malaria - prevalent areas in rok . multiple invasion and parasitemia were calculated by microscopic examination of 1,000 thin film fields at 1,000 magnification . parasitemia characteristics were elucidated by examination of the patients ' peripheral blood smears prepared when the disease was diagnosed . hematological and serological data at the time of diagnosis were also obtained from clinical records . any additional examination or blood collection was not performed for the sake of this study and all protocols were approved by the institutional review board of each hospital . statistical analyses were performed using the chi - square test without any assumption about the distribution of data using spss ( statistical products and service solutions , chicago , il , usa ) . continuous variables were compared by the student 's t test and mann - whitney u tests . of the 383 p. vivax malaria patients enrolled in this study , 283 ( 74% ) were male and the remaining 100 were female ( fig . median age of the patients was 39 yr ( range , 14 - 89 ) . more than half of the patients were in their 20 sec ( 104/383 ) and 40 sec ( 102/383 ) representing 27.2% and 26.6% of the study population , respectively . the next populated age group was in their 30 sec ( 19.8% , 76/383 ) . most patients ( 96% ) in this study presented at the hospitals between may and october ( fig . the mean parasitemia was 8,396/l with the minimum and the maximum of 12/l and 140,994/l , respectively ( table 1 ) . approximately half of the patients ( 50.9% ) showed parasitemia between 1,000 - 10,000/l , with the remainder showing either more than 10,000/l or less than 1,000/l . parasitemias were significantly higher in gametocyte ( + ) patients than in gametocyte ( - ) patients . intervals between the onset of the first symptom and diagnosis were also significantly longer in gametocyte ( + ) patients ( p = 0.001 ) . the anemia markers , red blood cell ( rbc ) counts , hemoglobin , and hematocrit levels , were significantly higher in gametocyte ( - ) patients . multiple invasions , which refer to infection of one rbc by multiple ring forms , occurred in 53.5% of the patients ( table 3 ) . the intervals between the onset of the first symptom and diagnosis were significantly longer in multiple invasion ( + ) patients . the anemia markers were significantly higher in multiple invasion ( - ) patients . among 205 patients with multiply infected rbcs , 151 patients showed rbcs with only two ring forms , and the maximum number of parasites per rbc was six in a patient . the mean proportion of rbcs infected by multiple ring forms among the total rbcs investigated in this study was 2.4% ( with a minimum of 0.19% and a maximum of 11.2% ; data not shown ) , which was less than the value ever observed in tropical zones ( 14 ) . among 353 gametocyte ( + ) patients , female gametocytes were found in 350 patients and male gametocytes in 194 patients ( table 4 ) . both female and male gametocytes were found in 191 patients ( group a ) , whereas only female gametocytes were found in 159 patients ( group b ) and only male gametocytes were found in three patients ( group c ) . the parasitemia was higher in group a. the parasitemias of groups a , b and c were 13,541 , 4,699 , and 2,533/l , respectively . the mean female gametocytemias of group a and b were 2,063/l and 595/l , respectively , and the mean male gametocytemias of group a and c were 169/l and 26/l , respectively . intervals between the onset of the first symptom and diagnosis of malaria in group a , of which the parasitemia and the gametocytemia were higher than the other group , were longer than those in group b and c. the intervals were 9.2 , 8.1 , and 5.7 days in groups a , b and c , respectively . during 2002 through 2006 when the patients were enrolled in this study , annual differences of gametocytemia were not significant ( data not shown ) . among the plasmodium that can cause human malaria , p. falciparum has been studied most intensively while p. vivax has received relatively little attention . moreover , the majority of studies on p. vivax were performed with tropical strains . for investigation of the parasitemia characteristics of p. vivax malaria in temperate regions , the korean peninsula is unique in that p. vivax malaria can be studied without confounding interactions with p. falciparum due to the complete absence of p. falciparum in this area . in rok , p. vivax malaria occurred on a large scale for the past 17 yr ( 6 , 8 - 10 ) , and chemoprophylaxis with chloroquine and primaquine has been performed in the military since 1997 ( 15 ) . the number of cases observed in the 6 hospitals from 2002 to 2006 ( 383 cases ) accounts for 5.4% of the total cases reported in rok during the same period ( 7,119 cases ) . most of the patients enrolled in this study were civilians without prior exposure to the chemoprophylaxis , providing a good opportunity to investigate the natural parasitemia characteristics without any significant intervention . interestingly , no child younger than 14 yr was enrolled during this study even though the 6 hospitals providing data were open to all ages . this made our study population distinct from tropical areas where p. vivax malaria was particularly prevalent in children and infants . the main reasons for paucity of children patients in korea may be unique population structure of korean rural areas with very low percentages of younger people and low exposure chance to infected mosquitoes in them . because children are far more populated in urban areas than in rural areas and they travel much less frequently to rural areas than adults do , they have less chance to be exposed to mosquitoes than adults . the mean parasitemia in this study was 8,396/l , which is 6.5 times higher than a previous study also performed in rok ( 16 ) . in that study , most of the enrolled patients were veterans who had received chemoprophylaxis before being discharged from the military , which might have resulted in suppression of parasitemia . the mean parasitemia of this study , however , was less than a half of p. vivax malaria in tropical zones ( 17 , 18 ) . the intervals between the first symptom onset and diagnosis were significantly longer in gametocyte ( + ) patients than in gametocyte ( - ) patients , which means that early case detection and treatment are important to block transmission . parasitemias were significantly higher in gametocyte ( + ) patients than in gametocyte ( - ) patients , presumably due to the longer intervals . the significantly lower levels of anemia indices in gametocyte ( + ) patients might be ascribed to the higher parasitemias , which means that the existence of gametocytes could be a coincident factor of the degree of anemia in p. vivax malaria regardless of their causal relationship . data in table 4 show that male gametocytes might appear in the hosts ' blood earlier than female gametocytes , which was in agreement with a previous study ( 12 ) . male gametocyte densities have been believed to be critical in the infectivity of p. vivax ( 19 ) . it might be ascribed to minor population of male gametocytes among total gametocytes . under this situation , it is more plausible that male gametocyte densities play a key role to determine fertilization efficacy in anopheles mosquitoes . in this study , the proportion of male gametocyte ( + ) patients among the enrolled patients and the ratio of male gametocytes to female gametocytes of group a were much lower than other p. vivax strains ( 12 ) . in group this ratio was 3 - 6 times higher than that of previous studies performed on p. vivax or p. falciparum ( 12 , 20 ) . under the sex ratio shown in this study , it is difficult that male gametocytes can make enough flagellated gametes to fertilize female gametes to form zygotes . these findings may be one of plausible explanation for low transmission efficacy of the p. vivax strain in rok . since the mid-2000s , epidemiological characteristics of p. vivax malaria have been changed in rok which raises the possibility of change of transmission efficacy ( 21 ) . it is necessary to monitor gametocytemia annually since gametocytemia is strongly related to transmission efficacy of plasmodium . most of the p. vivax malaria patients in rok after re - emergence have been primary infection cases without herd immunity against p. vivax , because rok was malaria - free for more than a decade . the diagnosis of malaria was usually determined at an early stage after illness in rok since the primary infection cases probably have apparent symptoms and healthcare system has been working well in rok . despite the early diagnosis in rok , the situation in north korea is presumed to be similar to this result , which provides chances of active p. vivax malaria transmission in the korean peninsula . multiple invasions have been common in p. falciparum malaria , but rarely described in p. vivax malaria ( 22 ) . in general , p. falciparum malaria shows higher parasitemia than p. vivax malaria , resulting in more mature schizonts , which can subsequently lead to more frequent multiple invasions ( 23 ) . in this study , however , multiple infected rbcs were observed in as high as 53.5% of the patients although the mean parasitemia was substantially lower than p. falciparum malaria . the ratio of patients with multiple infected rbcs in this study was also much higher than that observed in a tropical strain of p. vivax ( 24 ) , supporting the previous speculation that multiple invasion in p. vivax malaria might not be directly related to parasitemia but could be a strain - specific characteristic ( 25 ) . parasitemias and the proportion of asexual parasites among the total parasites were significantly higher in multiple invasion ( + ) patients , which was similar to the result of a previous study ( 23 ) . the parasitemia was significantly higher and the intervals between the first symptom onset and diagnosis were significantly longer in the patients with multiple invasions . the data in table 3 show that multiple invasion , like gametocytemia , is also a coincident factor of the degree of anemia in p. vivax malaria . in conclusion , the p. vivax strain in rok reveals relatively low parasitemia and low male to female gametocyte ratio . parasitemia characteristics of plasmodium vivax malaria patients in the republic of korea ae - jung huh , yee gyung kwak , eu suk kim , kkot sil lee , joon - sup yeom , yong - kyun cho , chang - seok kim , and jae - won park parasitemia characteristics of plasmodium vivax malaria in temperate regions may differ from those in tropical zones . however , most parasitological and clinical features of p. vivax malaria have been investigated in the latter . in this study , we investigated 383 malaria patients to clarify the parasitemia characteristics of a p. vivax strain in the republic of korea ( rok ) . the mean parasitemia ( 8,396/l ) was less than half of tropical p. vivax malaria , and multiple invasions of erythrocytes were not rare ( 53.5% of the patients , 2.4% of the total investigated rbcs ) , but less than the observations in tropical zones . the intervals between the first symptom onset and diagnosis were significantly longer in gametocyte ( + ) patients than in gametocyte ( - ) patients . only half of the total patients had both genders of gametocytes ( 191 out of 353 ) , and the male gametocyte density ( 169/l ) was lower than the p. vivax strain of a previous study . multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in p. vivax malaria . results of this study showed the low parasitemia density and considerable gametocytemia of p. vivax malaria prevalent in rok .

parasitemia characteristics of plasmodium vivax malaria in temperate regions may differ from those in tropical zones . however , most parasitological and clinical features of p. vivax malaria have been investigated in the latter . in this study , we investigated 383 malaria patients to clarify the parasitemia characteristics of a p. vivax strain in the republic of korea ( rok ) . the mean parasitemia ( 8,396/l ) was less than half of tropical p. vivax malaria , and multiple invasions of erythrocytes were not rare ( 53.5% of the patients , 2.4% of the total investigated rbcs ) , but less than the observations in tropical zones . the intervals between the first symptom onset and diagnosis were significantly longer in gametocyte ( + ) patients than in gametocyte ( - ) patients . only half of the total patients had both genders of gametocytes ( 191 of 353 ) , and the male gametocyte density ( 169/l ) was lower than that of p. vivax strains of a previous study . multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in p. vivax malaria . the present findings demonstrate the p. vivax strain in rok reveals relatively low parasitemia and low male to female gametocyte ratio . the low ratio may be related with low transmission efficacy .

orotracheal intubation is not suitable for assessing the dental relationship and occlusion , and nasotracheal intubation is contraindicated in patients with nasoorbitoethmoidal fractures or fractures of the base of the skull owing to potential complications such as cerebrospinal fluid leakage and meningitis . tracheostomy provides an alternative , surgical intervention , but can be associated with increased post - operative care , complication rates , and morbidity . we present two cases of submental intubation using a reinforced endotracheal tube with non - detachable universal connector in patients with complex maxillofacial trauma . an 18-year - old male patient ( 60 kg , 170 cm ) was scheduled for open reduction and internal fixation of a nasoorbitoethmoidal fracture ( fig . preliminary investigations indicated that he was otherwise healthy , with preanesthetic evaluation being unremarkable except for signs of sinus bradycardia ( 41 bpm ) . as intra - operative assessment of dental occlusion was required , orotracheal intubation was not indicated , with nasotracheal intubation also being inappropriate due to his nasoorbitoethmoidal fracture . in order to avoid tracheostomy , the patient was monitored with pulse oximetry , electrocardiography , and noninvasive blood pressure measurements . before anesthetic induction , after preoxygenation with 100% oxygen for 3 minutes , anesthesia was induced with propofol ( 100 mg ) and fentanyl ( 100 g ) . after successful ventilation with a facial mask , rocuronium ( 50 mg ) was administered intravenously . prior to intubation , the universal connector of a reinforced endotracheal tube ( ett ) was detached gently from the tube and reattached so that it could be easily disconnected during the procedure ( fig . orotracheal intubation was performed using a 7.5-mm - internal diameter reinforced ett ( lo - contour oral / nasal tracheal tube cuffed , mallinckrodt , ireland ) . anesthesia was maintained with sevoflurane and 100% oxygen to increase the apneic reservoir during the procedure . approximately 3 cm lateral to the mandibular midline , a submental horizontal incision ( of 1.5 cm ) was placed a fingerbreadth below the mandibular inferior border . blunt dissection toward the mouth floor was performed using a thin mosquito hemostat , forming an orocutaneous tunnel . a kelly forceps was then introduced through the tunnel from the outside to widen it . at that moment , the ett was disconnected from the ventilator circuit , and its connector was removed . the ett was then grasped by the tip of the kelly forceps and pulled out through the tunnel of the sub - mental incision , followed by the pilot balloon . after the ett was shifted , then , the ett was repositioned using magill forceps , and a stay suture was applied with silk in order to fix the tube to the skin to prevent accidental extubation ( fig . , the stay suture was removed , and the ett with a pilot balloon was retracted from the oral cavity , removed via the mouth , and brought back to the orotracheal position . the submental incision was layer - sutured , and wet gauze dressing was applied at the site of the wound at the mouth floor to facilitate secondary healing . following the procedure , extubation was performed , and the patient was transferred to the post - anesthetic care unit . the post - operative period was unremarkable , and there was negligible submental scarring at two months post - operation . a 48-year - old male patient ( 65 kg , 175 cm ) sustained bilateral orbital wall and maxillary fractures with a skull - base fracture after a motor vehicle accident . he had undergone brain surgery for epidural hematoma removal 11 days prior . as his level of consciousness was alert , intubation following the operation was unnecessary . the patient was monitored with pulse oximetry , electrocardiography , and noninvasive blood pressure measurements . after preoxygenation with 100% oxygen for 3 minutes , anesthesia was induced with propofol ( 80 mg ) and rocuronium ( 50 mg ) . following orotracheal intubation with a reinforced ett ( internal diameter , 7.5 mm ) , the tube was removed through the submental area using the aforementioned method . after the operation , the patient was transferred to the intensive care unit and was extubated at post - operative day 1 . an 18-year - old male patient ( 60 kg , 170 cm ) was scheduled for open reduction and internal fixation of a nasoorbitoethmoidal fracture ( fig . preliminary investigations indicated that he was otherwise healthy , with preanesthetic evaluation being unremarkable except for signs of sinus bradycardia ( 41 bpm ) . as intra - operative assessment of dental occlusion was required , orotracheal intubation was not indicated , with nasotracheal intubation also being inappropriate due to his nasoorbitoethmoidal fracture . in order to avoid tracheostomy , the patient was monitored with pulse oximetry , electrocardiography , and noninvasive blood pressure measurements . before anesthetic induction , after preoxygenation with 100% oxygen for 3 minutes , anesthesia was induced with propofol ( 100 mg ) and fentanyl ( 100 g ) . after successful ventilation with a facial mask , rocuronium ( 50 mg ) was administered intravenously . prior to intubation , the universal connector of a reinforced endotracheal tube ( ett ) was detached gently from the tube and reattached so that it could be easily disconnected during the procedure ( fig . orotracheal intubation was performed using a 7.5-mm - internal diameter reinforced ett ( lo - contour oral / nasal tracheal tube cuffed , mallinckrodt , ireland ) . anesthesia was maintained with sevoflurane and 100% oxygen to increase the apneic reservoir during the procedure . approximately 3 cm lateral to the mandibular midline , a submental horizontal incision ( of 1.5 cm ) was placed a fingerbreadth below the mandibular inferior border . blunt dissection toward the mouth floor was performed using a thin mosquito hemostat , forming an orocutaneous tunnel . a kelly forceps was then introduced through the tunnel from the outside to widen it . at that moment , the ett was disconnected from the ventilator circuit , and its connector was removed . the ett was then grasped by the tip of the kelly forceps and pulled out through the tunnel of the sub - mental incision , followed by the pilot balloon . after the ett was shifted , then , the ett was repositioned using magill forceps , and a stay suture was applied with silk in order to fix the tube to the skin to prevent accidental extubation ( fig . , the stay suture was removed , and the ett with a pilot balloon was retracted from the oral cavity , removed via the mouth , and brought back to the orotracheal position . the submental incision was layer - sutured , and wet gauze dressing was applied at the site of the wound at the mouth floor to facilitate secondary healing . following the procedure , extubation was performed , and the patient was transferred to the post - anesthetic care unit . the post - operative period was unremarkable , and there was negligible submental scarring at two months post - operation . a 48-year - old male patient ( 65 kg , 175 cm ) sustained bilateral orbital wall and maxillary fractures with a skull - base fracture after a motor vehicle accident . he had undergone brain surgery for epidural hematoma removal 11 days prior . as his level of consciousness was alert , intubation following the operation was unnecessary . the patient was monitored with pulse oximetry , electrocardiography , and noninvasive blood pressure measurements . after preoxygenation with 100% oxygen for 3 minutes , anesthesia was induced with propofol ( 80 mg ) and rocuronium ( 50 mg ) . following orotracheal intubation with a reinforced ett ( internal diameter , 7.5 mm ) , the tube was removed through the submental area using the aforementioned method . after the operation , the patient was transferred to the intensive care unit and was extubated at post - operative day 1 . although orotracheal intubation is the most frequently used route in securing the airway , it interferes with the surgical field and disturbs intra - operative assessment of dental occlusion . as an alternative to orotracheal intubation , nasotracheal intubation nasotracheal intubation is contraindicated in cases of skull - base trauma due to the incidence of accidental intracranial placement , possible cerebral spinal fluid leakage , and/or meningitis . tracheostomy is a good route to secure the airway in patients with complex maxillofacial injuries , particularly for those who need prolonged intubation . however , the procedure is associated with the risk of hemorrhage , pneumothorax , infection , and tracheal stenosis . , it has been used as an attractive option for intra - operative airway control in specific , complex maxillofacial injuries . submental intubation is also applied to bimaxillary orthognathic surgeries with simultaneous rhinoplasty or orthognathic surgeries in patients with large pharyngeal flaps or other anatomic anomalies precluding nasotracheal intubation . other possible indications for this technique are certain base of skull procedures or cancrum oris . submental intubation is contraindicated in patients who have severe neurological defects or those who require long - term airway support and maintenance . in these cases , tracheostomy should be considered . in patients with a history of severe keloid formation , this technique can be contraindicated . in the first case , endotracheal intubation was not indicated because of circumstances that prevented the intra - operative assessment of dental occlusion , with nasotracheal intubation also being inappropriate due to his nasoorbitoethmoidal fracture . as he was healthy , with the exception of his nasoorbitoethmoidal fracture , and did not require prolonged ventilatory support , the decision to choose submental intubation over tracheostomy had greater significance . in the second case , nevertheless , submental intubation was chosen because of his alert mental status in addition to his unrequired use of long - term airway support . flexible and kink - resistant ett is required to maintain airway patency despite the acute angle of the airway , particularly in the submental route . ball et al . reported submental intubation using the flexible tracheal tube supplied with the intubating laryngeal mask ( ilm , intavent , uk ) . however , it is rare in the market and expensive compared to our reinforced tube ( lo - contour oral / nasal tracheal tube cuffed , mallinkrodt , ireland ) . the universal connector of a standard reinforced ett , which we used , is bounded firmly to the tube to prevent accidental detachment . prior to intubation , it is important to detach and reattach the connector gently from ett using mosquito forceps so that it can be easily disconnected during the procedure . as discussed , tracheostomy is associated with increased postoperative care , complication rate , and morbidity . in comparison , the complications associated with submental intubation are less severe and lower in frequency . with respect to surgery , as maxillofacial trauma can often result in dental disarrangement , dental occlusion becomes clinically important . submental intubation removes the ett from the surgical field , thereby providing a clear surgical field that also assists in preventing injuries of the ett . additional advantages of submental intubation include its minimally invasive nature and the more esthetically acceptable nature of the resulting scar . the most serious complication is accidental extubation ; however , this can be prevented via a stay suture , as used in our cases . other complications are superficial skin infections , damage to the ett , tube dislodgement or obstruction , transient lingual nerve paresthesia , venous bleeding , and submental scarring . the submental intubation technique is a reliable alternative to tracheostomy in patients undergoing complex maxillofacial surgeries who do not require prolonged ventilatory support . it has minimal morbidity , a low complication rate , and avoids the potential complications associated with tracheostomy .

airway management in patients with complex maxillofacial injuries is a challenge to anesthesiologists . submental intubation is a useful technique that is less invasive than tracheostomy in securing the airways where orotracheal and nasotracheal intubation can not be performed . this procedure avoids the use of tracheostomy and bypasses its associated morbidities . a flexible and kink - resistant reinforced endotracheal tube with detachable universal connector is commonly used for submental intubation . herein , we report cases involving submental intubation using a reinforced endotracheal tube with a non - detachable universal connector in patients with complex maxillofacial injuries .

was introduced for two species described from tropical and subtropical habitats in africa and australia : a. gelatinosporus ( j.w . ( 2000 ) mainly on the basis of spore characters ( prominent spines on the spore surface within an inflated utricle ) and a pink to brown , gelatinized gleba , while hysterangium is characterized by a green - toned gleba with smooth , hyaline spores ( castellano et al . recent phylogenetic studies placed both hysterangium and aroramyces in the family hysterangiaceae ( hysterangiales ) and postulate that all members of this family are obligate ectomycorrhizal fungi with angiosperms and gymnosperms ( hosaka et al . the genus aroramyces had not been reported from north america until recent field trips to forests in central mxico from 2007 to 2011 yielded two undescribed aroramyces species . methods of collection , macroscopic , and microscopic study were generally those of castellano et al . dried specimens were hand cut and mounted in 5 % koh , melzer s reagent , or water for microscopic observation . spore measurements were made with a compound microscope at 1000x under oil immersion on 20 spores . etymology : balanosporus in reference to the shape of the spores that resemble an oak acorn formed by the irregular inflation of the utricle . diagnosis : this species differs from the two central africa and northern queensland species in the brownish spores with a distinct inflated utricle with embedded spines , and also spore size . type : mxico : state of jalisco : road to the volcano tequila , 20 49.9n 103 51.071w , hypogeous , 28 sept . 2009 , j trappe & m castellano 33802 ( itcv [ jos castillo tovar herbarium ] holotype ; osc isotype ) . basidiomes globose , sub - globose to irregular , 712 x 812 x 68 mm , peridial surface fibrillose or tomentose , often with cottony - like patches of bright white hyphae which encompass some soil debris and ectomycorrhizal roots scattered across the peridial surface , tomentum white with pale tan areas , numerous soil particles adhering to surface ; odour fungoid or indistinct ; taste not recorded . peridium separable and fragile , < 0.5 mm thick , white to pale tan in cross - section . gleba brown , gelatinized tramal veins , hard when dried , locules somewhat irregular or elongated . epicutis 38150 m thick , of hyaline to reddish brown , thin - walled , interwoven to repent or erect hyphae , 2.56 m broad , forming scattered caespitose groups of erect , branched , setal hyphae 2.55 m broad , with abundant crystalline structures adherent on hyphal walls , clamp connections present . mesocutis 125180 m thick , of reddish brown , isodiametric , angular , or irregular hyphae , occasionally interwoven , hyphae 2.542 m broad , walls to 2.5 m broad . subcutis 40125 m thick , of hyaline , repent or interwoven hyphae , 3 m broad . hyphae : occasionally with wide terminal prostrate hyphae to 48 m broad , broadly clavate , walls to 2.5 m broad , clamp connections present . trama of hyaline , interwoven to occasionally parallel hyphae , 28 m broad , immersed in a gelatinized matrix , clamp connections present . basidia clavate to broadly clavate , 1150 x 520 m , hyaline to pale brown , walls to 1 m broad , 4-spored . sterigmata 410 m long x 23 m broad , hyaline . basidiospores ellipsoid to occasionally broadly - ellipsoid , symmetrical , without utricle and hilar appendage ( 10)10.511(11.5 ) x 5.56 m , x = 10.7 x 5.7 m , q range = 1.712.17 , q mean = 1.89 ; with utricle and hilar appendage 12.513.5(15 ) x ( 7 )7.58 m , mean = 13.3 x 7.6 m , q range = 1.671.88 , q mean = 1.75 , numerous fine spines within the utricle , in koh hyaline to pale brown singly , reddish brown to dark brown in mass , hilar appendage 1 m long x 2.5 m broad , walls to 1 m broad , utricle inflated and to 3 m broad , hyaline , many spores with inflated utricle toward the distal part of the spore , inamyloid , nondextrinoid . distribution and ecology : mxico ( states of jalisco and michoacn ) . additional material examined : mxico : state of michoacn : in front of ooapas water - pump , road to tumbisca - ichaqueo , municipality of morelia , 29 nov . discussion : aroramyces balanosporus is recognized by its brownish spores with a distinct , inflated utricle with embedded spines . aroramyces radiatus from africa has smaller spores , 1012(13.5 ) x 67(8 ) m ( with hilar appendage ) , and associates with brachystegia spiciformis ( caesalpinioideae ) or uapaca sp . aroramyces gelatinosporus from australia has similar - sized spores but possesses a single - layered peridium and associates with eucalyptus spp . etymology : herrerae - in honor to tefilo herrera surez , one of the pioneer mycologists of mxico . diagnosis : differs from all other known aroramyces species in the distinctly inflated wing - like appearance of the utricle ( inflated to 6 m ) . type : mxico : state of michoacn : puerto madroo , 20 km south of morelia city , ejido atcuaro , municipality of morelia , hypogeous , 18 oct . basidiomes irregular , globose or subglobose , 518 x 6 - 15 x 510 mm , peridial surface white , pale tan to brownish , mottled dark brown with pale areas when handled and when dried , smooth when fresh , much wrinkled when dried , with some white mycelial strands , soil and organic matter adherent to surface , koh ( 5% ) brown to blackish on the surface of dried specimens ; odour acetone / ether solvent - like ; taste not recorded . gleba brown to dark brown in all stages , nearly black when dried , locules ellipsoid to elongate , stuffed with spores , columella dendroid , gelatinized , grayish . epicutis 45175 m thick , usually on the thinner side with occasional areas with wart - like protrusions , of septate , thin - walled , pale yellow - brown to yellow - brown , repent hyphae , 4.56.5 m broad , occasional cells inflated to 18 m broad , with interspersed small crystalline particles scattered across layer . subcutis 110135 m thick , of septate , thin - walled , hyaline , interwoven to subparallel or cross - weaved hyphae , 6.511(15 ) m broad . mycelial strands on peridium of dark brown , filiform , branched hyphae , 23 m broad , encrusted with small crystalline particles , clamp connection present . trama 37112 m thick , of hyaline , thin - walled , compactly interwoven to parallel hyphae , 25 m broad , in a gelatinized matrix , clamp connections present . basidiospores fusoid , symmetrical , without utricle and hilar appendage 10.512.5 x 5.57 m , mean = 11.3 x 6.0 m , q range = ( 1.63)1.722.00(2.17 ) , q mean = 1.91 , with utricle and hilar appendage ( 12.5)13 14 x 89.5 ( 10.5 ) m , mean = 13.5 x 9.0 m , q range = ( 1.25)1.461.60(1.78 ) , q mean = 1.51 , walls to 1 m thick , smooth when immature , numerous fine spines within the utricle when mature , utricle distinct , not encompassing hilar appendage to give spore a truncate appearance , utricle often not evenly inflated to protrude from one side or another , occasionally encompassing entire spore originating from the spore base , utricle laterally inflated to 5(6 ) m broad , in koh hyaline to yellow - orange singly , pale brown in mass , inamyloid , nondextrinoid . distribution and ecology : mxico , michoacn , in the trans - mexican volcanic belt . hypogeous , solitary , under quercus castanea , q. obtusata , q. magnilifolia , q. rugosa , pinus leiophyla , pinus pseudostrobus , and pinus michoacana at approximately 2160 m elevation , september and october . additional material examined : mxico : state of michoacn : puerto madroo , 20 km south of morelia city , ejido atcuaro , municipality of morelia,19 32 113 2011 , g guevara 1218 ( itcv , osc ) ; ichaqueo , municipality of morelia , 20 sept . discussion : aroramyces herrerae is recognised by its brown gleba and the distinctly inflated , wing - like appearance to the utricle ( inflated up to 6 m ) .

little is known of the truffle - like fungi of northern mexico . few mycologists have collected truffle - like specimens in this area . the wide diversity of habitat and potential mycorrhizal partners portend a unique and varied truffle - like mycota . in the conduct of recent field studies in this region we collected many interesting truffle - like specimens . we present two taxa that have unique characteristics , brownish spores with spines embedded within a distinctly inflated utricle surrounding each spore . aroramyces balanosporus and a. herrerae are described as new species . this is the first record of the genus aroramyces from north america .

the prevalence of human immunodeficiency virus ( hiv ) infection in india is 0.34%.(1 ) even though the survival of people living with hiv ( plwh ) has improved with the advent of anti - retroviral therapy ( art ) , their life is affected by social factors like stigma / discrimination . hence , quality of life ( qol ) which gives a holistic picture of their health status has gained importance.(2 ) further , knowing the factors affecting the qol of plwh would be helpful in making important policy decisions and health care interventions . literature on the factors influencing qol was not consistent as qol depends on the socio - cultural milieu in which the individual lives.(3 ) one such variation observed in hiv was with relation to gender . in chennai , it was found that females experienced poor qol in the sociological domain , and men in the psychological domain,(4 ) whereas , the multicentric preliminary study of whoqol with two centers in india found that women experienced less qol across all domains.(5 ) on the contrary , in africa , it was found that gender does not influence qol.(6 ) since india accounts for nearly half of the asia 's hiv prevalence,(7 ) there is a definite need to study qol and the factors influencing it in the indian setting . measuring qol would help to identify the most affected domain , and knowledge on factors influencing it will aid in taking appropriate intervention . it would also provide regional and objective data for planning interventions for the betterment of plwh . to assess the quality of life of plwh.to study the socio - cultural and demographic determinants of quality of life in plwh . to assess the quality of life of plwh . to study the socio - cultural and demographic determinants of quality of life in plwh . to assess the quality of life of plwh.to study the socio - cultural and demographic determinants of quality of life in plwh . to assess the quality of life of plwh . to study the socio - cultural and demographic determinants of quality of life in plwh . this descriptive study was conducted in puducherry , a union territory of india with a population of 9,74,345.(8 ) two tertiary care government hospitals , jawaharlal institute of postgraduate medical education and research ( jipmer ) and general hospital ( gh ) of puducherry , and three non governmental organizations ( ngo ) namely pondicherry network of positives ( pnp+ ) , community care centre ( ccc ) and shanthi bhavan were providing care for plwh in puducherry . the pilot study found that plwh utilizing the services of puducherry gh were also utilizing the services of the ngo . hence , the study was conducted at jipmer and the three ngo 's during 2005 - 07 . sample size of 200 was calculated using the mean domain score of 11.4 and standard deviation ( sd ) of 3.7 from the preliminary study of whoqol - hiv.(5 ) recruitment was continued till 200 subjects of > 18 years of age were interviewed . as plwh were utilizing the services of more than one centre , register was maintained to avoid duplication . based on feasibility , at each visit to these study centers , first three subjects were recruited from their registers . quality of life was assessed using hiv specific world health organization quality of life scale ( whoqol - hiv ) bref which was field - tested in six centers across the world , including two centers in india . it contains six domains namely physical , psychological , level of independence , social relationships , environment and spirituality / religiousness / personal belief ( srpb).(5 ) social support and hiv related stigma and was assessed using multidimensional scale of perceived social support and hiv stigma scale , respectively . all these scales had an excellent internal consistency.(5911 ) interview schedules also included information on socio - demographic , clinical and social characteristics . it was translated into the local language tamil , back translated and reviewed by a panel of two clinicians and five medical social workers . non - judgmental attitude and non - responsive body language was followed to minimize bias . the study was approved by the institute research council and institute ethics committee at jipmer . during the first one year of the study period , facility for checking cluster differentiation 4 ( cd4 ) count was not accessible to most subjects . hence , the performance scale in who classification system was used for staging the disease clinically which was as follows : stage i bedridden > 50% of the day during the last month.(12 ) data was analyzed using statistical package for the social sciences ( spss ) version 13 using the guidelines of the various scales . higher score in the multidimensional scale of perceived social support and hiv stigma scale indicated good social support and greater hiv related stigma , respectively . factors influencing qol were identified using backward stepwise multiple linear regression with the six domain scores as the dependent variables . age , gender , residence ( urban / rural ) , marital status , years of education , per - capita income , change of income after the diagnosis of hiv infection , duration since diagnosis , disease stage and intake of anti - retroviral therapy ( art ) , disclosure of hiv status , peer counseling , social support and hiv related stigma were the independent variables . mean age of the subjects was 35 years ( standard deviation - sd 8.6 ) . duration since diagnosis varied widely from 1 week to 12 years . most plwh contracted hiv through heterosexual route ( 153 , 76.5% ) and 71 ( 35.5% ) subjects were under art . among the subjects in stage hundred ( 50% ) subjects reported decrease in income after the diagnosis of their hiv status because of manifestations of the infection , decrease in work capacity and decrease in number of working days . but nine subjects earned more after the diagnosis of hiv as they felt the need to earn more money to meet their health care . psychological and srpb were the most affected domains [ mean = 12.7 , table 2 ] . level of independence was the least affected domain [ mean = 14.7 , table 2 ] . some common expressions of subjects regarding their present quality of life were i am not able to ask for my property , as my relatives are scaring me that they will disclose my hiv status . now i am alright , but if i start having diarrhea i can not even do my day - to- day activities . .when i see very thin persons attending the clinic , i am afraid that one day i may also become like them . these comments showed the helpless situation , fear / concern for uncertainties in future , and the fluctuating health status of these plwh . however , a few plwh commented positively ; after the diagnosis of hiv , i have tried to find a new meaning in my life . demographic characteristic of study subjects mean scores in the domains and the human immunodeficiency virus . specific facets of world health organization quality of life scale ( whoqol - hiv ) bref table 3 shows the influence of the various factors on the qol domains . the regression models explained between 20% and 57% of the variance in the different domains . the determination coefficients ( adjusted r ) were highest for the social relationship domain ( 57% ) followed by the psychological domain ( 51% ) . early stage of disease and better social support has significant positive influence on all domain of qol . disease stage was the most influential determinant of all domains except social relationship and environment . plwh experiencing greater hiv related stigma obtained lower scores in the psychological , environment and srpb domain . it is noteworthy to mention that decrease in income after the diagnosis of hiv negatively influenced psychological , social relationship and environment domain . females obtained lower scores in the psychological and the srpb domain . with time , the qol of plwh improved . other important significant determinants of qol identified by multivariate analysis were peer counseling , disclosure of hiv status , age and residence [ table 3 ] . in our study , multiple linear regression of factors influencing quality of life of people living with human immunodeficiency virus infection in the study the study revealed that psychological domain and srpb which includes facets such as self - esteem , negative feelings , purpose in life , fear of death etc . similar results were also found in preliminary study of whoqol.(5 ) schonnesson reported that though symptoms due to hiv decreased with art ; stigma / discrimination and uncertainties had not decreased thereby affecting them psychologically.(13 ) the score of the present study in level of independence , social relationship and srpb domains were significantly better than that reported from bangalore ( india ) . however the physical and psychological domains scores of the present study were lower than that from delhi ( india).(5 ) this indicates a wide variation in qol even within the country . decrease in income after the diagnosis of hiv was not well studied in the indian scenario . in our study , about half of the subjects earned less after the diagnosis of hiv due to manifestations of hiv . less income decreased their access to resources , affecting their living environment and social life.(14 ) they also faced challenges for meeting their increased health care cost . hence , vocational counseling and support for self employment provided through the ngo would improve the qol of plwh . our study and the preliminary study of whoqol hiv , showed that disease stage significantly influenced all the six domains.(15 ) hence , art which delays the progression of disease should be made accessible to all . similarly rivero - mendez et al . also found that art had a positive effect on physical well - being.(16 ) factors such as younger age,(15 ) female gender,(415 ) rural background and shorter duration of hiv status(17 ) were the high risk for poor quality of life , as identified in our study and also in literature . our study and study by molassiotis et al . showed the positive influence of peer counseling on qol.(18 ) hence counseling and support from peer counselors for these high risk groups would improve their qol . the influence of social support and hiv related stigma was consistent with other studies conducted across various cultural background.(1923 ) noteworthy , that in our study , social support had a major influence on all domains of qol , especially social relationships and environment domain . lower hiv related stigma had a positive impact on the environment , srpb and the psychological domain . however , the study being descriptive , temporal ambiguity of the associations exists , for example whether good social support led to good qol , or good qol made the plwh perceive the available support as good . peer counseling and individual counseling at every contact with the health care professional should be encouraged for the high risk group . the qol of plwh will also benefit the stake holders such as family , community and organizations working for plwh .

context : with anti - retroviral therapy ( art ) for human immunodeficiency virus infection ( hiv ) coming into picture , quality of life ( qol ) has gained importance . knowledge on the factors affecting qol would be helpful in making important policy decisions and health care interventions.aims:the aim of this study is to assess the quality of life of people living with hiv ( plwh ) and to identify the factors influencing their qol.materials and methods : the study was done among 200 plwh attending a tertiary care hospital , and three non governmental organizations at puducherry , india , from november 2005 to may 2007 . qol was assessed using hiv specific world health organization quality of life scale ( whoqol - hiv ) bref questionnaire which has six domains ( physical , psychological , level of independence , social relationships , environment and spirituality / religiousness / personal belief ) . social support and stigma were measured using multidimensional scale of perceived social support and hiv stigma scale , respectively , using likert scale . factors influencing qol were identified using backward stepwise multiple linear regression with the six domain scores as the dependent variables.results:male : female ratio was 1:1 and 58% were in early stage of the disease ( stage i / ii ) . psychological and srpb ( spirituality religiousness and personal beliefs ) domains were the most affected domains . all the regression models were statistically significant ( p<0.05 ) . the determination coefficient was highest for the social relationship domain ( 57% ) followed by the psychological domain ( 51% ) . disease stage and perceived social support significantly influenced all the domains of whoqol . younger age , female gender , rural background , shorter duration of hiv , non - intake of art and greater hiv related stigma were the high risk factors of poor qol.conclusion:interventions such as art , family , vocational and peer counseling would address these modifiable factors influencing qol , thereby improving the qol of plwh .

the growth in popularity of high - throughput technologies for identifying genomic variations such as single - nucleotide polymorphisms ( snps ) , insertions / deletions and copy number variants ( cnvs ) in large population based samples are providing researchers with large data sets containing information on millions of genomic variations for thousands of individuals ( 1,2 ) . genome - wide association studies ( gwas ) have gained increasing attention as it has become feasible and affordable to conduct studies involving thousands of samples and millions of variations per sample . despite this windfall of data one of the major challenges of gwas is to identify real causal variants and separate them from the millions of spurious variations , while also linking these variations to biological mechanism and disease pathogenesis by inference ( 310 ) . to achieve this goal , researchers often need to browse through thousands of candidate snps , link these snps to genes or other functional genomic elements such as regulatory regions near these loci , and then familiarize themselves with the existing knowledge about the function and related phenomena and diseases linked to the snps , genes and other elements . these efforts , while necessary , are inefficient , and impractical for studies involving more than a handful of variations . varietas is a web - based database portal that has been designed to aid researchers to easily retrieve information on a set of variations ( e.g. snps or cnvs ) , related genes and genomic elements in a batch like manner ( figure 1 ) . the retrieved information can be explored using a web browser , or downloaded as a tab - delimited text file for further processing . varietas also links out to several external resources that provide further information about the variations and genes of interest , such as the major genomic information resources pubmed ( 11 ) , dbsnp ( 11 ) , snpedia ( 12 ) and ensembl ( 13 ) . varietas can be especially useful when used as a starting point for interpreting gwas results , where the user can quickly enter a set of the top hits from the gwas and easily get the fundamental information about these variations , related genes , diseases , and follow links to further external resources . special consideration has been placed on keeping the user interface very simple , while still enabling users to have necessary control over the database queries . a major design feature is the ease of use such that no programming experience is needed to access and utilize varietas . users can enter variety of different features such as snps , genes , keywords or locations , or any combination of them . users can browse through the results using the web user - interface or download them as a tab - delimited text file . users can enter variety of different features such as snps , genes , keywords or locations , or any combination of them . users can browse through the results using the web user - interface or download them as a tab - delimited text file . varietas integrates data from and links out to various snp and genome databases and resources . data is currently integrated from the following resources : ensembl genome database , ncbi dbsnp database , the genomic association database ( gad ) ( 14 ) and snpedia . for example , disease data from online mendelian inheritance in man ( omim ) ( 15 ) and gene information from wikigenes ( 16 ) are included through gad and ensembl , respectively . query results from varietas contain links to external resources such as ncbi dbsnp , ncbi pubmed , ncbi entrez gene , ensembl , wikigenes and snpedia . data is periodically integrated through extractors that retrieve data from the respective data sources , and then integrate and store the data in a relational mysql database called varietasdb . variation information is primarily indexed and stored based on their dbsnp rs - numbers , allowing for other types of identifiers for variations that do not have assigned rs - number . gene information and gene related information such as omim disease information is indexed and stored based on ensembl gene identifiers and linked to variations using snp gene relationships from ensembl , including information about the relationships such as snps relative location ( e.g. exon , intron , downstream ) and consequence ( e.g. non - synonymous coding ) to the gene . if a single variation is linked to multiple data entries of the same type , e.g. consequence , phenotype or gene , queries will return a result set consisting of multiple rows indexed by the variation identifier and differing by the field(s ) containing multiple entries ( e.g. querying a snp that is located within two individual genes will return two rows that contain the same variation information but differ in their gene information fields ) . in situations where external data sources contain dissimilar information for a variation ( e.g. related phenotypes or linked genes ) all available information is still indexed and available in the database . users have the possibility to inspect the data to determine if the information is conflicting and what data sources are most reliable . information about the resource versions and extraction dates are available for varietas users in order to track information such as version of genome assemblies and data builds . varietas also archives and keeps online old versions of the integrated varietasdb and web user interfaces , enabling reproducible research and tracking of data changes between versions . varietas integrates data from and links out to various snp and genome databases and resources . data is currently integrated from the following resources : ensembl genome database , ncbi dbsnp database , the genomic association database ( gad ) ( 14 ) and snpedia . for example , disease data from online mendelian inheritance in man ( omim ) ( 15 ) and gene information from wikigenes ( 16 ) are included through gad and ensembl , respectively . query results from varietas contain links to external resources such as ncbi dbsnp , ncbi pubmed , ncbi entrez gene , ensembl , wikigenes and snpedia . data is periodically integrated through extractors that retrieve data from the respective data sources , and then integrate and store the data in a relational mysql database called varietasdb . variation information is primarily indexed and stored based on their dbsnp rs - numbers , allowing for other types of identifiers for variations that do not have assigned rs - number . gene information and gene related information such as omim disease information is indexed and stored based on ensembl gene identifiers and linked to variations using snp gene relationships from ensembl , including information about the relationships such as snps relative location ( e.g. exon , intron , downstream ) and consequence ( e.g. non - synonymous coding ) to the gene . if a single variation is linked to multiple data entries of the same type , e.g. consequence , phenotype or gene , queries will return a result set consisting of multiple rows indexed by the variation identifier and differing by the field(s ) containing multiple entries ( e.g. querying a snp that is located within two individual genes will return two rows that contain the same variation information but differ in their gene information fields ) . in situations where external data sources contain dissimilar information for a variation ( e.g. related phenotypes or linked genes ) all available information is still indexed and available in the database . users have the possibility to inspect the data to determine if the information is conflicting and what data sources are most reliable . information about the resource versions and extraction dates are available for varietas users in order to track information such as version of genome assemblies and data builds . varietas also archives and keeps online old versions of the integrated varietasdb and web user interfaces , enabling reproducible research and tracking of data changes between versions . varietas web user interface ( ui ) has been developed to present users with a very simple to use yet powerful tool ( figure 2 ) . basic search provides users with all of the main functionality of varietas while advanced search provides users with fine - tuning parameters for queries and returned results ( e.g. what fields to retrieve and how the results are displayed ) . the main functionality of varietas is to enter a batch of snps , genes , locations or keywords , and retrieve linked genomic variations , genes and related information such as gene and snp descriptions and information about linked diseases and publications . results can also be downloaded as a tab - delimited text file for further processing with the users favorite spreadsheet software and bioinformatics tools . figure 2.screenshot of varietas user interface showing partial results for basic query for a set of snps . queries can be performed based on given set of variations , genes , keywords or genomic locations . screenshot of varietas user interface showing partial results for basic query for a set of snps . queries can be performed based on given set of variations , genes , keywords or genomic locations . various resources for snp information retrieval and annotation exist , and they have been compared in detailed reviews ( 17,18 ) . when comparing varietas to existing resources , varietas adds new functionalities , improves existing ones and provides these services through a very simple and friendly ui that does not require specialized bioinformatics or programming skills from the users . when compared to existing genotype / phenotype databases such as snpedia , dbgap ( 19 ) , hgvbaseg2p ( 20 ) and similar databases ( 21 ) varietas also provides information about snps that are not yet identified in gwas studies , as well as information about linked genes and their phenotypes making it possible to predict novel phenotypic information for the variations . new and improved functionalities over existing tools include batch querying information from resources that do not have direct batch querying options ( e.g. snpedia ) , possibility to retrieve both combined snp and gene information with a single query instead of having to combine multiple queries and the possibility to combine query parameters such as snp and gene identifiers to free keywords that can include disease terms , gene descriptions and snpedia entries . these findings can then be further examined with more comprehensive genetic association and disease resources such as huge navigator ( 22 ) and omim . the main strengths of varietas are the easy to use web - based ui and the possibility to process large sets of snps to retrieve fundamental information about these snps , related genes and diseases . these results are gathered from sources that do not themselves allow batch queries . integrating data from snpedia , nhgri gwas catalog ( 23 ) and the european genome - phenome archive ( ega ) through ensembl allows users to find focused information for previously characterized individual snps , while integrated gene information allows making new hypotheses about the snp functions based on snps relations to genes , functions of those genes and related diseases . one of the more useful new applications for varietas is to use it to easily convert snps to gene sets , which can then be used for pathway and enrichment analysis using the wide variety of tools created for this purpose , such as gene set enrichment analysis ( gsea ) ( 24 ) . varietas is a novel snp database resource for researchers working with genomic variation data sets or genome variation studies . varietas includes a very simple and easy to use web - application that can be used to retrieve information about snps , related genes and diseases , based on data integrated from various genomic databases . in our own research projects varietas has proved to be an excellent starting point when beginning to interpret results from analysis of high - throughput genotype data , such as gwas . based on our experience , we believe that varietas can be useful for many other types of research as well . varietas enables users to quickly browse through large numbers of snps and provides links to external resources for further information retrieval , and can be very useful for researchers working with gwas and other variation data . we believe that when even greater volumes of genomic variation data becomes available , and our understanding of the links between genotypes and phenotypes improves through next - generation sequencing and large population based projects such as hapmap ( 2 ) and the 1000 genomes project ( 25 ) , the need for tools like varietas will be essential . finnish graduate school of molecular medicine ( to j.p . ) , and the saastamoinen foundation ( to j.p . and g.w . ) . funding for open access charge : university of eastern finland .

current high - throughput technologies for investigating genomic variation in large population based samples produce data on a scale of millions of variations . browsing through these results and identifying relevant functional variations is a major hurdle in these genome - wide association studies . in order to help researchers locate the most promising associations , we have developed a web - based database portal called varietas . varietas can be used for retrieving information concerning genomic variations such as single - nucleotide polymorphisms ( snps ) , copy number variants and insertions / deletions , while enabling users to annotate large number of variations in a batch like manner and to find information about related genes , phenotypes and diseases . varietas also links out to various external genomic databases , allowing users to quickly browse through a set of variations and follow the most promising leads . varietas periodically integrates data from the major snp and genome databases , including ensembl genome database , ncbi dbsnp database , the genomic association database and snpedia.database url : http://kokki.uku.fi/bioinformatics/varietas/

modern radiotherapy techniques such as intensity - modulated radiation therapy ( imrt ) or intensity - modulated arc therapy ( imat ) can provide better dose distribution for planning the target volume and sparing normal tissue [ 1 , 2 ] . however , these techniques require dosimetric quality assurance in every patient ( patient - specific quality assurance : sqa ) before treatment because of the very complex radiation delivery procedures required . according to the japanese guidelines , sqa is divided into two categories [ 38 ] : absolute dose measurement and dose distribution validation . in general , an ionization chamber is used for absolute dose measurement , whereas film is a standard dosimetric tool for validating dose distribution . radiographic films based on the silver halide reaction have been widely used for validation of the relative dose distribution in imrt phantom plans . however , the film processors are becoming obsolete because digital imaging technology now predominates in many hospitals . therefore , radiochromic films have recently been marketed as self - developing films that do not require a processor . these films have spatial resolution as high as radiographic films , are nearly tissue - equivalent , and have a well - defined dose response ; they can accordingly be used to accurately measure relative dose distributions . many investigators have reported the characteristics of radiochromic films and the scanning procedures for their use in therapeutic photon beam dosimetry . in particular , it is important to avoid systematic artifacts and dose uncertainty during the scanning procedure [ 913 ] . the gafchromic ebt2 ( ebt2 , isp ( wayne , nj , usa ) ) film is a transmission - type radiochromic film consisting of four layers , including a polyester substrate base . most users of ebt2 films measure the optical density of the film with a single channel acquisition ( sca ) technique . sca is the standard technique for measuring the film optical density using a color flatbed scanner . with this method one chooses a specific color range within the visible light spectrum transmitted through the film ; we normally use red because it best suits the characteristics of ebt2 film . on the other hand , the triple channel correction acquisition ( tcca ) technique is a model - based method for obtaining the characteristic curve of ebt2 film . the algorithm and fundamental characteristics of this method have already been reported by micke et al . . tcca has the benefit of separating the dose - dependent and dose - independent parts of the scanned signals , to compensate for a variety of anomalies , artifacts and other disturbances such as variations of film thickness ( e.g. active layer thickness ) , scanner non - linearity and process noise , and to allow entire available sensitivity range of the film to be used . in this study , we compared tcca with sca in performing ebt2 film dosimetry for imrt and imat . this study was composed of two parts : a fundamental evaluation and a clinical evaluation . a clinac ix medical linear accelerator ( varian medical systems , palo alto , ca , usa ) and a novalis tx medical linear accelerator ( brainlab , feldkirchen , germany ) were used in this study . for the fundamental evaluation , an entire sheet of ebt2 film ( 10 inch 12 inch ( 25.4 cm 30.48 cm ) ) was divided into a 3 4 grid of 6 6 cm pieces in the central part of the sheet . each film piece was inserted into a 30 30 30 cm solid - state water - equivalent phantom ( toughwater ; kyoto kagaku co. ltd , kyoto , japan ) . the films were irradiated with 0.25 , 0.50 , 0.75 , 1.0 , 1.25 , 1.50 , 1.75 , 2.00 , 2.50 , 3.00 , 3.50 and 4.00 gy using a 10-mv photon beam ; they were then scanned using a customized scanning protocol [ 5 , 16 ] , summarized in table 1 . films were scanned with a flatbed scanner ( v700 , seiko - epson corporation , nagano , japan ) 3 h after the irradiation . the spatial resolution was 150 dpi , with landscape orientation and the film positioned at the center of the entire scanning area , and a median ( 5 5 ) image processing filter was used ( see fig . 1 ) . the calibration curves for three color channels using seven dose - steps ( including five batches ) were then obtained by tcca and sca . we calibrated the monitor chamber of the linear accelerator according to the japanese standard measurement protocol before acquisition of the first calibration curve . after this process , we obtained all calibration curves under the same conditions , and curves were compared to evaluate their consistency . with tcca , the calibration curves were created using filmqapro 2010 software ( version 0 ; isp co. ltd , wayne , nj , usa ) . the data were then exported in american standard code for information interchange ( ascii ) format using an in - house program ( created using visual c language ) . on the other hand , the calibration curves for sca were created using omnipro software ( iba , bartlett , il , usa ) , and the data were exported as an ascii - format file via a function of this software . both ascii - format files were imported into excel software ( microsoft corporation , redmond , wa , usa ) to compare the calibration curves generated by the two methods . all calibration curves were plotted for analogue - to - digital conversion adc ) value against dose . table 1.summary of our customized protocol for film scanningthe details of scan settingthe film size of each piece for a field - by - field method6 6 cmthe film position on the glass plate of scannercenter position of entire scanning areathe scan orientation of filmlandscape directionthe spatial resolution of scan image150 dpithe gradation scale48 bit / rgbthe smoothing filtermedian filter ( 5 5)the interval time between irradiation and scanning3 hthe pass of transmission lightopposite to surface side of film table 2.comparison of pass rates for gamma analysis in sqapass rate : gamma analysis ( 3 mm , 3%)tcca methodsca methodplan 1 ( rapidarc ) ( n= 5)96.2 0.8%92.6 1.7%plan 2 ( rapidarc ) ( n= 5)96.8 0.7%92.9 1.2%plan 3 ( 7beams imrt ) ( n= 5)97.9 0.8%93.6 1.1%plan 4 ( 7beams imrt ) ( n= 5)97.3 0.7%92.4 0.9% experimental flowchart for this study . summary of our customized protocol for film scanning comparison of pass rates for gamma analysis in sqa for the clinical evaluation , we compared the planar dose distributions of imrt and imat ( rapidarc ) plans using tcca and sca . the imrt plan was designed using an i - plan ver.4.2 treatment planning system ( tps ) ( brainlab ) and the rapidarc plan using an eclipse ver.8.9 tps ( varian medical systems ) . we chose two programs ( filmqa pro 2010 prototype and omnipro - i'mrt ) for the analyses . however , the filmqapro 2010 prototype did not have enough functionality to allow a quantitative dose comparison . to compare the dose distribution obtained by tcca with that by sca with the same program , we therefore created an in - house program ( using visual c language ) to export the dose distribution data in ascii format from filmqapro . the ascii - file for tcca was exported to omnipro i'mrt via this in - house program . the agreement between the tps - created plan and the film dose distributions was quantitatively assessed on omnipro i'mrt software . the gamma analysis method was used with a tolerance level of 3% dose difference and 3 mm distance - to - agreement as criteria [ 3 , 4 , 7 ] . the calibration curves for adc value vs. dose are shown in fig . 2 . these curves were obtained by seven curves with 13 dose - steps for gafchromic - ebt2 film : a total of five batches were irradiated between may 2010 and july 2011 . the solid and dashed lines in the figure indicate the calibration curves obtained by tcca and sca , respectively . the error standard deviations ( 1 sd ) of the adc values among the five batches at each dose step are shown in fig . the optical density measured for sca was higher than that for tcca in all color channels . the maximum standard deviation was 11.1% at 0.25 gy with sca compared with 5.1% at this radiation dose with tcca . for doses from 0.25 to 1.00 gy , all sds were smaller for tcca than for sca , indicating that the consistency of the tcca calibration curves was superior . the most significant difference in the calibration curves between the two methods was observed in a low dose range ( 0.251.00 gy ) . 3.comparison of the standard deviation ( 1 sd ) of the calibration curve between tcca and sca . comparison of the standard deviation ( 1 sd ) of the calibration curve between tcca and sca . the results of the clinical evaluation are shown in table 2 and fig . the dose differences between the measured and planned doses in high - gradient regions were smaller with tcca than with sca . in fact , the average pass rates for gamma analyses were 97.2 0.8% ( n = 20/four cases ) for tcca and 93.0 1.2% ( n = 20/four cases ) for sca . 4.a case of patient - specific quality assurance for rapidarc delivery in a patient with prostate cancer . a case of patient - specific quality assurance for rapidarc delivery in a patient with prostate cancer . tcca balances the color channel with the highest sensitivity because the component with the highest derivative value is the dominant factor . the use of multiple wave ranges when scanning ebt2 film enables us to use the most sensitive range in the absorption spectrum of this film . the present results indicate that tcca allows us to reduce the systematic dose disturbance of ebt2 film in each batch . in the fundamental test , the consistency of the calibration curves among several batches was better for tcca than sca . we believe this relates to an advantage of tcca : subtraction of dose disturbance due to the film . because dose disturbance is subtracted , tcca resulted in not only a smaller standard deviation for each dose , but also a lower optical density on the calibration curve when compared with sca . although the consistency of the dose response in sca was worse than that in tcca , a calibration curve can be obtained with sca using a protocol suitable for clinical practice , to evaluate the dose distribution of imrt . in radiochromic film dosimetry , it is important to scan film accurately , and many investigators have reported methods to reduce uncertainty related to radiochromic film dosimetry . for example , moral et al . compared the fitting algorithm for gafchromic - ebt ( ebt ) film among four theoretical models . he concluded that the gamma - distributed single - hit model based on the percolation theory was suitable for fitting and smoothing of the calibration curve of ebt film . however , this algorithm is not able to account for density differences due to film thickness variation and/or scanner - dependent variation . in addition , the accuracy of this method was better in the dose range from 0 to 2 gy in radiochromic dosimetry , it is necessary to characterize the average relationship between the dose to which the film is exposed and the response of film . tcca can ensure the average value of the dose to which ebt2 films are exposed by means of a mathematical equation . hence , when creating a calibration table , tcca appears to have an advantage in obtaining the average relationship between the dose to which the film is exposed and the film response . in radiochromic film dosimetry reported the absolute non - uniformity correction method for radiochromic film dosimetry using a flatbed scanner . they estimated the total 2-sigma dose uncertainty at within 4% for doses between 1 and 3 gy and reaching a minimum at approximately 2 gy . their method might consequently reduce uncertainty and disturbance due to the film scanner ; however , it is limited to portrait orientation during scanning . reported the overall accuracy of ebt film absolute dosimetry in water . according to their evaluation , the overall random uncertainty in absolute dose with ebt film was in the order of 1.8% ( 1 sd ) . this result was obtained by scanning a limited area of film , and they recommended that a similar customized protocol for scanning of radiochromic film is important to avoid systematic artifacts . masi et al . reported a comparative study of different dosimetric tools in sqa for volumetric arc therapy . their results ( pass rate ) showed that ebt2 film had the highest associated uncertainty when compared with several other tools . however , they used ebt2 film with the ebt film protocol , and considered that the large difference ( uncertainty ) might have been caused by an inappropriate scanning protocol for ebt2 film . we previously established a customized protocol for scanning ebt2 film to reduce systematic uncertainty [ 13 , 14 ] , the details of which are given in table 1 . furthermore , this protocol is compatible with tcca , allowing the overall accuracy of ebt2 film dosimetry to be improved further . in sqa for imrt or other complex technologies , the composite validation of dose distributions is required to evaluate the doses both in the prescribed dose range and for organs at risk . inconsistency between the calibration curves might make it difficult to match the dose distribution in both dose ranges . in particular , if we normalize the dose distribution indicated by film so that it falls within the prescribed dose range , the dose difference from the organs - at - risk range might be unacceptable for sqa . tcca allows us to avoid significant dose difference due to non - uniformity of films in a low dose range because this method can accurately obtain the average film response even at such dose ranges . we therefore believe tcca is suitable for ebt2 film dosimetry to perform dose validation when the distribution has a high - gradient region , such as in imrt or rapidarc deliveries . on the other hand , we suggest that those performing radiochromic dosimetry with tcca should ensure that they evaluate slight differences such as those due to mlc leakage and the tongue and groove ( t&g ) effect . in particular , the t&g effect is noteworthy in terms of imrt dose distribution , and can generally be measured using film . the total transmission factor for the t&g effect ( ttg ) consists of the product of the transmission factors of tongue ( tt ) and groove ( tg ) structures ( eq . if tt is equal to tg , it is assumed that the tongue and groove are of equal thickness . by considering the transmission factors separately for t&g structures , the t&g effects on the dose distribution will be correctly included in the process of dose calculation . tcca requires a considerable area for smoothing ( at least 2.5 2.5 cm in 150 dpi ) , and if the area incorporating the t&g effect is much smaller than this large region in each color channel , the t&g effect will be sometimes ignored because of smoothing by tcca . hence the density difference due to t&g can not be corrected accurately for this large a region . in sqa , the dose comparison in the coronal plane includes a t&g evaluation . however , if the dose distribution has a high - gradient area in a small region , the slight density deviation due to t&g will be ignored as a noise area with tcca we showed that calibration curves obtained by tcca have better consistency than those produced by sca , particularly in the low - dose range . in the clinical evaluation , the potential advantage of the tcca method was observed in high - dose gradient regions in clinically relevant imrt and rapidarc cases . hence , ebt2 film dosimetry in combination with tcca could be useful in clinical practices such as sqa . it is noteworthy , however , that the correct handling procedure for ebt2 films is important to achieve accurate sqa results , even if tcca is used . in addition , the characteristics of tcca mean that this method sometimes ignores small deviations such as leaf transmission . we conclude that while tcca is useful for sqa , physicists should consider both its merits and demerits carefully .

the purpose of this study was to evaluate the triple channel correction acquisition ( tcca ) method for radiochromic film dosimetry performed with a flatbed scanner . the study had two parts : a fundamental and a clinical examination . in the fundamental examination , we evaluated the accuracy of calibration curves for gafchromic ebt2 ( ebt2 ) . the films were calibrated using a field - by - field method with 13 dose steps . seven calibration curves obtained by tcca were compared with those produced by a single channel acquisition ( sca ) method . for the clinical examination , we compared relative dose distributions obtained by tcca and sca for four cases of intensity - modulated radiation therapy ( imrt ) and intensity - modulated arc therapy ( imat ) . the fundamental examination showed that the consistency of the calibration curves was better for tcca than for sca , particularly for the dose range between 0.25 gy and 1.00 gy . the clinical examination showed that the dose differences between the measured and calculated doses in high - gradient regions were smaller with tcca than with sca . the average pass rates in gamma analysis for the tcca and sca methods were 97.2 0.8% ( n = 20 ) and 93.0 1.2% ( n = 20 ) , respectively . in conclusion , tcca can acquire accurate average dose values when creating the calibration curve . the potential advantage of tcca for ebt2 film dosimetry was seen in high - gradient regions in clinically relevant imrt and imat cases . tcca is useful to verify dose distribution .

metabolic syndrome refers to a cluster of metabolic abnormalities ( abdominal obesity , hyperglycemia , dyslipidemia , and hypertension ) related to a state of insulin resistance , often associated with an overweight or obese state . this clinical entity has been known to increase the risk of cardiovascular disease ( cvd ) , type 2 diabetes , chronic kidney disease ( ckd ) , and total mortality . metabolic syndrome is highly prevalent worldwide , with a prevalence ranging from 10 to 40% in different populations [ 13 ] . recently emerging data have suggested that metabolic syndrome is an important risk factor for ckd . better understanding of the underlying pathophysiology of metabolic syndrome related to ckd will help to identify potential treatment strategies to reduce ckd risk . the purpose of this paper is to explore the potential pathophysiology and treatment strategies related to metabolic syndrome and ckd by integrating available data from the literature . in 1923 , kylin first described a constellation of metabolic disturbances that included hypertension , hyperglycemia , and hyperuricemia . later scientists noted that , when these syndromes clustered together , they could have disastrous health consequences and referred to the clustering as syndrome x , insulin resistance syndrome , the deadly quartet , and obesity dyslipidemia syndrome [ 913 ] . between 1998 and 2005 , three definitions of metabolic syndrome had been developed and widely used ( table 1 ) . the three definitions stated that the primary components of the syndrome included central obesity , dyslipidemia , elevated blood pressure , and increased glucose . furthermore , previous studies have reported that all three definitions will identify persons at increased risk for diabetes , cardiovascular disease , and all - cause mortality [ 3 , 1416 ] . however , it is noteworthy that the ncep atp iii definition was a more powerful predictor of cvd and diabetes than the idf definition [ 1720 ] , while the idf definition identified more patients than the ncep atp iii definition [ 19 , 21 ] according to recent studies . therefore , ncep atp iii definition may have had more clinical impact . in 2009 , a global definition was developed by multiple organizations including the international diabetes federation ( idf ) , national heart , lung , and blood institute ( nhlbi ) , the world heart federation , the international atherosclerosis society , and the american heart association ( aha ) in an effort to harmonize clinical diagnosis of metabolic syndrome . their definition , summarized in table 2 , is the same as that by ncep atp iii with an exception that the criteria for elevated waist circumference are based on population- and country- specific definitions . in the last ten years , new research has examined the link between kidney disease and metabolic syndrome . in 2004 , chen et al . showed that metabolic syndrome was an independent risk factor of ckd . they examined the association of metabolic syndrome and risk of ckd in over 6000 subjects who participated in the third national health and nutrition examination survey ( nhanes iii ) and documented that metabolic syndrome was independently associated with risk of ckd . in 2005 , kurella et al . went further to include all metabolic syndrome traits in relation to risk for ckd . using data from the aric study , a prospective longitudinal study of cv disease risk factors in 10,096 middle - aged nondiabetic adults , they found that over a nine - year time span metabolic syndrome increased the risk of developing chronic kidney disease by approximately 50% . the multivariable - adjusted odds ratio of developing ckd in those with metabolic syndrome was 1.43 ( 95% ci 1.181.73 ) . they also looked at the individual traits associated with the syndrome and found that compared with an adult who has no metabolic syndrome traits , risk for ckd in someone with all five of the traits is two and a half times higher . in 2006 , ninomiya et al . they performed a slope analysis of the association between the glomerular filtration rate ( gfr ) slope and metabolic syndrome by using a multiple regression model . gfr decreased significantly faster in patients with 4 or more metabolic syndrome components compared with those who had 1 or no components . in 2007 , tozawa et al . conducted a prospective study to examine metabolic syndrome as a risk factor for ckd in an asian population . they examined ckd in 6,371 subjects without ckd or diabetes mellitus at baseline from 1997 through 2002 in okinawa , japan . during after adjusting for age , sex , current cigarette smoking , and alcohol drinking habits at baseline , the relative risk of developing ckd was 1.86 ( 95% confidence interval : 1.432.41 , p < 0.0001 ) in subjects with metabolic syndrome . insulin resistance has traditionally been defined by defective insulin action resulting in fasting hyperinsulinemia . yet , even before fasting hyperinsulinemia develops , postprandial hyperinsulinemia exists . the resultant hyperinsulinemia stimulates glucose uptake by muscle and suppresses endogenous glucose production in the liver . in insulin - resistant conditions , the ability of insulin to augment glucose uptake and inhibit hepatic glucose production is impaired . this creates a state of hyperglycemia that stimulates beta cells to secrete large amounts of insulin postprandially . high insulin concentration may overstimulate the cells of the arterial wall in the skeletal muscle . binding of insulin to the insulin receptor normally leads to activation of its tyrosine kinase activity and autophosphorylation of specific tyrosine residues of the receptor . the two major pathways for insulin signaling are the phosphatidylinositol-3 kinase ( pi-3k ) and the mitogen - activated protein ( map ) kinase pathways . the pi-3k pathway is initiated by tyrosine phosphorylation of a member of the insulin receptor substrate family , which is associated with the p85 regulatory subunit leading to activation of the enzyme . this results in activation of akt and downstream effector molecules that mediate metabolic response to insulin . this includes translocation of the glucose transporter type 4 ( glut4 ) into the membrane . the map kinase pathway begins with phosphorylation of insulin receptor substrate , which binds grb2 and activates ras . the erks mediate the mitogenic and proinflammatory responses of insulin signaling . in metabolic syndrome and type 2 diabetes , the pathways leading to activation of pi-3k are blocked , possibly through serine phosphorylation of the insulin receptor , leaving the map kinase pathway open . the activation of erk mapk pathway stimulates smooth muscle cell growth and proliferation , which maintains normal sensitivity to insulin even in insulin - resistant conditions . another key feature of metabolic syndrome is that free fatty acid production and release from adipocytes are not suppressed normally with the usual levels of insulin . adipocyte resistance to the antilipolytic effect of insulin and the consequent elevated plasma free fatty acid levels may play an important role in the development of insulin resistance in muscle and other target tissues . impairment in the pi-3k pathway could contribute to vascular endothelial dysfunction due to decreased nitric oxide [ 2729 ] . the study examined 2446 residents of a town in japan age 4079 without renal failure and had them undergo a series of physical and laboratory analyses including glucose tolerance test . the results were interpreted through correlation analysis and showed serum insulin , blood pressure , total cholesterol , low - density lipoprotein cholesterol , triglycerides , and body mass index were all negatively correlated with the reciprocal of serum creatinine level . in multiple regression analysis , the correlation between the sum of insulin levels and the reciprocal of serum creatinine remained significant even after controlling for age , sex , body mass index , blood pressure , total cholesterol , high - density lipoprotein cholesterol , low - density lipoprotein cholesterol , triglycerides , alcohol intake , and smoking habits . this study suggested that hyperinsulinemia was a significant relevant factor of renal function in the general population . renal dysfunction from insulin resistance and hyperglycemia is thought to be associated with the activation of the renin - angiotensin system ( ras ) leading to elevated angiotensin ii and aldosterone levels . the elevation affects the insulin / insulin - like growth factor-1 signaling pathways , causing oxidative stress leading to endothelial disruption , and even the development of cvd . insulin resistance and hyperinsulinemia are associated with decreased endothelial production of nitric oxide and increased oxidative stress which have been also implicated in the progression of diabetic nephropathy . when free fatty acids are released from the visceral fat , they drain into the portal circulation . it has been postulated that increases in this type of fat are directly associated with increases in risk for the sequel of metabolic syndrome . in addition , increases in abdominal subcutaneous fat would release lipolysis products into the systemic circulation and avoid direct effects on hepatic metabolism ( i.e. , glucose production , lipid synthesis , and secretion of prothrombotic proteins such as fibrinogen and plasminogen activator inhibitor 1 ) . furthermore , human adipocytes produce an as yet unidentified mineralocorticoid - releasing factor that stimulates adrenal aldosterone production by means of paracrine or endocrine mechanisms [ 35 , 36 ] . elevated levels of aldosterone promote insulin resistance and hypertension and therefore the development of the metabolic syndrome . when biopsies of obese patients are examined , focal and segmental glomerulosclerosis and glomerulomegaly are the most common morphological renal lesions . early changes noted upon review of biopsies seen in nondiabetic patients with only mild metabolic abnormalities and mild hypertension include increased glomerular cell proliferation , increased mesangial matrix , thicker basement membrane , and increased expression of glomerular transforming growth factor - beta . the mechanisms of obesity - induced renal injury likely result from a combination of hemodynamic and metabolic abnormalities . many factors contribute to the increase in both glomerular filtration rate ( gfr ) and rise in renal plasma flow ( rpf ) observed in obese patients . insulin resistance likely causes an increase in the efferent arteriolar pressure due to decrease of noradrenaline - induced efferent arteriolar constriction by insulin . insulin also stimulates the synthesis of igf-1 and igf-2 , both promoting glomerular hypertrophy . in 2011 , mathew et al . proposed that circulating cytokines ( leptin , adiponectin ) and inflammatory markers produced by adipose tissue are directly affecting cells in the renal glomeruli . moreover , elevated aldosterone in obesity promotes fibrosis and target - organ dysfunction by stimulating plasminogen activator inhibitor , transforming growth factor 1 , and reactive oxygen species ( ros ) [ 4143 ] . aldosterone also promotes loss of glomerular podocytes and a consequent decrease in the slit - pore membrane integrity , with consequent proteinuria [ 4447 ] . in addition , aldosterone increases renal tubular and interstitial oxidative stress and inflammation , processes that promote salt - induced tubuloglomerular injury , by means of rapid nongenomic effects . this condition is characterized by an increase in elevated triglycerides ( and increased vldl particle number ) , increased small ldl particles , and low hdl cholesterol . increased numbers of vldl and ldl particles lead to an increased level of total apo - b usually observed with atherogenic dyslipidemia . the ldl particles associated with the metabolic syndrome and atherogenic dyslipidemia tend to be small and dense . smaller ldls have been postulated to penetrate more easily into the arterial wall as well as be more prone to atherogenic modification . dyslipidemia seen in metabolic syndrome is postulated to cause ckd by inflammation and increased oxidative stress , which would cause endothelial damage and atherosclerosis diseases [ 5052 ] . used meta - analysis to postulate that elevated triglycerides and low hdl cholesterol in the plasma are independent risk factors for the development of chronic kidney disease . additionally , muntner et al . noted in the aric study that high triglycerides and low hdl cholesterol in plasma significantly increased the probability of developing renal dysfunction . it has even been examined that use of statins may slow the progression of chronic kidney disease . well - known complications of hypertension are chd , stroke , left ventricular hypertrophy , heart failure , and chronic renal failure . insulin is a vasodilator when given intravenously to people who are not obese . in the setting of insulin resistance , the vasodilatory effect of insulin can be lost , but the renal effect on sodium reabsorption preserved . in addition , fujita noted that , in obese rats , adipocyte - derived aldosterone releasing factors lead to hyperaldosteronism . hyperaldosteronism results in salt - sensitive hypertension as well as proteinuria in the obese hypertensive rats . fujita proposed that salt and aldosterone worked in synergy with the cardiovascular system through overproduction of oxidative stress . ros , induced by adipokines such as tumor necrosis factor - alpha , nonesterified fatty acids , angiotensinogen activated the mineralocorticoid receptor , in an aldosterone - independent fashion . the hypothesis proposed aldosterone and mineralocorticoid receptor activation may play an important role in the development of salt - sensitive hypertension , as well as the cardiovascular and renal injury seen in metabolic syndrome . some experts debate the clinical utility of aggregating individual risk factors into a specific diagnosis of metabolic syndrome when medically each risk factor is addressed separately . at this time the main strategy has been to reverse contributory factors such as an atherogenic diet , obesity , and a sedentary lifestyle . weight management and physical activity are recommended as first - line therapy in order to delay the progression of symptoms . epidemiological evidence suggests a lower prevalence of metabolic syndrome with dietary patterns that are rich in fruits , vegetables , whole grains , dairy products , and unsaturated fats . research from the dietary approaches to stop hypertension ( dash ) intervention studies demonstrates beneficial effects of an eating plan rich in low - fat dairy foods , fruits , and vegetables on blood pressure and lipids . a reduced calorie dash diet compared to a control and weight loss diet reduced most of the metabolic syndrome risks in both men and women and improved some components beyond that seen in a weight loss diet . the dash diet which is rich in calcium , magnesium , and potassium may also lower the risk of stroke and hypertension . fiber and other phytonutrients in fruit and vegetables may be protective by lowering cholesterol or markers of inflammation . some studies [ 65 , 66 ] suggest an inverse association between dairy consumption and risk for metabolic syndrome . in young overweight adults , the incidence of metabolic syndrome were lower by more than two - thirds among individuals in the highest category of dairy intake ( > 5 servings per day ) compared to lowest category ( < 1.5 servings a day ) . in addition , a dietary pattern that had higher intake of low - fat dairy has been associated with a lower risk of type 2 diabetes in middle aged or older women and with a 9% lower risk for type 2 diabetes in men . explored possible mechanisms underlying a dietary intervention and randomly assigned 180 patients ( 99 men and 81 women ) with the metabolic syndrome to either a mediterranean - style diet ( an increase in daily consumption of whole grains , vegetables , fruit , nuts , and olive oil ) or a cardiac diet with a decreased fat intake composed of less than thirty percent of total calories . only forty patients in the intervention group still had metabolic syndrome after two years compared with seventy - eight patients who consumed the control diet . given the increased blood pressure reactivity to dietary salt in patients with metabolic syndrome , a reduction in dietary salt may have a beneficial effect on lowering systolic blood pressure as suggested by hoffman 's study ( 8.2 g / day to 2.3 g / day of salt ) . recently , chen et al . reported that metabolic syndrome may enhance blood pressure response to sodium intake in nondiabetics . however , if low - sodium diet could lead reduction of metabolic syndrome - related morbidity and mortality remains to be investigated in clinical trials . in the diabetes prevention program trial , metformin reduced the risk of diabetes and the metabolic syndrome in individuals with impaired fasting glucose and impaired glucose tolerance . then assigned participants to metformin ( 850 mg twice daily ) or a lifestyle - modification program and followed them for 2.8 years . the lifestyle intervention reduced the incidence by 58% ( 95% ci , 48%66% ) and metformin by 31% ( 95% c , 17%43% ) , as compared with placebo ; the lifestyle intervention was significantly more effective than metformin . in a 10-year followup of this study , it was found that prevention or delay of diabetes with lifestyle intervention or metformin could persist for at least 10 years . in patients with the metabolic syndrome but normal glucose tolerance this is due to the fact that renal clearance of metformin and lactate is reduced , leading to increased levels of both and possibly causing lactic acidosis due to a buildup of lactic acid . rosiglitazone reduced the three - year incidence of type 2 diabetes by 60 percent in patients with impaired glucose tolerance or impaired fasting glucose who were taking the medication at the time of testing . the management of insulin resistance with thiazolidinediones ( tzds ) has resulted in greater attention to activators of the peroxisome proliferator - activated receptors ( ppars ) . tzds exert much of their effect on insulin resistance via activation of ppar - gamma . tzds not only improve glucose control but favorably affect both free fatty acid metabolism and insulin action . szapary et al . examined the effect of pioglitazone in patients with the metabolic syndrome and demonstrated a significant increase in hdl - c and favorable effects on lipid subfractions without an effect on triglycerides or low - density lipoprotein cholesterol ( ldl - c ) concentrations . tan et al . noted that pioglitazone therapy resulted in greater improvements in the atherogenic index of plasma and lowered triglyceride levels effectively while achieving greater increases in hdl - c when compared with rosiglitazone . the efficacy of statins in making marked reductions in ldl cholesterol , modest reductions in tg , and small increases in hdl cholesterol is well documented . atp iii recommended a goal serum ldl cholesterol of less than 100 mg / dl ( 2.6 mmol / l ) for secondary prevention in patients with type 2 diabetes [ 78 , 79 ] . patients in the 4s trial who met the lipid criteria for the metabolic syndrome were treated with simvastatin , 20 or 40 mg / d . it was noted that they had a 37.5% versus a 36.0% decrease , respectively , in ldl cholesterol , a 24.1% versus 6.7% decrease in tg , and a 10.3% versus a 0.6% increase in hdl cholesterol . rosuvastatin , 10 mg / d administered to patients with the metabolic syndrome , reduced ldl cholesterol by 47% , apolipoprotein b by 37% , and tg by 23% , while increasing hdl cholesterol by 10% . multiple statins have been shown to reduce cardiovascular events in patients with and without cvd , suggesting that this is a class effect of these drugs . some of the cvd risk reduction produced by statins may be attributable to nonlipid pleiotrophic effects of these drugs . treatment of patients with known coronary disease and the metabolic syndrome with atorvastatin 80 mg , compared to atorvastatin 10 mg , decreased the rate of major cardiovascular events at five years ( 9.5 versus 13 percent , hr 0.71 , 95% ci 0.610.84 ) . in 2009 , robinson et al . evaluated the lipid - lowering efficacy of ezetimibe / simvastatin 10/20 mg versus atorvastatin 10 or 20 mg , and ezetimibe / simvastatin 10/40 mg versus atorvastatin 40 mg in 1,128 patients with hypercholesterolemia and the metabolic syndrome . they noted that greater improvements in the levels of ldl cholesterol , non - high - density lipoprotein cholesterol , apolipoprotein b , and lipid / lipoprotein ratios resulted with ezetimibe / simvastatin compared with atorvastatin at all specified dose comparisons ( p < 0.001 ) . patients with hypertension and the metabolic syndrome have high risk of suffering from future cardiovascular and kidney disease . at present there are no large - scale , randomized trials to establish the antihypertensive drug of choice for patients with metabolic syndrome . most investigators have considered angiotensin - converting enzyme inhibitors ( acei ) as superior to beta - blockers and thiazide diuretics . the adverse potential metabolic side effects of thiazides and beta - blockers on increase of blood lipids and glucose have led to favoring of acei and calcium channel blockers ( ccb ) . beta - blockers also promote weight gain , and both thiazides and beta - blockers are associated with an increased incidence of diabetes , compared to ccb and acei . conducted subgroup analysis of the allhat study reporting that findings fail to support the preference for ccbs , alpha - blockers , or aceis compared with thiazide - type diuretics in patients with the metabolic syndrome . wright examined the metabolic and cardiovascular outcomes of the allhat trial in patients stratified according to race ( black versus nonblack ) and the presence or absence of the metabolic syndrome . among all the patients studied , chlorthalidone had the least favorable effects on blood glucose and cholesterol levels than lisinopril , amlodipine . in patients without the metabolic syndrome , both the acei and the ccb lowered the incidence of type 2 diabetes significantly , compared to the thiazide . wright also noted that black patients with the metabolic syndrome had worse outcomes with lisinopril , compared to chlorthalidone , with respect to every outcome measured , likely resulting from a 35 mmhg higher systolic pressure in the black patients on lisinopril , compared to those on chlorthalidone . wright concluded that acei should not be the first - line monotherapy for black patients with the metabolic syndrome . over the last decade , physicians have been examining the effects of resistance training on metabolic syndrome . reduced muscle mass as a result of normal aging and decreased physical activity have been postulated behind the high prevalence of this disorder . improved glycemic control , improved blood lipid profiles , and decreased bp are important for reducing microvascular and macrovascular complications in people with metabolic risk . as with increasing adiposity in aging and loss of muscle mass , the insulin - mediated glucose uptake and tg disposal in the skeletal muscle of elderly persons is reduced and the maintenance of a large muscle mass can contribute to the prevention of type 2 diabetes , which is associated with cardiovascular disease . resistance training is contributing to the decrease of major risk factors for the metabolic syndrome and should be recommended for the management of type 2 diabetes . although the number of studies on the effects of resistance training on blood pressure is small , strasser conducted meta - analysis confirming that resistance training does not increase blood pressure as was once thought and may even have potential benefits on resting systolic blood pressure . in the prospective controlled clinical study conducted by lee et al . , metabolic syndrome was prevalent in 52.2% of morbidly obese individual enrolled . significant weight reduction 1 year after surgery markedly improved all aspects of the metabolic syndrome and resulted in a cure rate of 95.6% . obesity surgery performed by laparoscopic surgery is recommended for obese patients with the metabolic syndrome that have not responded to conservative measures . while we are waiting for randomized clinical trial and even new drug development in treating metabolic syndrome to reduce risk of ckd , current key strategies should include prevention and treatment of obesity and insulin resistance . lifestyle modification particularly including low sodium diet and increasing physical activity would be important approaches . aldosterone antagonists would also be particular of interest to test in clinical trial in treating metabolic syndrome to reduce ckd risk . at the present , early identification of metabolic syndrome and treatment of individual components of metabolic syndrome may reduce the risk of ckd . however , these approaches need be further tested in large randomized clinical trial to verify their effect on reducing ckd risk .

metabolic syndrome is characterized by a clustering of cardiovascular risk factors , including abdominal obesity , elevated blood pressure and glucose concentrations , and dyslipidemia . the presence of this clinical entity is becoming more pervasive throughout the globe as the prevalence of obesity increases worldwide . moreover , there is increased recognition of the complications and mortality related to this syndrome . this paper looks to examine the link between metabolic syndrome and the development of chronic kidney disease .

hypertension is a chronic problem in the global health field and has resulted in a significant global responsibility in developed and developing countries . research has shown that the prevalence of this disease in all countries is increasing , with an annual death rate of 1.7 million people worldwide . hypertension is a common , asymptomatic disease that is usually easy to treat ; however , if left untreated , it often leads to fatal complications . although accurate statistics on the prevalence of hypertension are not available in iran ; the prevalence of hypertension has been reported to be 11% in isfahan , 17% in chaharmahal bakhtiari , 16.6% in gilan , 17.5% in zanjan , 6.26% in kermanshah , and 18.9% in arak . according to statistics from the ministry of health and medical education in iran , 20 of every 100 adults have high blood pressure ; half of these patients are not aware of their conditions , and only 5 of them are currently receiving treatment . in other words , this issue is not limited only to iran , but is also reported in developed countries . hypertension is defined as a systolic blood pressure higher than 140 mm hg and diastolic blood pressure above 90 mm hg . an individual s level of knowledge and his or her understanding of the risk factors of the disease as well as the motivation to reduce them can improve health and prevent disease through lifestyle changes . lifestyle has always been a focus of health education and promotion . the world health organization considers lifestyle as the specific patterns of behavior events that result from the interactions between personal characteristics , social relations , environmental conditions , and socioeconomic circumstances . studies have demonstrated that nutrition monitoring , physical activity , stress , smoking , weight control , rest , and sleep are particularly important lifestyle factors . an emphasis on the significance of lifestyle modification in blood pressure control and treatment is crucial ; accordingly , the seventh report of the joint national committee ( jnc7 ) on prevention , detection , evaluation , and treatment of high blood pressure considers inadequate attention to health education by health workers and a lack of health education coverage in society as the most important barriers to prevention , control , and treatment of hypertension . given that one of the basic tools in changing a patient s lifestyle is the presence of patient education programs as a part of the care system , ongoing training is regarded as an essential part of the monitoring and treatment of high blood pressure and might need to be repeated or modified in order to address changing circumstances and patient conditions . the importance of training programs depends on their effectiveness ; however , in the presence of appropriate theoretical support along with basic health needs , the effectiveness of these programs is greater . given the importance of healthy lifestyles and their considerable impact on disease prevention and health maintenance , this study aimed to survey the effect of educational intervention on lifestyle modifications in patients with hypertension in the city of aligudarz in lorestan province , iran , in 2014 . this study was quasi - experimental and was conducted from november 2013 to june 2014 , in the rural regions of ghaman sultan ( 30 people ) , doozan ( 28 people ) , and soor ( 28 people ) in the township of aligudarz in lorestan province , iran . the sample consisted of 86 hypertensive patients divided into case and control groups ( 43 individuals in each group ) using simple random sampling . the data were collected from interviews through a standard questionnaire of hplp ii , whose content validity and reliability ( = 0.87 ) were confirmed in a study by morovati et al . . demographic variables assessed with 30 questions , including questions on age , sex , marital status , occupation , and education ; 2 . data on lifestyle , including 52 questions with answers ranging from never , sometimes , often , and always , which measure the frequency of health - promoting behaviors in 6 components of health responsibility ( 9 items ) , spiritual growth ( 9 items ) , physical activity ( 8 items ) , nutrition ( 9 items ) , interpersonal communication ( 9 items ) , and stress management ( 8 items ) . the inclusion criteria for the study were comprised of : 1 . a diagnosis of hypertension by a physician , 2 . lack of access due to patient travel . in order to comply with research ethics , this study was approved by the ethics committee at ilam university of medical sciences , and the research purpose was delineated for the participants before its implementation stages . at the beginning , a pre - test was administered to both case and control groups ; then the case group was divided into three groups with 15 subjects each . two months after the fourth training session of the case group , both case and control groups received the post - tests . data were analyzed via statistical tests including the t - test and chi - square test as appropriate using spss software with a significance level of 0.05 . the mean and standard deviation of age in both case and control groups were 59.95 7.9 and 64.51 9.2 years ; respectively . the independent t - test showed no significant difference in the mean age between the two groups ( p = 0.47 ) . the mean and the standard deviation of blood pressure were 15.19 1.02 in the case group and 14.47 1.22 in the control one . the independent t - test revealed no significant difference in blood pressure between the two groups before intervention ( p = 0.13 ) . the chi - square test showed no significant difference between the two groups in terms of sex , marital status , education , occupation , family history of hypertension , or personal history of hypertension ( p > 0.05 ) ( table 1 ) . the chi - square test showed that the consumption of salt as well as fatty and fried foods considerably and significantly decreased after intervention in the case group ( p = 0.01 ) compared to the control group ( p = 0.17 ) . fruit and vegetable intake after educational intervention in the case group ( p = 0.05 ) showed a significant increase compared to the control group ( p = 0.6 ) . the results of the independent t - test between the case and control group revealed no significant difference between the mean scores of the two groups in terms of total lifestyle , physical activity , nutrition , or stress management before the training ( p > 0.05 ) ; this relationship was significant two months after the intervention ( p < 0.05 ) ( table 2 ) . the paired t - test results demonstrated that the mean scores of total lifestyle , physical activity , nutrition , and stress management were significantly increased two months after the intervention ( p < 0.05 ) ( table 3 ) . based on the findings of this study , educational intervention has an effect on lifestyle for patients with hypertension . in this study , the mean total score for lifestyle increased by 24 points . similar results were reported in a study by goodfrey katende et al . in a study by owji et al . , lifestyle and self - monitoring scores increased after the intervention ; blood pressure rates also increased . lifestyle should be viewed as a complex combination of actions and behavior habits in individuals and groups , especially in terms of cultural and socioeconomic conditions , social communications , and personality . teaching proper nutrition can help reduce death rates as well as disability from chronic hypertension . in this respect , , syria , japan , and iran indicate the existence of improper nutritional habits and insufficient knowledge of proper nutrition . in the present study , the mean score of nutrition in the case group rose by 16.6 after training . the results of a study by najimi et al . showed that blood pressure was reduced in the case group after three months of nutritional education . several studies , including a meta - analysis by gasperin et al . , showed that chronic stress and tension increase blood pressure . in this study , the mean score of stress management in the case group increased by 36.6 after training . the results of multiple studies conducted in patients with hypertension , including studies by tang et al . and age et al . , contributed to the results of the present study . after the training , the mean score of physical activity in the case group increased by 8.76 points . showed in their study that training programs help to broaden individuals awareness of physical activity . a study by stefani et al . showed similar results . according to the results of this study , salt intake in the case group decreased significantly in comparison with that of the control group . in addition , fruit and vegetable consumption in the case group were significantly increased compared with the control group . on the other hand , the consumption of fried and fatty foods in the case group showed a significant decrease compared with that of the control group . these results are consistent with the results of a study by lesan et al . . in this study , 51.2% of the subjects in the case group and 53.5% in the control group reported a family history of hypertension . these results are in line with the findings presented by tesfaye . in this study , a family history of hypertension was noted as a risk factor of high blood pressure . in the present study , there was no significant difference between body mass index and blood pressure in either group , which is consistent with the results of a study by shafieyan et al . . the results of this study revealed a correlation between lifestyle and hypertension . given the importance of lifestyle and its significant impact on disease prevention and health maintenance , the use of educational programs on nutrition , physical activity , and stress management is required to improve patient knowledge and modify their behaviors .

backgroundhypertension is one of the most common chronic health problems across the world , resulting in significant global responsibility in developed and developing countries . the aim of this study was to survey the effect of educational intervention on the lifestyle of patients with hypertension.methodsthis study was a case - control intervention study on 86 patients with hypertension that were selected by simple random sampling from the rural regions of aligoudarz county in lorestan province . before the intervention , both groups completed the standard questionnaire of hplp ii ; two months after the intervention , both groups completed the same questionnaire . the results were analyzed using spss software , t - test , and chi - square test.resultsthe mean age and standard deviation in case and control groups were 59.95 7.9 and 64.51 9.2 years , respectively . the mean of the total lifestyle scores was significantly increased in the case group compared with the control group ( p < 0.05 ) . in addition , the average scores for the three dimensions of physical activity , nutrition , and stress management after educational intervention in the case group compared to the control group showed a significant increase ( p < 0.05 , for all).conclusionbased on the relationship between lifestyle and hypertension , it seems that implementing educational programs in the fields of nutrition , physical activity , and stress management is essential to improvement in disease knowledge and behavior modification among patients with hypertension .

werlhof s disease or morbus maculosus haemorrhagicus was described by paul gottfried werlhof in 1753.1 this is likely to be the same condition mentioned in 1557 as morbus pulicaris absque febre by amatus lusitanus in his curationum medicinalium centuriae quatuor.2 the description by lusitanus of dark spots without fever is likely to have been a manifestation of immune thrombocytopenia ( itp).3 in 1658 , riverius postulated that the dark spots were due to thinness of the blood . the intervening years led to the discovery of blood granules that were later termed platelets . with the description of platelets by bizzozero in 1882,4 it was then possible to associate number of platelets with itp . in 1887 , denys5 observed that the platelet number dropped during the episode of purpura and that there was an increase in platelet count after the hemorrhagic episode . why were platelets low in itp ? as documented by bedson,6 the first antiplatelet sera was described by mf marino in 1905 . a decade later , in 1915 , jcg ledingham showed that anti - guinea pig platelet serum was noxious to guinea pigs and produced a condition analogous to itp in humans . the well - known harrington hollingsworth experiment clearly demonstrated that a factor in plasma was able to significantly reduce platelet number in healthy subjects transfused with plasma from itp patients.7 this evidence appeared to demonstrate that accelerated platelet destruction was the key initiating event in itp . these platelet - damaging factors in plasma are immunoglobulins that recognize abundant platelet receptors such as gpiib / iiia and gpib / ix . several studies have shown that a large proportion of itp patients have both platelet - associated and circulating antiplatelet autoantibodies.810 the coating of platelets by antiplatelet autoantibodies then leads to fc receptor - mediated phagocytosis.11 there is evidence for additional mechanisms that may cause a reduction in circulating platelets . for instance , destruction of autologous platelets by cytotoxic t - lymphocytes has been observed in chronic itp patients.12,13 lately , a new mechanism was described that may account , at least in part , for platelet destruction in itp . the loss of sialic acid moieties from platelet glycoproteins ( termed desialylation ) due to autoantibody activity increases platelet destruction by liver cells.14,15 thus , several mechanisms are responsible for platelet destruction in itp . however , as will be discussed ( and crucially for the mechanism of action of thrombopoietin receptor agonist [ tpo - ras ] ) , platelet destruction is only a partial explanation , and a reduction in platelet production by megakaryocytes is fundamental for the pathogenesis of itp . in 1890 , soon after bizzozero s description of the platelet , howell16 described the megakaryocyte and sometime later in 1906 wright17 proposed that these cells were the source of platelets . in the 1940s , it was shown that patients with itp had normal ( or slightly increased ) megakaryocyte numbers , but crucially a large proportion of these cells did not form platelets.18 this suggests that the observed reduction in platelet number is also a consequence of insufficient production . it is now clear that antiplatelet autoantibodies interact with glycoproteins on megakaryocytes.1921 the consequence of this interaction is detrimental to megakaryocytic progenitor cells22 and , as will be described , also to mature megakaryocytes.21,2325 observations in culture have indicated that itp autoantibodies have a negative impact on megakaryocyte differentiation , polyploidization , and proliferation.26,27 more recently , it was shown that antiplatelet autoantibodies from drug - induced itp inhibited megakaryocyte maturation , proliferation , and proplatelet formation.21 iraqi et al23 demonstrated that the treatment of cord blood - derived mature megakaryocytes with igg purified from itp plasma inhibited proplatelet formation and platelet release in culture . both inhibition of proplatelet formation and a reduction in proplatelet complexity have also been observed after treatment of megakaryocytes with anti - gpiib / iiia antibodies isolated from itp patients.24 together these studies indicate that antiplatelet autoantibodies interact with megakaryocytes and suppress platelet production . treatment of itp is initiated if the platelet count is under 3010/l or if signs of bleeding are present . depending on other factors ( comorbidities , risk of bleeding ) , a platelet count of < 5010/l may warrant intervention.28 the aim of the intervention is to reduce the likelihood of bleeding by maintaining an adequate platelet level . corticosteroids ( prednisone , dexamethasone ) are the first line of therapy28 and act by reducing phagocytosis and by decreasing autoantibody production . other first - line options are intravenous immunoglobulin ( ivig ) and anti - d . the effectiveness of ivig and anti - d in itp relate to the suppression of platelet destruction . ivig may decrease platelet destruction via the inhibition of fc receptors and by exerting a suppressive effect on the production of autoantibodies by b - cells . ivig may also compete with and promote the clearance of the autoantibody.29 these first - line treatments do not provide a permanent platelet response in most patients . the second line of itp therapy includes cyclosporine a , rituximab , splenectomy , and tpo receptor agonists.30 the depletion of b cells with anti - cd20 antibody ( rituximab ) is effective in itp . the initial study by cooper et al31 demonstrated a response in 54% of subjects and a lasting response in 32% . an analysis of 72 adults and 66 children who responded to standard rituximab administration found that 26% of patients achieved treatment - free response after 5 years.32 recently , a new triple therapy given over 4 weeks was developed in our hospital.33 this intervention consists of high - dose dexamethasone , cyclosporine a , and low - dose rituximab . the trial resulted in relapse - free survival of 76% after 2 years.33 this indicates that by using current drugs a lasting remission might be attainable in a large percentage of cases and merits further investigation . the hypothesis was that itp was caused by excessive destruction of platelets by the spleen . the first splenectomy performed in an itp patient was successful and resulted in a rapid increase in platelet number and resolution of the purpura.3 this established the use of splenectomy as the therapy of choice for cases of severe itp and it remained as the sole therapeutic choice until the 1950s ( a second therapeutic option only became available in the middle of the 20th century with the introduction of corticosteroids ) . to date , splenectomy remains a central option for adults that do not respond to steroid treatment.34 it should be noted that splenectomy is an effective treatment and is the only intervention that leads to a complete response in a large number of patients.35 a systematic review of the literature ( 2,623 adults ) found that there was a stable complete response in two thirds of itp patients34 ( median follow - up 28 months ) . mikhael et al,36 who reviewed 23 studies conducted between 1991 and 2008 , reached a similar conclusion . they reported a failure rate in 28% of cases 5 years after splenectomy.36 there are complications associated with the procedure , including the possibility of postoperative bleeding , sepsis , and thrombosis . factors that affect decision - making include impact of itp in the patient s life , personal opinion of the likely success of the intervention , and consideration of splenectomy as a treatment of last resort.37 there may also be some reluctance to splenectomy by practitioners given that it is an invasive procedure that does not eliminate the possibility of relapse.38 it has been proposed that splenectomy could be delayed for up to 3 years due to the increased likelihood of remission in that period.39 moreover , removal of the organ causes loss of functions such as antibody production and effective elimination of nonfunctional blood cells . in fact , the rate of splenectomy as a treatment for itp appears to be in decline.40 the rebound in platelet levels following thrombocytopenia was ascribed to a substance termed thrombopoietin ( tpo or thpo ) in the 1950s ( see review by kaushansky41 ) . isolation and eventual cloning of tpo was a demanding undertaking and took decades to accomplish . cloning and biological evaluation of murine tpo in 199442 was followed by the demonstration that tpo sustained megakaryocyte colony formation and promoted differentiation and polyploidization both in vitro and in vivo.43 tpo activity is mediated via activation of its receptor , c - mpl . the cloning and characterization of tpo led immediately to proposals regarding its potential therapeutic use . the first recombinant preparations to be evaluated were human thrombopoietin ( rhtpo ) and pegylated human megakaryocyte growth and development factor ( peg - rhmgdf ) . the former was the full - length protein produced in mammalian cell culture and the latter was expressed in bacteria and contained the tpo receptor binding domain ( these compounds are now called first - generation tpo - ras ) . these agents were promising in their capacity to increase platelet counts in conditions such as itp,44 myelodysplastic syndrome,45 cancer,46,47 and hiv - associated thrombocytopenia.45 the observation of severe thrombocytopenia in some subjects treated with peg - rhmgdf ended the clinical application of the first generation of tpo - ras . the drop in platelet counts was caused by the development of antibodies against peg - rhmgdf that cross - reacted with and neutralized endogenous human tpo.48 to circumvent potential detrimental immune reactions , efforts were directed toward the development of molecules structurally unrelated to tpo . several tpo - ras underwent development and two of these , romiplostim and eltrombopag , were approved for clinical use in 2008.49,50 here , we will look specifically at studies using romiplostim . romiplostim is a peptibody consisting of four c - mpl binding peptides ( 14 amino acids long ) linked to two fc domains of igg1 . the peptide has no sequence homology to tpo but competes for binding to its receptor.51 engagement of romiplostim with c - mpl leads to receptor activation , intracellular signaling , and increased platelet production.52 the lack of sequence homology suggests that if anti - romiplostim antibodies were developed , these would not interact with and neutralize endogenous tpo . indeed , a study of 225 itp patients treated with romiplostim found that 11% developed anti - romiplostim antibodies , but only one patient had neutralizing antibodies.53 importantly , none of these antibodies cross - reacted with endogenous tpo.53 of note , 17 out of 225 patients investigated had preexisting anti - romiplostim antibodies , but this did not affect the activity of the drug . the effectiveness of romiplostim ( also known as amg 531 , amp-2 , or nplate , amgen inc . , thousand oaks , ca , usa ) as a potential therapy for itp was first shown in a dose - finding clinical trial.54 amg 531 was found to be effective in increasing platelet counts in itp patients and did not produce significant adverse side effects.54 in this study , most patients ( 79% in phase i and 67% in phase ii ) had undergone splenectomy,54 but the response data were not collated based on this criterion . a study by kuter et al55 examined the activity of romiplostim in both splenectomized and nonsplenectomized itp patients . the durable platelet response ( defined as a platelet count of 5010/l during at least 6 of the last 8 weeks of treatment ) was more pronounced in nonsplenectomized patients ( 56% ) versus 38% in the splenectomized cohort.55 the overall response ( both transient and durable ) was 88% and 79% for nonsplenectomized and splenectomized patients , respectively . on the other hand , a japanese phase iii trial of 10 splenectomized and 12 nonsplenectomized itp patients found no differential effect on weekly platelet response.56 even though treatment with tpo - ra requires ongoing drug administration and is not a single intervention like splenectomy , the potential adverse effects of splenectomy would argue against its preferential use as a second line of therapy . the adverse effects of romiplostim include headaches , fatigue , and nasopharyngitis , while for splenectomy adverse outcomes may include hemorrhage , infection , sepsis , and , in rare cases , death.30 so far , the adverse effects of prolonged romiplostim use seem minor . an analysis of over 1,000 patients receiving romiplostim for a mean of 76 weeks found no increase in adverse effects including thrombosis , bone marrow reticulin , or malignancy.57 the fact that no increase in thrombosis was reported is important since itp , despite low platelet counts , could be considered to be a prothrombotic condition.58 in fact , splenectomy would be more concerning in this case since it is associated with an increase in thrombotic events.59 analysis of 13 clinical trials ( 653 patients ) treated with romiplostim for extended periods ( up to 5 years ) found that the treatment was tolerated and there were no salient safety concerns in terms of hematopoietic malignancies , bone marrow reticulin or thrombotic events.60 a study of 234 nonsplenectomized patients receiving either romiplostim or standard of care reported better platelet response , improved quality of life , and lower rates of splenctomy ( 9% versus 36% ) in patients receiving romoplostim.61 it should be noted , however , that a meta - analysis of 15 studies with 3026 thrombocytopenic patients concluded that there was an increased risk of thromboembolism in patients treated with tpo - ras relative to controls ( frequency 3.69% versus 1.46% , respectively).62 however , the statistical significance was principally obtained from non - itp thrombocytopenic patients such as those with chronic liver disease . this study lends some support to the european guidelines , which allow romiplostim treatment in nonsplenectomized patients only if splenectomy is not an option.63 the guidelines recommend the use of romiplostim in splenectomized patients if they are refractory to standard treatments . the american society of hematology ( ash ) guidelines regard splenectomy as an intervention that achieves remission in most patients . therefore , the ash guidelines would consider splenectomy as an option following failure of corticosteroid therapy.64 the international consensus group , on the other hand , gives equal consideration to splenectomy as to other second - line treatments such as rituximab , cyclosporine a , and tpo - ras.30 splenectomy remains the only option for patients that are refractory to both first- and second - line therapies , but the procedure is advisable only if satisfactory platelet counts are not obtained for some time.28 refractoriness refers to the proportion of itp patients that does not respond to treatment . in the case of splenectomized patients , it refers to persistent low platelet counts and the requirement for additional therapy to maintain adequate levels.65 why would refractory patients respond to tpo - ras ? the dual nature of itp , ie , increased platelet destruction and decreased platelet production , suggests that if platelet production is sufficiently suppressed , then interventions that prevent platelet destruction , such as corticosteroids , ivig , rituximab , and splenectomy , would not be adequate to overcome the platelet deficit . therefore , only patients with higher levels of platelet production would be expected to be more responsive to inhibition of platelet destruction . tpo - ras are the only option that deals with suboptimal platelet production and are likely to benefit preferentially patients with impaired platelet formation . the fact that splenectomized patients responded well to romiplostim treatment55,56 indicates that lack of platelet production was a major underlying cause in responders . dual therapy with ivig and romiplostim in patients unresponsive to either therapy has been successfully used to raise the platelet levels prior to splenectomy.66 it is likely that in these patients splenic platelet destruction was severe and could not be overcome by the activity of romiplostim alone . an unexpected benefit of tpo - ra administration is the sustained remission achieved by certain patients after cessation of therapy.67,68 remission seems to be independent of splenectomy status , sex , or age.65 a potential mechanism is the restoration of immune tolerance by tregs.69 why are not all itp patients responsive to tpo - ras ? nonresponsiveness to tpo - ras may be due to intrinsic properties of the antiplatelet autoantibodies . it is clear that the known activity of tpo - ras ( ie , induction of megakaryopoiesis ) still occurs in nonresponders , but platelet release is somehow blocked by the effect of the autoantibody.70 there might be several reasons for deficient platelet release by megakaryocytes , for instance , defective signaling pathways or mutations ; however , it appears that the activity of different classes of autoantibodies , such as anti - gpib / ix antibodies , might prevent platelet release.25 experimental evidence using cultured megakaryocytes indicates that , in the presence of itp autoantibodies , tpo - ras are capable of increasing the proportion of megakaryocytes producing proplatelets from an existing population.23 this implies that there is an additional mechanism in which tpo - ras operate on mature megakaryocytes , overcome the harmful activity of the antiplatelet antibody , and promote proplatelet formation . importantly , tpo - ras were not effective in the presence of some of the autoantibodies tested,23 suggesting that the nature of the autoantibody ( specificity , binding site , affinity ) is a substantial determinant of the outcome . future work will substantiate this proposed property of tpo - ras . for itp patients refractory to other treatments , splenectomy remains the only therapy that provides the prospect of complete remission . the development of romiplostim has seen the addition of a platelet - boosting drug to the treatment of itp . romiplostim administration generates a stable platelet response in a majority of patients without significant adverse effects and results in a reduced number of patients undergoing splenectomy . ongoing observation is required to establish its safety and effectiveness after long - term use .

romiplostim is a thrombopoietin receptor agonist ( tpo - ra ) used for the treatment of adult primary immune thrombocytopenia ( itp ) . itp is an autoimmune condition characterized by low platelet counts due to increased destruction and reduced platelet production . first - line interventions include corticosteroids , anti - d , and intravenous immunoglobulins , while second - line therapies comprise splenectomy , rituximab , cyclosporine a , and tpo - ras . the recognition that compromised platelet production is a critical part of the pathogenesis of itp prompted the development of therapeutic strategies based on the stimulation of the tpo receptor . tpo - ras enhance megakaryocyte proliferation , increase platelet production , and lead to a reduction in bleeding episodes in itp patients . this review will summarize current data on the tpo - ra romiplostim , with a particular focus on its relation to splenectomy .

recent studies have observed the reduced blood flow in both ocular and peripheral districts in patients suffering from retinitis pigmentosa ( rp ) . the drop of the blood flow has been detected not only in retinal and choroidal vessels but also in retroocular vessels [ 24 ] . also , a reduced baseline peak flow in the cutaneous capillary of fingers has been measured and a significantly longer recovery time has been found after cold provocation . in the early stages of rp before detecting any ophthalmoscopic fundus modification , an increase of the arteriovenous passage time has been observed in the retina . using colour doppler imaging , cellini and coworkers demonstrated reduced peak systolic velocities in both ophthalmic arteries and posterior ciliary arteries [ 3 , 4 ] . they also observed a certain augmentation of endothelin-1 ( et-1 ) plasma levels in patients with early - stage rp , even though some investigators took issue with this observation [ 68 ] . a sort of dysregulation of both blood supply and vessel caliber seems to occur often in rp patients . konieczka has suggested that the primary vascular dysregulation syndrome , mainly characterized by an impaired vascular tone , might describe a wide range of local and systemic signs , such as ocular blood flow reduction with subsequent augmentation of et-1 plasma levels in the eye and peripheral vasospasm in response to cold , emotional stress , or low blood pressure [ 1 , 913 ] . disturbed autoregulation of ocular perfusion provokes an irregular blood flow , which means an unstable retinal blood supply , and a sort of attenuation of retinal vessels with reduced neurovascular coupling [ 14 , 15 ] . therefore , a large number of rp patients show a high prevalence of primary vascular dysregulation syndrome as the primary manifestation of ocular blood flow dysfunction . even if the exact pathophysiological mechanism is still unknown , the dysfunction of both autonomic nervous system and endothelial cells is currently investigated . it is characterized by alterations of photoreceptors ( prs ) and retinal pigment epithelium ( rpe ) resulting in progressive retinal degeneration . the most frequent symptoms are night - blindness and the growing impairment of visual field , perceived as tunnel vision , that may lead to legal blindness . clinical signs found at ophthalmic fundus examination are characteristic : bone - spicule pigment deposits in the mid periphery along with rpe atrophy , attenuation of retinal vessels , waxy pallor of the optic disc , and relatively spared macula surrounded by a perimacular ring of depigmentation [ 16 , 17 ] . the age of onset , the rate of progression , and the severity of rp are extremely variable depending not only on the genetic background but also on some influencing factors . symptoms may start in childhood as well as in the early or mid - adulthood . even though the progression of the disease is unpredictable , severe visual impairment typically occurs by the age of about 4050 years . the prevalence of all different forms of rp is reported to be about 1 : 35005000 individuals and , nowadays , it is estimated that there are almost two million affected people around the world [ 18 , 19 ] . the group of rp is characterized by a complex association between tremendous genotypic multiplicity and great phenotypic heterogeneity . the severity of the clinical manifestation depends on the penetrance of the disease gene , but also interactions between the gene expression and the environmental factors are of great importance . specific biochemical and molecular signaling pathways keep monitoring the perfusion pressure , originally controlled by the heart pump , and the local resistances , depending on the vessel caliber . vasospasm stands for inappropriate constriction of an artery . in the presence of excessive vasoconstriction and/or altered vasodilation , the overall condition is described as vascular dysregulation and it is often accompanied by a barrier dysfunction . if this condition is associated with symptoms or signs , it is referred to as vascular dysregulation syndrome . the diameter of the vessels is regulated by contraction ( vasoconstriction ) or relaxation ( vasodilation ) of both smooth muscle cells , forming the arterial or venous wall , and pericytes , encircling the capillaries . the smooth muscle cells are very sensitive to modifications of tension ( myogenic regulation ) , numerous metabolic factors originating from surrounding tissues , and biochemical signals coming from the autonomic nervous system ( neurohumoral regulation ) . the local perfusion pressure , depending on the cardiac output , and the local resistance to flow , depending on the rate of local autoregulation , are the two main aspects of the tissue blood flow . the endothelium is a very thin layer of cells lining vessels inwards . beyond its function as mechanical barrier , it plays crucial roles in immune and inflammatory responses , in haemostasis , and in vascular tone regulation . also it has autocrine , paracrine , and endocrine functions [ 24 , 25 ] . the first supplies the iris , the ciliary body , the choroid , and also , by diffusion , the outer retina , including the photoreceptors . the retinal vascular network is characterized by low perfusion rate , high vascular resistance , and high oxygen extraction . conversely , the choroidal network shows a high perfusion rate , a low vascular resistance , and a low oxygen extraction . autoregulation , actually effective within certain limits of perfusion pressure , is the intrinsic capacity to maintain constant flow despite changes in perfusion pressure . the neurovascular coupling is the tight link between the caliber of retinal vessels and the neuroretinal activity . a signal from the central nervous system , closely connected to the retina , is able to evoke small changes in the retinal blood flow through complex pathways involving neurons , glia , and endothelial cells . furthermore , the retinal vascular system features the blood retinal barrier which regulates the flux of ions , proteins , hormones , and water , even monitoring the infiltration of immune competent cells . it provides oxygen and other metabolites , controls eye temperature , and mostly contributes to the fine - tuning of accommodation by regulating choroidal thickness . the endothelin ( et ) system encompasses three active peptides ( et-1 , et-2 , and et-3 ) , two g protein - coupled receptors ( eta and etb ) , and activating peptidases including the et - converting enzymes ( ece-1 and ece-2 ) , . knock - out mice have been used as models to study pathophysiological role of the et system . some et components are essential for the development of both tissues , such as cardiac or craniofacial tissue , and systems , such as enteric and nervous system [ 2931 ] . endothelin-1 ( et-1 ) , widely distributed in human tissues , is produced by vascular endothelial cells and also by many other cells . endothelin-2 ( et-2 ) , with equally high affinities for both eta and etb , is largely expressed in the gastrointestinal tract and it serves as local and paracrine / autocrine mediator . very little is known about the function of endothelin-3 ( et-3 ) , except for the fact that it seems to be secreted somewhere near the relevant target cells , such as enteric neuroblasts expressing the etb . first identified in 1988 , et-1 is the most powerful endogenous vasoconstrictor of both small and large vessels [ 34 , 35 ] . it is a peptide with 21 amino acid residues and it is mostly released by endothelial cells of arteries , veins , and lymphatic vessels . et-1 acts as a modulator of the secretion of renin , vasopressin , and aldosterone , but at the same time it inhibits platelet aggregation . indeed , it has been observed in patients suffering with high blood pressure , arteriosclerosis , acute myocardial infarction , and diabetes mellitus [ 3841 ] . also hypoxic or oxidative stress , systemically and locally , has been identified as strong stimulus to raise primarily the levels of hypoxia - inducible factor 1 ( hif-1 ) and subsequently the levels of et-1 . in the eye , local synthesis and secretion of et-1 are performed by many tissues , such as cornea , uveal tissue , retinal microvascular pericytes , rpe cells , and optic nerve , suggesting that the et system could have an important role in the pathophysiology of some eye diseases [ 4246 ] . glaucoma , diabetic retinopathy , retinal vein / artery occlusion , proliferative vitreoretinopathy , and inherited retinal dystrophies are featured by impaired ocular blood circulation and they all present abnormal et-1 plasma levels [ 4751 ] . et-2 is a protein encoded by the endothelin-2 gene ( edn2 ) and is a member of the et system . it is referred to as macrophage chemoattractant and it is strongly induced in prs in the course of retinal diseases and injury , being involved as stress signal to mller cells through etb [ 52 , 53 ] . supposedly , a wide range of retinal disorders , including the inherited retinal dystrophies such as rp , converge on a relatively small number of molecular pathways going towards the cellular repair or death . mller cells , the most abundant glial cells in the retina , seem to monitor the status of retinal neurons , being generally activated after prs ' degeneration or death [ 55 , 56 ] . there is evidence that injured or degenerated prs start to release et-2 stimulating the activation of the mller cells fitted with etb . these glial cells increase both the production of glial fibrillary acidic protein and the sensitivity to et-2 by upregulating the etb as part of their repairing response [ 53 , 57 ] . et-1 can be used as biochemical marker to assess ocular hemodynamics . to measure intraocular et-1 blood concentration , investigations showed the significant correlation between et-1 plasma choroidal and plasma systemic concentrations . in cases of rp , cellini and coworkers have reported that a general increase in plasma levels of et-1 elicits vasoconstriction both systemically and locally in the eye , causing retinal and choroidal hemodynamic impairment involving both ophthalmic artery and posterior ciliary arteries [ 2 , 5 , 6 , 58 , 59 ] . the role of the et system in the pathogenesis of several ocular diseases , including the rp , is unquestionably important . however , at present there are some discordant studies about et-1 plasma levels in people suffering from inherited retinal dystrophies . cellini and colleagues have repeatedly observed high concentrations of et-1 in these patients , whereas ohguro and colleagues reported low levels of et-1 [ 3 , 8 , 51 , 60 ] . regardless of their opposite experimental observations , common evidence is the decreased intraocular blood flow , initially in both choroid and optic nerve head and later in retina , due to abnormal plasma levels of et-1 [ 61 , 62 ] . a proper biochemical pathway responsible for the reduced intraocular blood flow is still not ascertained ; however , there are some hypotheses under investigation . the first is about the migration of rpe cells to the inner layers of the retina after prs ' death . local secretion of et-1 from the proliferated and migrated rpe cells may stimulate repairing processes by deposition of extracellular matrix just underneath the retinal vessels . as a consequence , thinning and fenestration of retinal vascular endothelial cells the second assumption considers the fact that the more the prs die the less the ocular blood supply is required . another aspect to take into consideration is the low oxygen consumption by the degenerated and dying prs . the subsequent vasoconstriction , mediated by increased levels of et-1 , is referred to as mechanism for limiting higher oxygen levels locally [ 65 , 66 ] . table 1 supplies a brief and clear summary of the crucial role of et-1 in rp . on the basis of recent investigations , in subjects with rp there is a statistically significant correlation between the augmentation of et-1 plasma levels and the drop of the peak systolic velocity in both the ophthalmic artery and the posterior ciliary arteries . the genetics are definitely remarkable , but the impaired retinochoroidal blood supply might be considered as an important modifier of the progressive loss of prs . furthermore , a sort of systemic dysfunction of the microcirculation seems to be in people with rp . in fact , cellini and coworkers have measured , by means of laser doppler flowmetry , a lower baseline cutaneous capillary blood flow and a longer warm recovery time in rp patients with respect to healthy subjects . it has been observed that biochemical and metabolic alterations of the endothelial cells of retinal vessels and the augmentation of the intraocular oxygen concentration are triggering factors for the retinal production of et-1 . this molecule provokes high levels of intracellular ca in glial and neuronal cells and increases both neuronal activities and neuronal responses to glutamate . also , through eta receptors et-1 may have a synergistic effect on the glutamate - induced neurotoxicity in the retina . other studies have also highlighted the pivotal role of et-1 in relation to the apoptosis of the retinal ganglion cells through etb receptors . to date , big efforts are still to be made to assess a fine correlation between genetic mutations and clinical manifestations in the rp . the most of mutations occur in genes coding for proteins involved in the cycle of vision , at the level of rods , cones , and rpe cells . in the course of the disease , the prs go towards apoptosis , so that the outer retinal nuclear layer flattens . the pigment deposits , described as bone - spicule pigmentation , result from both rpe cell degeneration and migration into the neural retina after prs ' death . there is evidence that oxidative stress and related conditions play an important role in the inherited retinal dystrophies . another important aspect of rp is the reduced blood flow in both ocular and peripheral districts along with a substantial increase in levels of inflammation [ 1 , 22 ] . strobbe and coworkers demonstrated a statistically significant correlation between ocular inflammation , detected by measuring aqueous flare with the noninvasive laser flare - cell meter , and high et-1 plasma levels in patients suffering from rp . this condition features alterations in choroidal thickness and blood flow leading to a boost in free radicals and oxidative stress . several general conditions , such as hypoxia , chronic oxidative stress , vascular dysregulation , or systemic inflammation , provoke the increase of et-1 plasma levels . this aspect is well - recognized in patients with rp who have low and impaired ocular blood flow , also characterized by progressive choroidal thinning and attenuation of retinal vessels . some authors have observed that rp subjects feature an imbalance of the antioxidant - oxidant status in the peripheral blood . thus , the subclinical general inflammation and the substantial oxidative stress trigger the production of high levels of et-1 , which in turn bring about vascular dysregulation and diffuse hypoxic stress . this vicious circle contributes to activating and amplifying the inflammatory response . in the eye , vasospasm and altered intraocular perfusion bring about relative ischemia and , as a consequence , degeneration of the prs . further investigations are needed to confirm the link between vascular dysregulation and subclinical inflammation in rp . however , in the next future research probably focuses on the development of novel drugs as antagonists of et-1 and new antioxidants in order to better improve the vascular function systemically as well as locally .

retinitis pigmentosa is a clinical and genetic group of inherited retinal disorders characterized by alterations of photoreceptors and retinal pigment epithelium leading to a progressive concentric visual field restriction , which may bring about severe central vision impairment . haemodynamic studies in patients with retinitis pigmentosa have demonstrated ocular blood flow abnormalities both in retina - choroidal and in retroocular vascular system . moreover , several investigations have studied the augmentation of endothelin-1 plasma levels systemically in the body and locally in the eye . this might account for vasoconstriction and ischemia , typical in vascular dysregulation syndrome , which can be considered an important factor of reduction of the ocular blood flow in subjects affected by retinitis pigmentosa .

it is vital to understand the meanings and dynamics of language because it is the currency through which ideas and social norms are consolidated ( cameron & kulick , 2003 ; kiesling , 2007 ) . research documents the prevalence of homophobic language in sporting settings , where homophobia limits the life experiences of sexual minorities , polices gendered behaviors , and stigmatizes nonmasculine youth ( messner , 1992 ; parker , 1996 ) . for example , anderson ( 2002 ) demonstrates how the prevalence of homophobic language has been instrumental in many gay athletes decisions to remain closeted , because these athletes viewed homophobic language as indicative of a hostile climate toward sexual minorities . however , more recent research documents less damaging forms of homosexually - themed language in sport settings , and even suggests that it can have positive social effects ( anderson , 2011 ; mccormack & anderson , 2010a ) . accordingly , it is necessary to explore the multiple meanings of homophobic and homosexually - themed language in order to understand the regulation and stratification of sexuality , within sport as well as in the wider culture ( plummer , 1999 ) . as homophobia becomes increasingly condemned in western cultures ( mccormack , 2011a ; weeks , 2007 ) , and increasing numbers of people talk about homosexuality in positive ways , it is necessary to explore the full variety of meanings of language concerning homosexuality . however , even though a diverse body of literature exists on the topic ( mccormack & anderson , 2010a ; pascoe , 2005 ; rasmussen , 2004 ) , understandings are all too frequently based on a simplistic conceptualization of whether language is or is not homophobic . this can lead to an exaggeration of the prevalence of homophobia because many people have been socialized into a culture where almost all colloquial language relating to homosexuality has been homophobic , making people at risk of hearing homophobia in language whether it is there or not . this is problematic because fear of homophobia ( even when this fear is unwarranted ) can cause gay people to stay in the closet ( anderson , 2002 ) . in order to clarify what makes language homophobic , i argue that the literature documents two requisite features : 1 ) it is said with pernicious intent ; and 2 ) it has a negative social effect . the first requirement of homophobic language , pernicious intent , recognizes that the speaker is intending to degrade or marginalize a person or behavior by use of the association with homosexuality . armstrong ( 1997 ) argued that hostility and devaluation of homosexuality are implicit in the usage of homophobic terms ( p. 328 ) , and thurlow ( 2001 ) highlighted the use of intent by examining he found that homophobic language was accompanied by an intensifier more frequently than any other form of insult , for example , by saying you fucking queer rather than you queer . further evidence of the pernicious intent of homophobic language is provided by considering its role in bullying . rivers ( 1996 ) found that verbal abuse was the most common form of bullying practice leveled at gays and lesbians , and homophobic language has also been frequently used by bullies of heterosexual students ( epstein , 1997 ; rivers , 1995 ) . given that bullying is the marginalization of a person by more powerful peers , the frequent use of homophobic language in bullying behaviors is clear evidence of its pernicious intent . bullying also evidences the second component of homophobic language : it maintains negative social effect . gay and lesbian adults often speak of the emotional trauma caused by homophobic bullying in their youth ( flowers & buston , 2001 ; plummer , 1999 ) , and research also highlights the negative social impact this has on students and athletes , including elevated rates of absenteeism , social isolation , and higher dropout rates in school ( warwick , aggleton , & douglas , 2001 ) , as well as lower rates of sporting participation and ostracization within sporting teams ( brackenridge , allred , jarvis , maddocks , & rivers , 2008 ; pronger , 1990 ) . furthermore , homophobic bullying has led to elevated levels of suicide among gay youth ( d'augelli , hershberger , & pilkington , 2001 ) , although more recent research suggests that suicide rates of lesbian , gay , bisexual , and transgender individuals are in fact comparable to urban heterosexual youth ( mustanski , garofalo , & emerson , 2010 ) . even when homophobic language is intended to marginalize a behavior or action rather than a person , it still reproduces homophobia because users intend to stigmatize same - sex desire ( hillier & harrison , 2004 ; rubin , 1984 ) . for example , using explicitly anti - gay epithets to regulate heterosexual athletes who do not conform to orthodox gender stereotypes reproduces the hierarchical stratification of all masculinities , as well as harming the recipient of the abuse . accordingly , homophobic language can contribute to a hostile sports culture for all male youth ( hekma , 1998 ; messner , 1992 ) . while pernicious intent and negative social effect are the two key factors that have been used to determine if language is homophobic , implicit in most academic research is an assumption that this homophobic language is said within a homophobic environment ; that is , homophobic language has occurred in settings where people have homophobic attitudes and gay and lesbians are closeted or marginalized . some scholars documented the homophobic culture ( messner , 1992 ) , while others assumed its presence ( jackson & dempster , 2009 ; smith , 2007 ) . the assumption of a homophobic environment is understandable given that the vast majority of the research on homophobic language occurred between 1980 and 2000 , when british and american cultures were homophobic ( anderson , 2009 ; loftus , 2001 ) . however , the marked decrease in levels of homophobia of recent years necessitates that this assumption be made explicit for the cultural context to be recognized . accordingly , i propose an additional factor for analyzing homophobic language a homophobic environment . in figure 1 , i provide a pictorial framework for understanding the components of homophobic language . this linking of environment with effect and intent helps to historically contextualize the conceptualization of homophobic language that so accurately captured the social dynamics of the 1980s and 1990s ( see anderson , 2005 ; griffin , 1998 ; messner , 1992 ) . however , more recent research on the use of homosexually - themed language has highlighted complexities that do not readily fit into this framework of homophobic language . in 2005 , pascoe introduced the concept of fag discourse into discussions of homophobic language . building on thorne and luria 's ( 1986 ) notion of fag talk , fag discourse conceptualized a gendered form of homophobia . here , the purpose was not necessarily to regulate sexuality , but instead to regulate boys behaviors . importantly , pascoe distinguished the use of the word fag from the use of anti - gay pejoratives such as queer and poof because fag no longer had explicit associations with sexuality for many of the participants in her study . while the observation about the gendered nature of homophobia built on the work of british scholars of masculinity ( epstein , 1997 ; mac an ghaill , 1994 ) , an important difference was that pascoe ( 2005 , 2007 ) documented the ways in which fag was used as a pernicious insult that regulated gender without intending to marginalize same - sex identities . for example , pascoe ( 2005 ) highlighted that some boys took pains to say that accordingly , fag discourse conceptualizes the use of anti - gay epithets because of antipathy toward gender nonconformity , not homosexuality . pascoe ( 2007 ) also highlighted that the intent was not necessarily pernicious , even if it frequently was . fag talk and fag imitations serve as a discourse with which boys discipline themselves and each other through joking relationships ( p. 54 ) . while there is always intent with fag discourse to regulate something ( be it sexuality or gender , a person or a behavior ) , the precise intention varies . for example , it can be to wound someone , but it can also be used to castigate a behavior or even just competitively joke with friends . indeed , use of the word fag seems almost habitual ; a compulsive ( p. 86 ) part of boys interactions . it should be clear that this nuance is not recognized in the pernicious intent component of homophobic language . pascoe 's ( 2007 ) work was considered important for the significance it had in understanding the gendered nature of homophobia . however , most academics failed to appreciate the significance of her work in documenting the changing nature of homosexually - themed language . because pernicious intent was still sometimes evident , and because the social effect was often extremely negative , they overlooked the changes in the use of language that fag discourse conceptualized . accordingly , despite the subtle changes in intent and effect , scholars continued to label fag discourse as part of the traditional framework of homophobic language ( bortolin , 2010 ; kimmel , 2008 ) . this would have been appropriate in the 1980s and 1990s , when the word fag was used to demonstrate disgust of homosexuality in a broader culture of extreme homophobia ( anderson , 2009 ) , but it did not accurately capture use of the word in a different cultural context . it is easy to read high levels of homophobia in the school where pascoe collected data , and there were certainly highly homophobic aspects of the setting . yet , there were also several openly gay students , as well as heterosexual students who maintained pro - gay attitudes . indeed , pascoe ( 2007 ) also documented that many students elected not to use both homophobic and fag discourse . i was stunned at the myriad opportunities to levy the epithet and the seeming refusal by these boys , gay and straight , to invoke it this is something not documented in previous research ( see mac an ghaill , 1994 ) and it is evidence of a less homophobic environment . accordingly , i conceptualize fag discourse as distinct from homophobic language , as seen in figure 2 . a framework for understanding fag discourse . ( color figure available online . ) pascoe 's ( 2007 ) work has not been the final development in understanding homosexually - themed language . this is because homophobia has continued to decrease at a rapid rate since pascoe 's study ( mccormack , 2011a ; weeks , 2007 ) . while many researchers acknowledge this ( pringle & markula , 2005 ; swain , 2006 ) , my research has been the first to examine how homosexually - themed language operates in a pro - gay environment . in an attempt to address this lack of engagement with changing cultural attitudes , i first coauthored an article with eric anderson that sought to understand how the effect of homosexually - themed language varied according to the social context ( mccormack & anderson , 2010a ) . in this article , we developed the concept of gay discourse to understand the use of language that had a homosexual theme but was not homophobic as described by the frameworks above . this concept emerged from our ethnographic data with heterosexual rugby players who espoused pro - gay attitudes and had openly gay friends but , nonetheless , used phrases like do n't be gay and that 's so gay . they asserted that this position was consistent because gay had two meanings : it referred to sexuality in some contexts and meant rubbish in others ( see lalor & rendle - short , 2007 ) , and they argued that the two meanings were wholly independent of each other . some scholars continue to argue that the phrase that 's so gay is homophobic despite decreasing cultural homophobia ( see depalma & jennett , 2010 ; sanders , 2008 ) . yet , they do this without critical investigation of the attitudes of those using the language . as a result , they tend to misattribute the phrase as homophobic because they do not engage with attitudes toward homosexuality . equally problematic , they tend not to engage with how students interpret this discourse ( see adams , anderson , & mccormack , 2010 and lalor and rendle - short , 2007 for notable exceptions ) . in this rugby research , anderson and i explained this use of language by viewing the phenomenon through ogburn 's ( 1950 ) lens of cultural lag . cultural lag occurs when two related social variables become dissociated because their meanings change at different rates . in this case , adolescents employ this language without consideration or even knowledge of what it once conveyed . in other words , these rugby players use of language lagged behind their pro - gay attitudes . accordingly , we felt the need to develop a new way of understanding their language , one that did not position the participants as implicit homophobes . our concept , gay discourse , ameliorated this issue by arguing that while implicitly privileging heterosexuality , the language did not have the negative social effects of either homophobic language or fag discourse . figure 3 conceptualizes this in a way that draws out the difference from homophobic language . with a more inclusive environment and an absence of pernicious intent , this framework was useful in understanding the dynamics and implications of the phrase that 's so gay . in order to understand the limited extent of this negative effect , it is important to recognize that the word gay has been used as an expression of displeasure without intent to reflect or transmit homophobia in many contemporary youth settings ( adams , anderson , & mccormack , 2010 ; lalor & rendle - short , 2007 ; mccormack , 2011c ; mccormack & anderson , 2010b ) . this means that when young people hear the phrase , they do not automatically associate it with homosexuality and it is not necessarily the case that the expression of dissatisfaction translates to negative feelings about same - sex desires or gay people . anderson 's ( 2011 ) most recent examination of the experiences of openly gay male athletes finds that they do not attribute anti - gay sentiment to the phrase that 's so gay . indeed , one of his respondents argued that , you ca n't judge homophobia that way the word has different meanings and most of the time it 's not got anything to do with gay ( p. 260 ) . furthermore , all of his participants argued that use of the words gay and fag were not indicative of homophobia . they also did not use the prevalence of the word as a determining factor when deciding to come out . accordingly , the negativity from the use of homosexually - themed language has been expunged in these instances . a new framework for understanding gay discourse . while gay discourse was a powerful concept to understand the prevalence of that 's so gay , it was less effective in explaining another use of homosexually - themed language . in my coauthored article on rugby players ( mccormack & anderson , 2010b ) , we found that many of these heterosexual men used homosexually - themed language as a form of social bonding . in greeting one another we argued that this could continue to privilege heterosexuality because of the framework of homosexual stigma that used to exist in rugby . unfortunately , we were falling back on the same assumption of context that we accused others of doing in labeling that 's so gay as homophobic : a position that was aided by the fact that there were no openly gay athletes to judge this use of language . still , we did not have evidence that this privileged heterosexuality and should not , therefore , have drawn the conclusions that we did . i address this issue in my forthcoming book ( mccormack , 2011a ) , where i examine the use of homosexually - themed language between gay and straight students and explore the social effects of this language . in this book i conceptualize pro - gay language as a way of understanding homosexually - themed language that has a positive social effect . i defined it as the use of homosexually - themed language that is used to bond people together in socio - positive ways or to demonstrate pro - gay attitudes . in this research on three high schools in the united kingdom , i documented that gay and straight male students used pro - gay discourse as a way of bonding . one example of this occurred in an english lesson , when openly gay student , max , was working with his heterosexual friends , cooper and james . while cooper was doodling in his book , he looked up and asked , is this really gay what i 'm doing ? max started laughing , and said , this was just one example of pro - gay discourse not only bonding students together , but also expunging some of the negativity from these words . interestingly , this again supports the heterosexual rugby players contention that their language use was not homophobic ( mccormack & anderson , 2010a ) : at the time , anderson and i argued that the negative social effect of their banter was questionable because no gay peers were around to interpret their language , yet , this finding would suggest that any negative impact is minimal . in my book ( mccormack , 2011a ) , i also documented a second form of pro - gay language , one where heterosexual male students casually call their close friends lover or students enacted this language out of homosocial affection , without any discernible attempt to consolidate their heterosexual standing . proclaiming close friends as boyfriends was understood by the students as a way of demonstrating emotional intimacy . importantly , these students did not think that labeling each other this way would homosexualize them . the ability for boys to express their emotions in such an open way is clearly a positive development . moreover , just as homophobic language once contributed to a homophobic school environment , this form of pro - gay language now helps promote gay friendly cultures of inclusivity . in this section , i present an empirically grounded model for conceptualizing all forms of homosexually - themed language . in my new model , i foreground the importance of the cultural context in understanding language . this is because the social environment is pivotal in discerning the intent of language , how it is interpreted and the social effects it has . to explain this for example , when i was a closeted school student i heard the phrase that 's so gay frequently , and it was one of the reasons i decided to stay in the closet : i interpreted the phrase as deeply homophobic and it had negative emotional effects on me . yet , i attended a highly homophobic school where the phrase was heard alongside homophobic pejoratives such as poof , however , students at the same school today will experience the same phrase very differently ( mccormack , 2011a ) . the point is that the phrase that 's so gay is not necessarily homophobic , nor does it have to be part of gay discourse the categorization of language will depend on the cultural context . in order to explore the importance of context in more depth two key factors affect the level of homohysteria : the homophobia of a culture and the awareness that homosexuality exists within the culture . more specifically , in highly homohysteric cultures , there is an awareness that anyone can be gay . in homohysteric settings , the stigma attached to homosexuality results in men deploying homophobia in an attempt to prove their heterosexuality . anderson ( 2009 ) argues that as homohysteria declines , boys no longer care about being socially perceived as gay and they are afforded a greater range of gendered behaviors . this is something i documented in three british school settings ( mccormack , 2011b , 2011c ) . homohysteria is a useful concept here because it explains how homophobia is central to construction of masculinity in particular historical times and social contexts ( ibson , 2002 ) . accordingly , i argue that homohysteria is the key factor in determining the type of social environment , and i now develop my model in relation the homohysteria of a setting . in figure 4 , i situate cultural context as central to understanding and categorizing types of homosexually - themed language . first , in a highly homohysteric culture , boys use homophobic language to consolidate their own heterosexual identity and masculine standing ( plummer , 1999 ) . in this stage , homosexually - themed language is indeed homophobic , as it is used with pernicious intent and has a very negative social effect . the second framework , fag discourse , occurs in settings that are slightly less homohysteric . here , it is likely that many gay people have negative experiences in sport and schools , and the setting will be homophobic . but it is also likely that there will be people who support gay rights . in this stage , some young men who use fag discourse will insist that it is not meant to stigmatize homosexuality , while others will use it with pernicious intent . however , it will continue to have negative social effects , including the regulation and restriction of acceptable masculine behaviors , because the intent of language use is not always clear ( anderson , 2002 ) . in the third framework , gay discourse occurs in settings where young men are not particularly concerned about whether they are socially perceived as gay . in settings of low homohysteria , boys say phrases like that 's so gay as expressions of dissatisfaction and frustration . importantly , there is no intent to marginalize or wound people with the use of this language . while this is not necessarily pro - gay , young men maintain that the word gay does not connote same - sex desire in this context . a model of homosexually - themed language . ( color figure available online . ) finally , in gay friendly cultures ( see mccormack , 2011a ) , men are not part of a homohysteric culture . while they might prefer to be thought heterosexual , they do not police their behaviors to live up to a heteromasculine ideal . here , homosexually - themed language is used in a way that has positive social effects . sometimes pro - gay language is said without any specific intention , but it is also used as a mechanism for bonding students by demonstrating emotional intimacy or inclusion of openly gay students . the fun and fundamentally friendly way this language is used that is , the ease these students have with gay peers helps contribute toward a gay friendly environment . there will be some overlap between types of language , as well as exceptions to the framework . for example , while a person can use homophobic language in a gay friendly setting , this would be an anomalous result and would not fit with general conceptualizations of homophobic language in the wider literature . for example , if a student were to shout you fucking poof at another boy on a gay friendly sports team , it is highly likely that athlete would be reprimanded by both his fellow teammates and the coach ; and , apart from the impact it would have on the recipient , it would have marginal if any negative social effect on the broader culture . likewise , saying that 's so gay in a highly homophobic setting would probably be interpreted as homophobic , while it would not in a gay friendly setting . in understanding this form of language , context is all - important ( see davies , 1999 ; mccormack & anderson 2010a ) . it should also be noted that figure 4 does not provide an exhaustive list of words or phrases with a homosexual theme . it does , however , provide a framework by which to judge other forms of language . originating in hip - hop culture , it has received commentary in the media from leading scholars such as pat griffin and michael kimmel ( see also , brown , 2011 ) . no homo is used when men transgress traditional heteromasculine boundaries as a way of consolidating their heterosexual identities this is something i call heterosexual recuperation ( mccormack & anderson , 2010b ) . in her blog , griffin ( 2009 ) is particularly scathing about this use of language , viewing it as homophobic language and saying , these words can become weapons that provoke fragile peers to suicide or murder . however , drawing on pascoe 's ( 2007 ) work , kimmel ( 2009 ) suggests that no homo is a sign of progress , writing that no homo reflects the significant decline in homophobia among straight men in the united states today . in my model as kimmel ( 2009 ) argues , there is no actual intent to stigmatize feminine behaviors or homosexuality just a recognition that a particular behavior might code a person as gay . indeed , it can even be used as a technique to expand heteromasculine boundaries ( see mccormack & anderson , 2010b ) , effectively enabling someone to say i 'm straight , but i love you . indeed , one could argue whether it is homosexually - themed at all , that it is not instead just a statement of one 's own heterosexuality ( an issue i have examined elsewhere [ mccormack , 2011a ] is how pro - gay heterosexual men demonstrate their heterosexuality without being homophobic ) . but this nitpicking aside , while no homo privileges heterosexuality , it is hard to see it as homophobic . accordingly , this model can be used as a framework to understand new manifestations of homosexually - themed language as they arise . in this article , i have examined the use of homosexually - themed language as it is discussed in the academic literature . by comparing the language used in settings of homophobia with that of sites where homophobic language is absent , i developed a four - stage model to historically and spatially situate the use of language and how it is and should be interpreted . it is my hope that this new model will help teachers , students , and scholars understand and identify different forms of homosexually - themed language . hopefully , by categorizing it appropriately , we can ensure that we are all more able to appropriately judge the extent to which particular settings ( be they sport based , educational , or more general ) are homophobic , gay friendly , or somewhere in between .

in this article , i present a new model for understanding homosexually - themed language . by detailing how old conceptualizations of homophobic language no longer maintain heuristic utility in explaining the social dynamics of many sport and educational settings , i situate other conceptualizations of homosexually - themed language depending on the cultural context . i argue that whether language is considered homophobic , or whether it is better conceptualized as fag discourse , gay discourse or pro - gay language , is primarily dependent on the homohysteria of a setting . this model should enable scholars and educators to understand the operation of homosexually - themed language in society and properly evaluate the homophobia of a setting .

as the global suicide rate increases by 60% in 50 years in the underdeveloped and developing countries , paraphenylenediamine ( ppd ) poisoning is emerging as an important etiological factor reported from india . ppd ingestion is a very common form of poisoning in the indian subcontinent as this compound is a component of hair dyes and is easily available . it is brown or black colored solid substance easily soluble in hydrogen peroxide and not in water . it is a good hydrogen donor and metabolized by electron oxidation to an active radical by cytochrome p450 peroxidase to form a reactive compound called benzoquinone diamine . this can be further oxidized to a trimer known as brandowaski 's base , a well - known compound reported to cause anaphylaxis and mutation . it is traditionally used for dyeing palm , sole along with henna , and to dye the hairs . ingestion of ppd causes rapid development of edema of the face , neck , pharynx , tongue , and larynx initially and rhabdomyolysis later . finally , acute renal failure supervenes as renal tubular necrosis occurs due the deposits of the toxic metabolites of ppd . a 24-year - old young man who had ingested ppd pellets ( exact amount could not be calculated ) with suicidal intention . he developed a generalized itching and intermittent lacrimation for a few days and had a symptomatic treatment from a nearby medical practitioner concealing the history of consumption of ppd pellets . he presented after 6 days with complaints of pain and stiffness of both lower limbs and passage of chocolate colored urine followed by anuria . his blood urea and serum creatinine were 210 mg / dl and 12.4 mg / dl , respectively . he was managed symptomatically with diuretics , phosphate binders , sodium bicarbonate , oral calcium , and alkalization of urine . in view of his persistent oliguria and deranged metabolic parameters , the patient required 12 sessions of hemodialysis and his urine output and metabolic parameters improved . he was observed for 1 month in the hospital for any recurrence of the symptoms . although the cervicofacial edema is also a common manifestation , it was not a prominent clinical feature in this case . the alteration in the prominent clinical features may be due to the symptomatic management in the initial stages . however , the awareness must be created to know that the major cause of mortality is respiratory distress due to edema , complications related to myocarditis , and renal failure to make a protocol towards the management . the cause of acute tubular necrosis in ppd poisoning , independent of rhabdomyolysis is due to concentration of ppd in the renal tubules . this occurs due to the aromatic structure of ppd which makes it readily absorbable and concentrated in the tubules . with regards to treatment gastric lavage with 2% sodium bicarbonate is also effective . due to low molecular weight and hydrophilic nature of ppd acute renal failure is managed with adequate input and output charting , treating metabolic complications due to renal failure such as hyperklemia , hypocalcemia , hyperphosphatemia , and metabolic acidosis by hemodialysis . renal biopsy can be taken when there is undue delay in recovery of renal function . apart from intentional or accidental excessive exposure to ppd which results in above manifestations , the routine use of this compound on the regular basis for a long time can also lead to toxic effects . association between the personal hair dye use and non - hodgkin 's lymphoma , multiple myeloma , leukemia and other bladder cancer have been reported but have not been consistently observed . however , the united states environmental protection agency has not classified ppd as a carcinogen . therefore , no warnings of toxicity have been printed on boxes of hair dye . however , in view of easy availability and potential toxicity , awareness about the toxic effects of this compound may be useful . in the prospective study in future , the triad of strider due to upper airway edema , rhabdomyolysis and acute renal failure has to be considered for ppd poisoning . then , the early airway intervention , alkaline diuretics and hemodialysis are the three strategies in the management of ppd poisoning may be helpful in reducing the mortality .

the commonest constituent of all hair dyes is paraphenylene diamine ( ppd ) being used by the people to color their hair all over the world . hair dye poisoning is emerging as one of the emerging causes of intentional self - poisoning to commit suicide . in this article , the importance of clinical manifestations and of hair dye poisoning is discussed due to the lack of specific diagnostic tests . since there is no specific antidote for ppd poisoning , the early supportive treatment modalities are discussed .

( 1 ) single - molecule assays were performed as described previously . additional description of protein and dna constructs , single molecule assays , data collection and analysis are given in supplementary information . ( 2 ) if dttp and atp compete for binding to helicase according to the kinetic pathway outlined in fig . 2d , then the resulting unwinding rate is : vtot=(vmaxatp[atp]kmatp+vmaxdttp[dttp]kmdttp)/(1+[atp]kmatp+[dttp]kmdttp ) , where for each type of nucleotide km = k1+k2k1 and vmax = sk2 with s being the step size ( in nt ) ( see supplementary discussion ) . ( 3 ) in the presence of dttp and atp , if n helicase subunits coordinate in their chemo - mechanical activities and dna binding , then the resulting distance between slips ( processivity ) is : dprocessivity = c(vmaxatp[atp]kmatp+vmaxdttp[dttp]kmdttp)/([atp]kmatp[atp]kmatp+[dttp]kmdttp)n1 ( supplementary discussion ) , with c being a proportionality constant .

helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication , repair , and recombination1,2 . bacteriophage t7 gene product 4 is a model hexameric helicase which has been observed to utilize dttp , but not atp , to unwind dsdna as it translocates from 5 to 3 along ssdna26 . whether and how different subunits of the helicase coordinate their chemo - mechanical activities and dna binding during translocation is still under debate1,7 . here we address this question using a single molecule approach to monitor helicase unwinding . we discovered that t7 helicase does in fact unwind dsdna in the presence of atp and the unwinding rate is even faster than that with dttp . however unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events , ultimately preventing unwinding over a substantial distance . this behavior was not observed with dttp alone and was greatly reduced when atp solution was supplemented with a small amount of dttp . these findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination . we found t7 helicase binds and hydrolyzes atp and dttp by competitive kinetics such that the unwinding rate is dictated simply by their respective vmax , km , and concentrations . in contrast , processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations . this does not agree with an uncoordinated mechanism where each subunit functions independently , but supports a model where nearly all subunits coordinate their chemo - mechanical activities and dna binding . our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide - ligated and thus interact with the dna to ensure processivity . such subunit coordination may be general to many ring - shaped helicases and reveals a potential mechanism for regulation of dna unwinding during replication .

a consensus regarding the appropriate treatment , as well as the timing of treatment , for children with empyema continues to elude us . treatment options are varied , ranging from antibiotics , instillation of fibrinolytics , thoracentesis , thoracoscopy , to open thoracotomy . video - assisted thoracic surgery ( vats ) offers the distinct advantage of minimizing operative morbidity , and as technology and surgical skill have improved , so has its promise in regards to the treatment of empyema in children . we have previously published our results of the use of early vats in managing pediatric empyemas and have formulated an algorithm . therefore , we reviewed our experience using this algorithm to manage pediatric empyema in a prospective fashion in nonselected consecutive patients . we reviewed the records of 22 consecutive children who were diagnosed with empyema from november 1997 to april 2001 . the presumptive diagnosis of empyema was made preoperatively based on clinical examination , chest radiography , and imaging using ultrasound or computed tomography ( ct ) . treatment algorithm for pediatric empyema . if a free - flowing fluid collection was found , thoracentesis was undertaken . if an empyema was present ( ie , frank purulence , positive gram stain , ph < 7 , ldh > 1000 iu / dl , glucose < 40 mg / dl ) , a chest tube was placed . however , if a loculated fluid collection was seen on imaging studies , the patient underwent vats for the empyema without any other preoperative interventions . chest tubes were removed when the patient was clinically improving and drainage was less than 50 ml / day . our technique for vats in managing pediatric empyema in brief is carried out with either a single - lumen endotracheal tube with selective bronchial intubation or a double - lumen endotracheal tube . the patient is placed in a lateral decubitus position with trocar placement dictated by the location of the empyema . one trocar is placed for the telescope and 2 subsequent incisions are made under direct visualization . these are placed in such a manner that they can be included in a formal thoracotomy incision if necessary . all purulent and fibrinous material is suctioned , and the visceral and parietal pleura are completely dbrided . if circumstances preclude effective dbridement ( ie , excessive bleeding , poor visualization , inability to tolerate single lung ventilation ) , the procedure is converted to a mini - thoracotomy . the majority of children were transferred to the pediatric floor where chest tubes were removed when the lung was fully expanded and drainage was less than 50 ml / day . intravenous antibiotics were started preoperatively on all patients and continued postoperatively until the patient was afebrile . all patients were discharged home on oral antibiotics and follow - up included a chest x - ray . demographic data , days to diagnosis , days to surgery , length of hospital stay , postoperative chest tube days , complications , and follow - up results were recorded and are displayed where appropriate as means standard deviation . thoracentesis was performed in 9 children preoperatively ( 3 at outside institutions ) with 6 positive cultures ( streptococcus pneumoniae , 3 ; streptococcus pyogenes , 3 ) . the 6 children who underwent thoracentesis at our institution had them performed by a pediatric intensivist prior to our consultation . subsequent workup in all but one of these cases revealed loculated fluid collections and therefore only 1 chest tube was placed . this child went on to develop a loculated effusion that failed chest tube drainage , and the child subsequently underwent successful vats . two chest tubes were placed preoperatively ( one at an outside institution without a preceding thoracentesis ) . all children were imaged with chest x - rays ; ultrasound was used in 17 ( 77% ) and ct in 13 ( 59% ) . in 2 children , loculated fluid was seen on initial lateral and decubitus chest x - rays , and no further studies were obtained prior to surgery . in 7 children , ct was equivocal for loculations , but in all 7 cases , loculated fluid collections were confirmed by chest ultrasound examination . one child who has an immunodeficiency required a second vats for a second empyema , which developed 1 month later , with successful resolution of her empyema . one child had a residual left upper lobe loculation , which resolved with the placement of a ct - guided percutaneous drain during the same hospitalization . it was removed 3 days later , and the patient was discharged uneventfully the following day . one child underwent mini - thoracotomy secondary to difficulty with ventilation and was found to have an abscess with a necrotic right upper lobe that required resection . four operative cultures grew bacteria ( streptococcus pyogenes , 1 ; streptococcus pneumoniae , 3 ) . nineteen children presented for follow - up ( 86% ) at a mean of 5 months postoperatively . all 19 were clinically stable and free from symptoms at the follow - up office visit . most empyemas arise as a result of preceding pneumonia but less than 1% of pneumoniae result in an empyema . a thickening of the pleural exudate and development of a peel with resultant loculations characterize the fibrinopurulent stage . the final stage is one of organization , where the loose peel becomes more tenacious with fibrosis and scar formation . traditionally , administration of prolonged intravenous antibiotics with or without drainage ( thoracentesis or chest tube ) has been the treatment algorithm , and only when progression to the final organizing stage of empyema has occurred is open thoracotomy undertaken . the perceived associated morbidity of an open thoracotomy is often the reason for prolonged nonoperative management of empyemas . surgeons are increasingly advocating early rather than late surgical drainage to reduce length of hospital stay and need for long - term antibiotics . the increasing utilization of thoracoscopy and rapidly improving technology has compelled many to replace an open decortication with a thoracoscopic approach . our algorithm is based on the presence or absence of radiological signs of loculations rather than operating within a specific time frame . the majority of these children presented to our service having already progressed to the fibrinopurulent stage . , chest sonography is the initial imaging modality of choice , and it detected loculations in 100% of the cases in which it was used . several groups have found it to be better than ct in evaluating pleural fluid collections . a chest ct scan may also be indicated to evaluate the lung parenchyma if clinical or radiological suspicion of a lung abscess exists . a mean length of hospital stay of up to 27 days has been reported by others while our experience was 13 days . chest tubes were removed an average of 3 days after surgery , avoiding the morbidity of prolonged indwelling chest tubes . follow - up was available in 19 children , and none developed recurrences of their empyemas , aside from 1 patient with a relatively normal chest x - ray but who had an immunodeficiency and developed a recurrent empyema 1 month following discharge from the hospital . few would argue that the ideal treatment of an empyema would involve minimal morbidity with a speedy recovery and very low recurrence rate . our experience using an image - guided treatment algorithm accomplishes this via a simple , safe , and effective process . it joins the growing body of evidence supporting vats in the treatment of pediatric empyema .

introduction : the management of pediatric empyema remains controversial . we contend that early thoracoscopic intervention results in shorter hospital stays , decreased morbidity , and superior outcomes . we propose an algorithm using early image - guided thoracoscopy as an effective treatment for pediatric empyema.methods:consecutive pediatric empyemas treated from november 1997 to april 2001 using a prospective management algorithm were reviewed . demographic data , days to diagnosis , days to surgery , length of stay , chest tube days , complications , and follow - up were recorded.results:twenty-two children with 24 empyemas were treated using this algorithm . their mean age was 49 months . mean days to diagnosis was 11 and from diagnosis to surgery was 3 . imaging included chest radiography ( cxr ) in all , ultrasound in 17 ( 77% ) , and computed tomography ( ct ) scan in 13 ( 59% ) . one thoracoscopy was converted to a mini - thoracotomy because of difficulty with ventilation . chest tube removal averaged 3 days with an average length of stay of 13 days . one patient required a second thoracoscopy for recurrent empyema , and 1 patient developed a contralateral empyema . no other complications or deaths occurred . follow - up in 19 of 22 ( 86% ) children at 5 months revealed no recurrences or mortality.conclusions:this treatment algorithm , using early image - guided thoracoscopy , is a safe and effective means of managing pediatric empyema , while shortening hospital stay and avoiding the morbidity of thoracotomy .

total knee arthroplasty ( tka ) has been widely accepted as an effective treatment for end stage osteoarthritis demonstrating satisfactory outcomes and survivorship . still , restoration of normal range of motion ( rom ) after tka remains as a challenge ; in general , the rom of the knee after conventional tka ( range , 110 to 120 ) is less than that of a normal knee2 ) . in particular , squatting , sitting cross - legged , and kneeling with deep flexion of the knee joint that involve > 125 of knee flexion the femoral component of the high - flexion design has a 24 mm extended and thicker posterior condyle that does not necessitate additional bone cut to maintain the contact area during deep knee flexion67 ) . in addition , the larger posterior condyle of the femoral component decreases the risk of impingement between the posterior cortex of the femur and the polyethylene insert8 ) . the new prosthesis has a modified cam and post mechanism with increased jump distance to avoid dislocation under deep flexion angles9 ) . the anterior recess in the tibial polyethylene insert reduces impingement on the patella and the patellar tendon during deep knee flexion . the new high - flexion concept lospa knee system ( corentec , seoul , korea ) has recently become available . it is designed to achieve ligament isometry from 0 to 90 using a single radial axis of the femoral component . the single radius of the femoral component contributes to stability in the mid - flexed state10 ) . cam - post engagement occurs at 75 , which prevents unnecessary cam - post contact during level walking and has an advantage of posterior translation of the femoral condyle . the high - flexion design with more rounded femoral contour and deepened patellar groove facilitates deep flexion by reducing joint capsule overstuffing . this prosthesis has a 10-mm thick posterior condyle of the femoral component that replaces the 10-mm posterior femoral bone cut , which contributes to increased surface contact and bone support through a relatively larger posterior radius than the conventional design ( fig . 1 ) . compared to nexgen lps - flex ( zimmer , warsaw , in , usa ) , another high - flexion design implant , that requires 12-mm posterior femoral bone cutting , lospa prosthesis is more advantageous for bone preservation . some studies demonstrated the superiority of the high - flexion prosthesis with regard to the postoperative rom and function411 ) while others showed the opposite712 ) . a concern has been raised that high - flexion tka accelerates early aseptic loosening compared to conventional tka due to the increased shear force between the femoral component and the femur during high flexion of the knee1314 ) . cho et al.13 ) and han et al.14 ) noted a high rate of aseptic loosening of the femoral component in their patients after high - flexion tka . in this study , we retrospectively evaluated the clinical and radiographic results of tka performed using the newly introduced lospa knee system for a minimum 2-year follow - up . we examined the following two hypotheses : 1 ) the rom of the knee with the lospa prosthesis would be comparable to that with other high - flexion concept prostheses of the recent literatures in korean patients and 2 ) the implantation of the lospa prosthesis would be stable without aseptic loosening at minimum 2- year follow - up . a total of 198 patients ( 215 knees ) underwent tka with the posterior - stabilized ( ps ) lospa total knee prosthesis from may 2011 to august 2013 at our institution . of these , we retrospectively reviewed 191 knees of 177 patients ( 88% ) who were available for a minimum 2-year follow - up . inclusion criteria were patients with indications for primary tka : kellgren - lawrence classification grade 34 severe osteoarthritis , osteonecrosis15 ) , and rheumatoid arthritis . the mean follow - up period was 2.6 years ( range , 2 to 4 years ) . all surgeries were performed by a senior author ( in ) using the ps lospa knee system . all procedures were performed through a subvastus approach under general anesthesia with tourniquet inflation to 300 mmhg . postoperatively , none of the patients did continuous passive motion exercises , but they were encouraged to perform active exercises according to our protocol of rehabilitation . quadriceps - strengthening exercises were started immediately after surgery and patients began walking with use of a walker on the first postoperative day . on the second postoperative day , the patients started active rom exercises under the guidance of the medical team . clinical and radiographic data were evaluated at postoperative 6 weeks , 3 months , 6 months , and 1 year and then yearly thereafter . each knee was evaluated according to the knee society scoring system ( kss)16 ) and the western ontario and mcmaster universities osteoarthritis index ( womac)17 ) by one of the authors ( kim ) . at the time of each follow - up , active rom of the knee was measured using a standard 60-cm goniometer with the patient placed in supine position by one of the authors ( kim ) who was blinded to the implant used . weight - bearing anteroposterior , lateral , and merchant radiographs were taken at each follow - up visit . radiolucent lines were examined at different zones around the femoral and tibial components using the knee society tka radiographic evaluation and scoring system18 ) . all radiolucent lines were recorded and the radiographic sign of loosening was defined as the presence of a radiolucent line more than 2 mm in width at the cement - bone or cement - prosthesis interface19 ) . implant position in relation to the limb anatomic axis and component alignment was evaluated and the presence and location of the radiolucent lines were assessed using the kss18 ) . preoperative and postoperative clinical outcomes ( kss and womac scores ) and radiographic alignment were compared using the paired t - test . a p - value of < 0.05 was considered significant , and the analysis was performed using the ibm spss ver . 21.0 ( ibm co. , armonk , ny , usa ) . the rom at the final follow - up increased significantly compared to the preoperative rom ( table 2 ) . the mean kss improved from 121.4 ( range , 42 to 185 ) preoperatively to 174.0 ( range , 130 to 200 ) at the last follow - up ( p<0.001 ) . the mean womac score also improved from 56.1 ( range , 23 to 88 ) preoperatively to 16.4 ( range , 0 to 85 ) postoperatively ( p<0.001 ) ( table 3 ) . the mean kss and womac scores were 165.8 ( range , 105 to 200 ) and 28.2 ( range , 2 to 65 ) , respectively , at the 6-month follow - up , 178.9 ( range , 130 to 200 ) and 22.8 ( range , 3 to 57 ) , respectively , at the 1-year follow - up , 173.6 ( range , 130 to 200 ) and 17.9 ( range , 0 to 85 ) , respectively , at the 2-year follow - up , and 174.2 ( range , 130 to 200 ) and 14.8 ( range , 0 to 85 ) , respectively , at the 3-year follow - up . the mean femorotibial angle was a varus of 3.74.8 ( range , varus of 16.8 to valgus of 12.3 ) preoperatively and a valgus of 5.71.8 ( range , valgus of 0 to valgus of 8.4 ) postoperatively ( p<0.001 ) . the coronal angle of the femoral component ( ) was 96.61.6 ( range , 92.1 to 99.9 ) , the coronal angle of the tibial component ( ) was 89.71.7 ( range , 84.2 to 95.9 ) , the flexion angle of the femoral component ( ) was 2.81.3 ( range , 0.1 to 5.5 ) , and the tibial slope angle ( ) was 85.82.1 ( range , 82.1 to 90.0 ) postoperatively . the mean posterior femoral condylar offset was 29.62.5 mm ( range , 19.8 to 36.8 mm ) preoperatively and 29.92.8 mm ( range , 20.5 to 36.9 mm ) postoperatively ( p=0.073 ) . radiolucent lines were found in 15 knees ( 14 patients , 7.9% ) : two in the medial tibial condyle ( zones 1 and 2 ) , 2 in the anterior femoral condyle ( zones 1 and 2 ) , and 11 in the posterior femoral condyle ( zones 3 and 4 ) ( fig . no case of osteolysis was detected during the 2- to 4-year follow - up period . none of the knees developed aseptic loosening of the femoral , tibial , or patellar component . two major complications required surgery in two knees . a 70-year - old female with a history of lymphedema developed a deep infection in the extremity of the operated side 1 year postoperatively . the patient had received a radical hysterectomy with total lymph node dissection for uterine cervical cancer . methicillin - sensitive staphylococcus aureus was cultured from the joint fluid , and a two - stage revision operation was performed for the infected joint . a patellar fracture occurred in one patient and was treated with open reduction and internal fixation . one patient received brisement due to < 90 rom limitation at 4 weeks after the operation and the final flexion angle was 125. two patients demonstrated wound dehiscence 2 weeks postoperatively . symptomatic deep vein thrombosis , pulmonary embolism , and hyperextension ( recurvatum ) were not observed during the follow - up . the purpose of this study was to evaluate the rom and early stability following tka performed using the new high - flexion prosthesis during a short - term follow - up . the most important finding of the present study was that the newly introduced high - flexion lospa prosthesis resulted in no early aseptic loosening of the component and provided outcomes comparable to those used in high - flexion tka in korean patients ( table 4 ) . postoperative rom is an important factor for patient satisfaction after tka , particularly among asians20 ) . thus , manufacturers have modified and upgraded the design of knee prostheses to allow for high flexion after tka . many studies have investigated the efficacy and safety of tka using a high - flexion design prosthesis performed by korean surgeons71113142122232425 ) . however , there has been some puzzling inconsistency among studies on the efficacy of high - flexion tka designs471112 ) . there is still a paucity of data on the rom of the knee following tka using this new high - flexion implant ; therefore , we investigated postoperative rom in patients who received the newly designed high - flexion prosthesis . the lospa knee system demonstrated improved clinical and radiographic outcomes at 2- to 4-year follow - up . the outcomes of our study were comparable to those reported by other korean studies on high - flexion tka . seo et al.26 ) compared the lospa ps total knee prosthesis with another high - flexion ps prosthesis . in their study , the mean postoperative flexion angle of the lospa group was 126.9. the mean postoperative rom of the patients was similar to that of the current study . theoretically , a knee flexion angle of 140 is possible with the high - flexion lospa system , but it appears not to be attainable in vivo . in the present study , the postoperative rom of the knee was less than our expectation ( up to 140 ) and the superiority of the prosthesis compared with conventional tka in terms of rom was not observed71122 ) . several factors may contribute to the rom after tka including patient factors ( preoperative rom , obesity , postoperative quality of rehabilitation , patient motivation , and preoperative mental illness ) , intraoperative technical issues ( resection of back osteophytes , ligament balancing , component positioning , altered joint line , and posterior tibial slope ) , and perioperative complications27 ) . it has been generally considered that the preoperative rom is the most predictable factor for the postoperative rom28 ) . our study showed that the new high - flexion lospa prosthesis would not improve the postoperative rom in patients with a preoperative rom of < 120. a controversy still exists regarding the relationship between the posterior condylar offset and the rom629 ) . bellemans et al.6 ) reported that the final knee flexion angle was significantly correlated with the posterior femoral condylar offset in tka patients . they insisted that restoring posterior condylar offset would be very important to achieve a greater flexion angle before impingement of the posterior tibial insert against the posterior femur . in contrast , hanratty et al.29 ) found no correlation between the range of knee flexion and posterior condylar offset after tka . in our study , an additional 2-mm posterior femoral bone resection was not necessary because of the 2 mm extension of the posterior condyle of the femoral component . therefore , we found it difficult to determine the relationship between the posterior condylar offset and knee the flexion angle in the present study . concerns have been raised about aseptic loosening of the high - flexion knee systems during the early postoperative phase . cho et al.13 ) reported that progressive radiolucent lines around the femoral component were detected in 13.8% of 218 knees that received the nexgen lps - flex tka at a mean follow - up of 51 months . in their study , radiolucent lines were observed around the anterior and posterior flanges of the femoral component . han et al.14 ) noted aseptic loosening in 38% of cases at a mean follow - up of 32 months after using the same high - flexion total knee prosthesis and performed revision in 21% of cases at a mean of 23 months postoperatively . they reported radiolucent lines were most common in the anterior flange of the femoral component . in high - flexion tka , the tibial insert only articulates with the femoral component , resulting in the absence of load sharing effect and application of greater shearing force on the femoral component . conventional tka requires a distinctly higher loosening force compared to high - flexion tka . among the high - flexion designs , those with an open geometry of the internal femoral component are more vulnerable to loosening than those with a closed geometry . peg fixation is essential to good fixation of a component and provides stability against the loosening force30 ) . the lospa total knee system requires a 10-mm posterior femoral condylar bone cut , which is more conservative compared to the 12 mm required by the nexgen lps - flex system . in addition , peg fixation of the lospa prosthesis provides resistance to the loosening of component , but open femoral component geometry is not beneficial for prevention of loosening30 ) . our results showed a 7.8% incidence of radiolucent lines around the anterior flange of the femur , the posterior femoral condyle , and the medial tibial condylar area . however , no symptoms or signs of aseptic loosening were detected in patients with radiolucent lines . han et al.14 ) reported that the mean flexion angle was 136 in the loosening group and 125 in the well- fixed group . the proportion of patients who were able to perform high - flexion activities such as squatting and kneeling was also higher in the loosening group . the femur contacted with the posterior end of tibia at 133 of flexion angle . in our study , the mean flexion angle of the knee was 127 , which was similar with the angle of the well - fixed group and below 13314 ) . we did not thoroughly investigate the percentage of patients who were able to perform high flexion activities , but we recommended the patients not to be involved in high - flexion activities as much as possible . it was an attempt to prevent early loosening of component following high - flexion tka13 ) . the knees with appearance of radiolucent lines require further follow - up to assure the clinical course . we did not have a control group to compare the knee rom and radiographic and clinical outcomes . second , the minimum 2-year follow - up period was relatively short , so we can not determine the long - term clinical and radiographic outcomes . the evaluation of patients who underwent tka with the high - flexion lospa prosthesis revealed improved clinical and radiographic results and low complication rates after a minimum 2-year follow - up . however , a long - term follow - up is required to determine whether the new high - flexion concept design demonstrates a comparable survivorship . in conclusion , the new high - flexion total knee prosthesis resulted in no early aseptic loosening of the component and improved rom comparable to other high - flexion tka prostheses at 2- to 4-year follow - up .

purposethe purpose of this study was to evaluate minimum 2-year follow - up results of total knee arthroplasty ( tka ) performed using a new high - flexion prosthesis design ( lospa).materials and methodsthe 2- to 4-year results of 191 consecutive tkas ( 177 patients ) with the lospa posterior - stabilized prosthesis were evaluated . the patients were assessed clinically and radiographically using the knee society scoring system ( kss ) and the western ontario and mcmaster universities osteoarthritis index ( womac).resultsthe mean range of motion ( rom ) increased significantly from 117.4 ( range , 75 to 140 ) preoperatively to 126.7 ( range , 80 to 144 ) postoperatively ( p<0.001 ) . the mean kss and womac scores improved significantly from 121.4 ( range , 42 to 185 ) and 56.1 ( range , 23 to 88 ) preoperatively to 174.0 ( range , 130 to 200 ) and 16.4 ( range , 0 to 85 ) postoperatively , respectively ( both , p<0.001 ) . one knee required revision for deep infection . no knee had aseptic loosening or osteolysis . radiolucent lines were noted in 15 knees ( 7.9%).conclusionsthe new high - flexion total knee prosthesis resulted in no early aseptic loosening of the component and improved postoperative rom comparable to other high - flexion tka prostheses at 2- to 4-year follow - ups .

by examining the effects of various experiences on cognitive performance , we can gain a better understanding of the plasticity of the mind and brain . this understanding can , in turn , be used to develop high - quality educational and rehabilitation programs . here , we consider two common experiences that may improve certain aspects of cognition : learning how to speak a second language ( becoming a bilingual ) and learning how to play a musical instrument ( becoming a musician ) . in previous work , bilinguals and musicians were found to have increased domain - general executive control , as evidenced by superior performance on nonlinguistic , nonmusical , visual - spatial tasks that involved attending to a relevant and informative feature of a stimulus while ignoring an irrelevant and misinformative feature [ 1 , 2 ] . however , the evidence for increased executive control is mixed in bilinguals [ 35 ] and limited in musicians [ 6 , 7 ] , suggesting a need for more research to confirm or deny these cognitive benefits . it is also unknown how the combination of bilingualism and musicianship affects executive control . in the current study , we investigate these issues by testing bilinguals , musicians , bilingual musicians , and controls on a simon task that assesses interference suppression ( the ability to ignore an irrelevant and misinformative cue ) . bilinguals gain experience using interference suppression during language processing because of their need to prevent the nontarget language from interfering while using the target language . for example , during speech comprehension , both english - spanish bilinguals and english monolinguals mentally activate similar - sounding words in the target language , such as the english word carton , when hearing the english word carpet [ 8 , 9 ] . bilinguals , however , also activate similar - sounding words in their other language , such as the spanish word cartera ( i.e. , wallet ) , when hearing the english word carpet [ 911 ] . this parallel activation is instantiated in bilingual models of both spoken word comprehension ( e.g. , blincs ) and written word comprehension ( e.g. , bia+ ) . similarly , during speech production , both languages become activated in parallel , consistent with green 's inhibitory control model . due to activation of both languages during comprehension and production , experience - dependent plasticity , this practice may lead to better domain - general interference suppression . as evidence , a recent study tested bilingual and monolingual younger adults on a numerical stroop task , in which participants viewed items on a screen ( e.g. , the number 11 ) and had to indicate how many distinct items were presented ( in this case , 2 , because each digit is a different item ) . in some trials , called incongruent trials , the quantity expressed by the items was incongruent with the number of items ( e.g. , 33 ) . interference suppression was required on these trials in order to ignore the irrelevant feature ( i.e. , the quantity expressed by the items ) , which conflicted with the correct response . in other trials , called neutral trials , letters appeared on the screen ( e.g. , gg ) and interference suppression was not required , because the presented items did not express a quantity that was at odds with the number of items . in still other trials , referred to as congruent trials , the expressed quantity and number of items matched ( e.g. , 333 ) and therefore interference suppression was not required on these trials either . to measure the ability to suppress interference on the incongruent trials , an interference effect was calculated by subtracting response times on neutral trials ( a baseline condition ) from response times on incongruent trials . the interference effect was smaller for bilingual younger adults relative to monolingual younger adults , indicative of better interference suppression in bilinguals . a bilingual advantage in interference suppression has been observed in other studies as well [ 17 , 18 ] . furthermore , better interference suppression in bilinguals compared to monolinguals has been linked to neural differences in regions associated with executive control . however , some studies have failed to find better interference suppression in bilinguals , particularly in young adults , but also in children and older adults [ 4 , 5 , 2022 ] . one possible reason why some studies may fail to find a bilingual effect is the use of inadequate measures of interference suppression . many commonly used executive control tasks include incongruent trials and congruent trials but do not include neutral trials ( i.e. , baseline control trials ) . when neutral trials are included , interference suppression can be accurately calculated by subtracting response times on neutral trials ( i.e. , baseline control trials ) from response times on incongruent trials ( yielding an interference effect ) . otherwise , interference suppression is calculated by subtracting response times on congruent trials from response times on incongruent trials ( yielding a simon effect ) . the simon effect calculation does not provide a pure measure of interference suppression because it is influenced by how much participants benefit from the helpful congruent cue ( i.e. , congruent facilitation ) . bilinguals sometimes benefit slightly more from a congruent cue than monolinguals [ 16 , 23 ] , which has the numerical effect of increasing the simon effect score ( indicative of poorer performance ) . the simon effect measure may therefore mask a true bilingual advantage in interference suppression , and so it is critical that assessments of cognitive advantages include a neutral baseline condition . similar to bilingualism , musicianship may also enhance interference suppression through experience - dependent plasticity , though the evidence is significantly more limited , and the source of this potential enhancement is less clear . one possibility is that musical experience enhances interference suppression partly through the same type of mechanism as bilingualism . recently , theories of music comprehension have drawn parallels to language comprehension and have posited that , as a melody unfolds , other melodies that are consistent with initial notes of the target melody become activated ( similar to activation of similar - sounding words in language processing ) [ 24 , 25 ] . music comprehension would therefore involve a need to ignore misinformation ( i.e. , activated but incorrect melodies ) , a notion that is supported by studies indicating activation of frontal executive areas during music listening [ 26 , 27 ] . this practice inhibiting interference from nontarget melodies during music comprehension may lead to enhancements in interference suppression . evidence for enhanced interference suppression in experienced musicians comes from a recent study , in which professional musicians were found to have smaller stroop effects than amateur musicians . additionally , in a study with older adults , smaller stroop effects and simon effects were observed in musicians relative to nonmusicians (; see also for a similar finding in younger adults ) . furthermore , musicians do not show the same age - related declines in executive control areas ( e.g. , dorsolateral prefrontal cortex and inferior frontal gyrus ) as nonmusicians do . these results suggest that musicians may have better interference suppression than nonmusicians , but more research is needed to confirm this finding , given the small number of studies . in the current study , we provide additional data on musicians ' executive control abilities . to our knowledge , no previous studies have considered the combined effect of bilingualism and musicianship on interference suppression . if both the bilingual and musician advantages do exist , they may be additive , resulting in even stronger benefits in bilingual musicians . however , bilingualism and musicianship were found to combine nonadditively in a study assessing task - switching abilities . this finding suggests an alternative hypothesis ( that the enhancements in interference suppression may not be larger in bilingual musicians ) . advantages in interference suppression due to bilingualism or musicianship alone could already place younger adults at their cognitive peak , precluding any extra gains from acquiring both experiences . in line with this reasoning , bilinguals who have other executive control enhancing traits ( e.g. , video game experience or high socioeconomic status ) do not show further gains over bilinguals who do not have these experiences [ 31 , 32 ] , suggesting that bilinguals may reach a ceiling level that is resistant to further plasticity . , previous research suggests that , through experience - dependent plasticity , bilinguals and musicians may develop enhanced interference suppression , but the evidence is mixed for bilinguals and limited for musicians . moreover , it remains unknown how the combination of bilingualism and musicianship affects interference suppression . in the current study , we examined interference suppression in bilinguals , musicians , and bilingual musicians . based on bilingual and musical proficiency , four groups were formed : bilinguals , musicians , bilingual musicians , and controls ( nonbilinguals and nonmusicians ) . each group performed the simon task , which is a nonlinguistic , nonmusical , visual - spatial task that can be used to assess domain - general executive control abilities in bilinguals and musicians [ 1 , 29 ] . the simon task involves responding to the color of a rectangular box ( pressing a key on the right if blue and a key on the left if brown ) , while the irrelevant location of the box is misinformative ( incongruent trials ) , informative ( congruent trials ) , or uninformative ( neutral trials ) . the difference in response time between incongruent and neutral trials , called the interference effect , was used to assess interference suppression . we also considered the facilitation effect ( difference between neutral and congruent trials ) and simon effect ( difference between incongruent and congruent trials ) , both of which may be influenced by bilingual and/or musical experience [ 16 , 28 ] . participants were 219 young adults ( mean age = 21.9 ) with a range of linguistic and musical backgrounds . these participants were recruited through classes and flyers at a university in the united states and were then tested in a university research lab . after data collection , the participants were divided into four groups : bilinguals ( high bilingual proficiency , low music proficiency ; n = 43 ) , musicians ( low bilingual proficiency , high music proficiency ; n = 42 ) , bilingual musicians ( high bilingual proficiency , high music proficiency ; n = 69 ) , and controls ( low bilingual proficiency , low music proficiency ; n = 65 ) . groups were formed using median splits based on bilingual proficiency ( defined as self - rated proficiency in understanding their second best language on a 010 scale ) and music proficiency ( defined as self - rated proficiency in playing their best instrument on a 010 scale ) . ratings were obtained using the language experience and proficiency questionnaire ( leap - q ; ) and a music questionnaire ( , originally adapted from ) . the question used to measure language proficiency was on a scale from zero to ten , please select your level of proficiency in understanding this language and the question used to measure music proficiency was what is your skill level in playing this instrument / singing ? with the scale in both cases being 0-none , 1-very low , 2-low , 3-fair , 4 slightly less than mediocre , 5-mediocre , 6-slightly more than mediocre , 7-good , 8-very good , 9-excellent , 10-perfect . ( proficiency in understanding the second language was used to represent bilingual proficiency because previous research suggests that receptive abilities ( potentially more so than expressive abilities ) play a critical role in the development of the bilingual advantage in executive control ( e.g. , ) . for example , preverbal bilingual infants show advantages despite a lack of expressive skills ( e.g. , ) . in addition , significant correlations between receptive language tasks and executive control performance have been found in younger bilingual adults ( e.g. , ) . it should also be noted that , in the current sample , the correlation between first and second language receptive and expressive proficiency was very high ( r = 0.95 ) . ) mean bilingual and music proficiencies for each of the 4 groups are displayed in figure 1 . mean bilingual proficiency for each group was as follows : bilinguals = 8.65 ( se = 0.16 ) , musicians = 1.83 ( se = 0.32 ) , bilingual musicians = 8.57 ( se = 0.12 ) , and controls = 1.63 ( se = 0.24 ) . mean music proficiency for each group was as follows : bilinguals = 2.26 ( se = 0.29 ) , musicians = 7.30 ( se = 0.15 ) , bilingual musicians = 7.45 ( se = 0.11 ) , and controls = 2.08 ( se = 0.23 ) . a one - way anova with bilingual proficiency as the dependent measure revealed an effect of group , f(3,215 ) = 350.10 , p < 0.05 , with bilinguals and bilingual musicians reporting significantly higher bilingual proficiencies than musicians and controls ( ps < 0.05 , bonferroni corrected ) . a one - way anova with music proficiency as the dependent measure also revealed an effect of group , f(3,215 ) = 234.77 , p < 0.05 , with musicians and bilingual musicians reporting significantly higher music proficiencies than bilinguals and controls ( ps < 0.05 , bonferroni corrected ) . similarly , for current percentage of time using a second language , bilinguals ( mean = 24.8% ) and bilingual musicians ( mean = 23.7% ) reported more current usage than musicians ( mean = 2.1% ) and controls ( mean = 4.5% ) , f(3,215 ) = 45.66 , p < 0.05 , pairwise comparison ps < 0.05 , bonferroni corrected . likewise , for current hours per week of playing an instrument , musicians ( mean = 3.1 ) and bilingual musicians ( mean = 2.7 ) reported more current usage than bilinguals ( mean = 0.1 ) and controls ( mean = 0.4 ) , f(3,215 ) = 8.95 , p < 0.05 , pairwise comparison ps < 0.05 , bonferroni corrected . the approach of using median splits of proficiency to categorize participants into groups for the analyses was chosen over an approach of using continuous proficiency scores , for two reasons . the first is that both bilingual proficiency and music proficiency were bimodally distributed ( into a high and low group ) , as determined by hartigan 's dip statistic ( bilingual proficiency , hds = 0.11 , p < 0.05 ; music proficiency , hds = 0.07 , p < 0.05 ) . the second is that , by dividing participants into groups , our findings can be more easily connected to other research in the field , as most research in this field uses a categorical group approach ( e.g. , [ 1 , 16 , 23 ] ) . nevertheless , we supplement this categorical approach with a continuous approach by conducting regression analyses of bilingual proficiency and music proficiency scores ( see section 2.3 ) . bilinguals , musicians , and bilingual musicians had experience with a large variety of languages and/or instruments . this diverse group of participants represents the wide range of linguistic and musical experiences that exists in the real world , thereby increasing the external validity of the study . the languages reported by bilinguals and bilingual musicians included english ( n = 112 , 100% of bilinguals and bilingual musicians ) , spanish ( n = 53 , 47.3% ) , korean ( n = 15 , 13.4% ) , mandarin ( n = 14 , 12.5% ) , chinese ( unspecified ) ( n = 8 , 7.1% ) , arabic , cantonese , and polish ( each n = 3 , 2.7% ) , french ( n = 2 , 1.8% ) , bengali , czech , german , gujarati , hebrew , japanese , lithuanian , marathi , russian , tamil , and urdu ( twenty ( 17.9% ) bilinguals and bilingual musicians learned english as the first language , 61 ( 54.4% ) learned english as the second language , and 31 ( 27.7% ) learned english and another language simultaneously . mean age of acquisition of the second language ( or both languages in the case of simultaneous bilinguals ) was 4.39 ( range = 014 ) years . musicians and bilingual musicians listed a variety of instruments , including piano ( n = 65 , 58.6% ) , voice ( n = 45 , 40.1% ) , guitar ( n = 34 , 30.6% ) , violin ( n = 26 , 23.4% ) , flute ( n = 20 , 18.0% ) , drums / percussion ( n = 11 , 9.9% ) , clarinet ( n = 10 , 9.0% ) , saxophone ( n = 8 , 7.2% ) , bass ( n = 7 , 6.3% ) , trumpet ( n = 6 , 5.4% ) , viola ( n = 4 , 3.6% ) , cello , recorder , and xylophone ( each n = 3 , 2.7% ) , guzheng , oboe , and ukulele ( each n = 2 , 1.8% ) , and banjo , bassoon , erhu , french horn , trombone , and tuba ( each n = 1 , 0.9% ) . mean age of acquisition of the first learned instrument was 8.41 ( range = 018 ) years ( one participant indicated voice training within the first year ) . on average , musicians and bilingual musicians played 2.32 ( range = 15 ) instruments and had taken 11.26 ( range = 031 ) years of lessons . table 1 provides each group 's demographic information with respect to male - to - female gender ratio , age , nonverbal iq ( performance subtests of the wechsler abbreviated scale of intelligence ; wasi ) , and short - term memory ( digit span subtest of the comprehensive test of phonological processing ; ctopp ) . ( the following background data are unavailable for a subset of participants : gender ( n = 1 ) , nonverbal iq ( n = 5 ) , and digit span ( n = 3 ) . ) measures of iq and short - term memory were included in order to determine whether groups differed on other variables that are known to correlate with interference suppression [ 38 , 39 ] . the wasi nonverbal iq was derived from the block design and matrix reasoning subtests . in the block design in the matrix reasoning subtest , participants saw a pattern with a missing element and chose the response option that best completed the pattern . the short - term memory score was derived from the ctopp digit span task . in the digit span task , participants heard a series of numbers and had to repeat those numbers in the same order in which they heard them . a chi - square analysis indicated no group differences in male - to - female gender ratio , (3 , n = 218 ) = 2.81 , p > 0.05 . a one - way anova with age as the dependent measure revealed an effect of group , f(3,215 ) = 4.95 , p < 0.05 , with the bilingual musicians being younger than the bilinguals and controls ( ps < 0.05 , bonferroni corrected ) . an effect of group was also found in one - way anovas on short - term memory performance ( f(3,212 ) = 3.80 , p < 0.05 ) and nonverbal iq ( f(3,210 ) = 2.65 , p = 0.05 ) , with participants in the bilingual group having lower digit spans than the other three groups ( ps < 0.05 , bonferroni corrected ) and marginally lower iqs than musicians ( p = 0.067 , bonferroni corrected ) . because the groups differed in age , digit span , and iq , additional analyses of the simon data were conducted to control for differences in these measures ( described in section 2.3 and presented at the end of section 3 ) . a visual - spatial simon task the simon task was chosen because it is well - validated , uses nonlinguistic and nonmusical stimuli , and has an effective control condition ( i.e. , when neutral trials are included as a baseline control condition , the task consistently elicits neutral response times that are slower than congruent response times and faster than incongruent response times ) . in the simon task , a blue or brown rectangle was presented on the left , center , or right side of the computer screen . participants were asked to push a button on the left side of the keyboard ( the a key marked with a blue sticker ) when the blue rectangle was presented and a button on the right side of the keyboard ( the l key marked with a brown sticker ) when the brown rectangle was presented , regardless of the spatial location of the rectangle . when the rectangle appeared on the same side as the response button ( i.e. , a blue rectangle on the left side of the screen or a brown rectangle on the right side of the screen ) , the trial was classified as congruent . when the rectangle appeared in the middle of the screen , the trial was classified as neutral . when the rectangle appeared on the side opposite to the response button ( i.e. , a blue rectangle on the right side of the screen or a brown rectangle on the left side of the screen ) , the trial was classified as incongruent . participants completed 126 experimental trials ( 42 congruent trials , 42 neutral trials , and 42 incongruent trials ) in a random order that was fixed across participants . in each trial , a fixation cross appeared for 350 ms , followed by a blank screen that was displayed for 150 ms , followed by a colored rectangle that was presented for 1500 ms , followed by a 850 ms blank screen serving as the intertrial interval . when an error was committed , an x was displayed on the screen for 1500 ms . when a correct response was made , no feedback was provided and the next trial began immediately . prior to completing the 126 experimental trials , participants completed 24 practice trials , 8 of each trial type . simon trials that were responded to incorrectly , that were not responded to within the 1500 ms response window , or that had a response time greater than 2.5 standard deviations from a participant 's mean were removed from the dataset . this procedure led to the omission of 6% of all trials . after removing these trials , response time data were submitted to a 4 3 mixed - design anova , with group ( bilinguals , musicians , bilingual musicians , and controls ) as the between - subjects independent variable and congruency ( congruent , neutral , and incongruent ) as the within - subjects independent variable . ( in a preliminary analysis , a 2 ( language status : a bilingual , not a bilingual ) 2 ( music status : a musician , not a musician ) 3 congruency ( congruent , neutral , and incongruent ) anova was conducted ; this analysis yielded a three - way interaction , f(2,430 ) = 3.92 , p < 0.05 , providing a statistical justification for dividing participants into our 4 groups ( bilinguals , musicians , bilingual musicians , and controls ) . ) in the event of an interaction between group and congruency , follow - up anovas were conducted on the interference effect ( as well as the facilitation effect and simon effect ) . the interference effect is calculated by subtracting response time on neutral trials from response time on incongruent trials . the facilitation effect is calculated by subtracting response time on congruent trials from response time on neutral trials . note that the facilitation effect is difficult to interpret , as it may reflect a better ability to utilize the irrelevant but informative stimulus location on congruent trials [ 16 , 23 ] or a worse ability to inhibit the irrelevant but informative stimulus location on congruent trials [ 41 , 42 ] . the simon effect is calculated by subtracting response time on congruent trials from response time on incongruent trials . the simon effect is also difficult to interpret because it conflates facilitation and interference effects . nevertheless , simon effect and facilitation effect data are presented to enable readers to compare our results to the results of other studies . follow - up pairwise comparisons on the interference , facilitation , and simon effects were conducted with bonferroni corrections . first , ancovas with iq , digit span , and age as covariates were conducted due to group differences in these measures . second , an analysis was conducted on a subset of the participants who were matched on iq , digit span , and age . in this analysis , subsets were selected by randomly sampling 42 participants per group ( n = 42 was chosen because the smallest group contained 42 participants ) until the four groups did not differ significantly on iq ( f = 2.62 , p > 0.05 ) , digit span ( f = 2.48 , p > 0.05 ) , or age ( f = 2.30 , p > 0.05 ) . this random sampling procedure was conducted by first assigning a number to each participant in a group and then using a random number generator to select 42 participants from each of those groups . we repeated this procedure until one of the samples yielded p values above 0.05 for all three anova comparisons ( iq , digit span , and age ) . in a third analysis , we entered neutral response times as a covariate ( as a proxy of processing speed ) in ancovas , because processing speed may affect the magnitude of interference and facilitation effects and because there were nonsignificant trends of group differences in raw response times . in a fourth analysis , we conducted separate multiple linear regressions for the interference effect , the facilitation effect , and the simon effect , with music proficiency , bilingual proficiency , and an interactive term of music and bilingual proficiency as the predictor variables ; these analyses treat music and bilingual proficiency as continuous variables and provide a more fine - grained analysis than categorical analyses that collapse across individual differences in proficiency . in the fifth analysis , anovas were conducted on accuracy on the simon task in order to rule out a speed - accuracy trade - off . a group ( bilinguals , musicians , bilingual musicians , and controls ) by congruency ( congruent , neutral , and incongruent ) anova conducted on response time data yielded a significant main effect of congruency , f(2,430 ) = 453.58 , p < 0.05 , p2 = 0.68 , a marginally significant main effect of group , f(3,430 ) = 2.50 , p = 0.06 , p2 = 0.03 , and a significant interaction between group and congruency , ( table 2 presents the raw rts for each group at each level of congruency . ) the main effect of congruency demonstrated the validity of the simon task and reflected that neutral trials were faster than incongruent trials ( i.e. , there was an interference effect ) , that congruent trials were faster than neutral trials ( i.e. , there was a facilitation effect ) , and that congruent trials were faster than incongruent trials ( i.e. , there was a simon effect ) ( all ps < 0.05 , bonferroni corrected ) . the marginally significant main effect of group indicated that groups might differ in overall response time , but bonferroni corrected pairwise comparisons indicated no significant or marginally significant differences ( all ps > 0.1 ) . the significant interaction between group and congruency suggested differences between groups in the interference effect , facilitation effect , and/or simon effect . to follow up on the interaction between group and congruency , one - way anovas were conducted on the interference effect as well as the facilitation effect and simon effect . an anova performed on the interference effect yielded a significant difference among groups , f(3,215 ) = 6.21 , p < 0.05 , p2 = 0.08 , with pairwise comparisons indicating that bilinguals , musicians , and bilingual musicians had smaller interference effects than controls , indicative of better interference suppression ( all ps < 0.05 , bonferroni corrected ) . an anova performed on the facilitation effect yielded a significant difference among groups , f(3,215 ) = 2.93 , p < 0.05 , p2 = 0.04 , with pairwise comparisons indicating that bilinguals had a significantly larger effect than bilingual musicians ( p < 0.05 , bonferroni corrected ) and a marginally significantly larger effect than controls ( p = 0.067 , bonferroni corrected ) . an anova performed on the simon effect also yielded a significant difference among groups , f(3,215 ) = 3.23 , p < 0.05 , p2 = 0.04 . pairwise comparisons revealed a smaller simon effect in bilingual musicians relative to controls ( p < 0.05 , bonferroni corrected ) . five additional analyses were then conducted to confirm the above results ( see section 2.3 for detailed explanations of how and why these analyses were conducted ) . first , we conducted ancovas with iq , digit span , and age as covariates in order to control for group differences in these measures ; these analyses yielded a significant group difference in the interference effect , f(3,207 ) = 5.91 , p < 0.05 , with bilinguals , musicians , and bilingual musicians producing smaller interference effects than controls , reflecting enhanced interference suppression ( all ps < 0.05 , bonferroni corrected ) . there was also a significant group difference in the facilitation effect , f(3,207 ) = 3.03 , p < 0.05 , with bilinguals producing larger facilitation effects than bilingual musicians ( p < 0.05 , bonferroni corrected ) ( and marginally larger effects than controls , p = 0.097 , bonferroni corrected ) , as well as a significant group difference in the simon effect , with bilingual musicians producing smaller simon effects than controls ( p < 0.05 , bonferroni corrected ) . next , an analysis was conducted on a subset of the participants who were matched on iq , digit span , and age . analyses carried out on this subset yielded similar results ( i.e. , significant group differences in the interference effect , f(3,164 ) = 8.80 , p < 0.05 , with bilinguals , musicians , and bilingual musicians producing smaller ( i.e. , better ) interference effects than controls , all ps < 0.05 , bonferroni corrected ; significant group differences in the facilitation effect , f(3,164 ) = 2.81 , p < 0.05 , with bilinguals producing larger facilitation effects than bilingual musicians , p < 0.05 , bonferroni corrected ; and significant group differences in the simon effect , f(3,164 ) = 4.10 , p < 0.05 , with bilingual musicians producing smaller simon effects than controls , p < 0.05 , bonferroni corrected ) . in the next analysis , neutral response time was entered as covariate ( as a proxy of processing speed ) in an ancova in order to control for the potential effects of processing speed on the interference effect and facilitation effect . this ancova replicated the interference effect results ( i.e. , significant group differences , f(3,214 ) = 6.41 , p < 0.05 , with bilinguals , musicians , and bilingual musicians producing smaller ( better ) interference effects than controls , all ps < 0.05 , bonferroni corrected ) . ( another way to control for processing speed is to calculate proportional interference effects ( interference effect divided by response time on neutral trials ) . analyses of proportional interference effects yielded the same results ( i.e. , significant group differences , f(3,215 ) = 6.63 , p < 0.001 , with bilinguals , musicians , and bilingual musicians producing smaller interference effects than controls ) . ) however , when taking processing speed into consideration , there were no significant or marginally significant differences in the facilitation effect , f(3,214 ) = 1.83 , p > 0.1 . next , we conducted linear multiple regression analyses of the interference effect , facilitation effect , and simon effect , with bilingual proficiency , music proficiency , and a bilingual proficiency / music proficiency interaction term as predictor variables . when the interference effect was entered as the dependent variable , bilingual proficiency was a significant predictor ( beta weight = 0.32 , p < 0.05 ) and music proficiency was a marginally significant predictor ( beta weight = 0.21 , p = 0.059 ) , while the interactive term was not significant ( beta weight = 0.03 , p > 0.1 ) . these results confirm that higher bilingual proficiency and higher music proficiency were associated with smaller ( i.e. , better ) interference effects ( but that bilingual and music proficiency do not have additive effects ) . when the facilitation effect was entered as the dependent variable , bilingual proficiency was a significant predictor ( beta weight = 0.34 , p < 0.05 ) and the interactive term was a marginally significant predictor ( beta weight = 0.04 , p = 0.057 ) , while music proficiency was not significant ( beta weight = 0.03 , p > 0.1 ) . when the simon effect was entered as the dependent variable , none of the predictor variables reached significance ( ps > 0.1 ) . accuracy was high overall ( mean congruent accuracy = 98.23% , mean neutral accuracy = 97.71% , and mean incongruent accuracy = 93.23% ) . an anova yielded a significant main effect of congruency , f(2,430 ) = 100.60 , p < 0.001 , p2 = 0.32 , reflecting that incongruent trials were responded to less accurately than congruent and neutral trials ( ps < 0.05 , bonferroni corrected ) , but no main effect of group , f(3,430 ) = 1.63 , p > 0.1 , p2 = 0.02 , or interaction between group and congruency , f(6,430 ) = 1.03 , p > 0.1 , p2 = 0.01 . the lack of a difference between groups in accuracy suggests that group differences in interference effect response times were not due to a speed - accuracy trade - off . in the current study , we examined the effects of bilingual and musical experience on executive control using a nonlinguistic , nonmusical , visual - spatial simon task . our results revealed lower interference effects in bilinguals , musicians , and bilingual musicians ( relative to controls ) , indicative of enhanced interference suppression . these results were observed across all analyses and lend support to the idea that experience can drive plasticity in cognitive functions . in addition to the interference effect , we reported participants ' simon effect score , which is often used to assess interference suppression . the simon effect may not provide a pure measure of interference suppression ( because it is influenced by congruent facilitation ) ; consequently , this measure may conceal true bilingual and musician advantages in interference suppression . indeed , the simon effect results masked the enhancement in bilinguals and musicians because of variability in the facilitation effect across groups . aside from our primary finding of better interference suppression in bilinguals , musicians , and bilingual musicians , three secondary results are worth noting . first , in some analyses , bilinguals produced larger facilitation effects than both the controls and bilingual musicians . larger facilitation effects in bilinguals relative to controls have previously been reported in the literature . however , in the current study , facilitation differences did not hold up after factoring out the effects of processing speed , age , iq , and short - term memory . thus , in our study , the facilitation results may have been due to cognitive and demographic differences among the groups . another result of interest is that bilingual musicians had smaller simon effects than controls , while bilinguals and musicians did not differ from controls . this result may suggest that the combination of bilingual and music experience ( relative to bilingual or music experience alone ) is necessary to develop advantages on some aspects of the simon task . however , simon effects are difficult to interpret because they conflate the ability to utilize congruent cues ( i.e. , facilitation effects ) with the ability to ignore incongruent cues ( i.e. , interference effects ) and thus further research in needed to clarify this finding . a third result relates to overall response speed on the simon task . although not significant , there was a numerical trend toward bilinguals responding more slowly than other groups ( particularly , musicians and bilingual musicians ) . this slight , but not reliable , delay in bilinguals ' response times contrasts with some previous work reporting significantly faster overall response times for bilinguals ( e.g. , ) but is consistent with several other studies ( e.g. , [ 23 , 43 , 44 ] ) . this trend appears to be due in part to the bilingual sample 's lower iq and short - term memory scores ; indeed , the differences in overall response time are considerably smaller when controlling for iq and short - term memory . importantly , the primary finding of a bilingual enhancement in interference suppression does not seem to be due to differences in response time , as this enhancement is still observed after controlling for response time in our analyses . bilinguals ' enhanced interference suppression ability observed in the present study may be due to their need to reduce competition from cross - linguistic activation of the nontarget language during comprehension and/or production . in support of this explanation , blumenfeld and marian reported a correlation between the degree of activation of cross - linguistic similar - sounding words during speech comprehension ( e.g. , activation of the spanish word cartera when hearing the english word carpet ) and the level of enhanced interference suppression in bilinguals . however , the reasons why musicians show better interference suppression ability are less clear than in bilinguals . as noted earlier , one possibility is that the musician advantage derives in part from a similar mechanism as the bilingual advantage . that is , analogous to how bilinguals activate similar - sounding words in the nontarget language during language comprehension , musicians may activate similar - sounding nontarget melodies during music comprehension [ 24 , 25 ] . this melody activation is not specific to musicians in the same way that word activation is not specific to bilinguals . however , just as bilinguals have a larger number of words to suppress , musicians have a larger number of melodies to suppress ( and likely higher frequency of certain melodies ) . the additional melodies that musicians activate could make the task of melody identification more difficult and may therefore serve as a good exercise for suppression mechanisms . consistent with the notion that melody identification is more difficult for musicians , musicians were shown to identify melodies at a later point in time than nonmusicians . this challenging practice of inhibiting interference from nontarget melodies during music comprehension may improve musicians ' cognitive control abilities , similar to how management of nontarget words may enhance bilinguals ' cognitive control abilities . while there may be similarities in the mechanisms behind bilingual and musician advantages for example , during speech production , bilinguals need to select a single language at a time because both of their languages use the same output modality ( the mouth ) and thus can not be produced simultaneously ( e.g. , the word , cat , and the spanish word for cat , gato , can not be produced concurrently ) . the need to limit speech production to one language at a time may train executive control abilities and contribute to a bilingual advantage . when this restriction of one output modality is removed , as in speech - sign bimodal bilinguals who use two different output modalities ( the mouth and the hands ) , the benefits to executive control are reduced . ( note , however , that bimodal bilinguals still face inhibitory demands during comprehension like unimodal bilinguals , which may result in some executive control benefits ; [ 46 , 47 ] . ) like bimodal bilinguals , musicians are able to produce in their language and instrument at the same time ; even voice musicians can simultaneously incorporate both melody and words in their productions . while musicians may not have the same constraints on production as unimodal bilinguals , they nevertheless develop similar enhancements in interference suppression . this finding suggests that the source of enhanced interference suppression in musicians may differ in some ways from that of unimodal bilinguals . one potential source of musicians ' enhancement comes from the opera ( overlap , precision , emotion , repetition , and attention ) hypothesis of musical training . according to this hypothesis , musical training involves focused attention , for example , in selectively attending to the fine acoustic details of sound sequences . increased selective attention can contribute to better interference suppression , as reducing interference from irrelevant cues can be accomplished through selective attention to the relevant cue and/or inhibition of the irrelevant cue . it is possible then that improved interference suppression in musicians may arise from the need to attend to fine acoustic details while limiting interference from factors that may impede attention to the details of sound . similar to bilinguals and musicians , bilingual musicians also demonstrated better interference suppression ability than controls . however , bilingual musicians did not outperform bilinguals or musicians , suggesting that the benefits of bilingualism and musicality may not be additive . a possible reason why bilingual musicians did not show further gains over bilinguals or musicians is that bilingualism and musicianship alone may reach the upper limits of interference suppression in many younger adults , precluding any further benefit of experience with both . one limitation of the current study is that strong claims about a causal relationship between linguistic / musical experience and executive control can not be made , given that participants were not randomly assigned to groups . random assignment to life - long bilingual , musical , or bilingual - musical experience is not feasible . nevertheless , due to a lack of random assignment , it is possible that the bilinguals , musicians , and bilingual musicians in the current study were cognitively advantaged before their extensive training in a second language and/or a musical instrument . however , considering our cognitive measures , this possibility seems unlikely for both musicians and bilinguals : musicians did not outperform controls on measures of iq and short - term memory , and bilinguals were actually disadvantaged relative to controls in short - term memory . because ses was not directly measured in the current study , it is possible that the groups differed in ses and that this difference contributed to the results ( see for a discussion ) . while ses was not directly measured , iq test performance was measured , and iq test performance has been shown to correlate highly with ses . group differences in iq were controlled for in the present study through both matched - groups analyses and analyses of covariance . a third limitation of the current study is the way in which bilingual and musical proficiency were measured . because we tested participants with a diverse set of linguistic and musical backgrounds , it was not feasible to collect objective proficiency measures for each language and instrument . instead we used subjective measures , which have been shown to correlate significantly with objective measures [ 33 , 50 ] . nevertheless , objective measures are more precise and should be used in future studies to confirm the current findings . future studies should also address some of the questions that arise from the current finding of enhanced interference suppression in bilinguals , musicians , and bilingual musicians . one such question is the extent to which bilingual and musician advantages derive from similar mechanisms . another is what may be some of the reasons why bilingual musicians do not have interference suppression abilities that are above those of bilinguals and musicians . in closing , the current study took a behavioral approach to cognitive plasticity by assessing whether adults with second language and/or musical experience have advantages in executive control . the results indicate enhanced interference suppression in bilinguals , musicians , and bilingual musicians relative to monolingual nonmusicians . we conclude that learning a second language or playing a musical instrument has benefits that extend beyond the specific domains of language and music to more general nonlinguistic cognitive function , including core skills like executive control . because executive control abilities are related to a broad range of competencies [ 51 , 52 ] , these findings have implications for education practices by encouraging support for second language and music instruction .

learning how to speak a second language ( i.e. , becoming a bilingual ) and learning how to play a musical instrument ( i.e. , becoming a musician ) are both thought to increase executive control through experience - dependent plasticity . however , evidence supporting this effect is mixed for bilingualism and limited for musicianship . in addition , the combined effects of bilingualism and musicianship on executive control are unknown . to determine whether bilingualism , musicianship , and combined bilingualism and musicianship improve executive control , we tested 219 young adults belonging to one of four groups ( bilinguals , musicians , bilingual musicians , and controls ) on a nonlinguistic , nonmusical , visual - spatial simon task that measured the ability to ignore an irrelevant and misinformative cue . results revealed that bilinguals , musicians , and bilingual musicians showed an enhanced ability to ignore a distracting cue relative to controls , with similar levels of superior performance among bilinguals , musicians , and bilingual musicians . these results indicate that bilingualism and musicianship improve executive control and have implications for educational and rehabilitation programs that use music and foreign language instruction to boost cognitive performance .

over the last century , the surgical management of ureteropelvic junction obstruction ( upjo ) has dramatically evolved . various open surgical techniques have been described based on the cause , location , and length of the upjo . the most popular repair is the anderson - hynes dismembered pyeloplasty , which has universal application and is accepted as the gold standard of treatment . with the development of endoscopic techniques and equipment , minimally invasive approaches have come into favor in the treatment of patients with primary and secondary upjo . more recently , with advancing laparoscopic skills and the introduction of robotic - assisted surgery , many centers have moved to laparoscopic pyeloplasty ( lp ) as first - line therapy . improved suturing skills and the use of robotic assistance have greatly facilitated laparoscopic dismembered pyeloplasty for primary and secondary repairs . the use of ureteral stents following pyeloplasty ensures adequate drainage , particularly in the presence of postoperative edema . for similar reasons , stents are commonly used after ureteroscopy although some controversy still exists regarding its necessity after uncomplicated ureteroscopic stone removal . in the pediatric population the advantages of stent placement following pyeloplasty include lowering the risk of urinoma formation following upj repair and providing support and alignment of the fresh suture line . the importance of the stent is highlighted when the anastamosis is not watertight , or after endopyelotomy , allowing healing of the defect while urine is diverted by the stent . however , ureteral stents are not free from risk , and potential problems include migration , encrustation , retained or forgotten fragments , irritative urinary symptoms , exposure of the upper urinary tract to high pressure during urination , flank pain , and increased risk of infection . in a porcine model , soria et al evaluated whether it is possible to reduce the duration of ureteral stenting following endopyelotomy , and thus reduce side - effects . ureteral stent placement for 1 week was found to be insufficient to assure correct healing and evolution of the upj following endopyelotomy . stenting for 3 weeks was shown to be effective , and it was therefore not necessary to extend stenting time to 6 weeks . in the senior surgeon 's experience ( als ) , 17 stented lps have been performed to date . excellent outcomes have been previously reported with the use of the lapraty clip during collecting system reconstruction . although the role of stents has been well described after endopyelotomy , its role after laparoscopic pyeloplasty , where watertight closure can be achieved , is unclear . this is an institutional review board approved , prospective collection and retrospective analysis of data obtained from patients specifically undergoing lsp . between november 2004 and february 2006 , 6 consecutive patients were treated without the placement of a postoperative stent , all of whom were operated on by the same surgeon ( als ) . in addition , all patients had undergone retrograde ureteral stenting by their local urologist before being seen at our center . briefly , after cystoscopy and ureteral stent removal followed by repeat retrograde urography , patients were placed in a lateral decubitous position . after port placement , the colon was reflected medially followed by identification of the ureter either at the level of the lower pole or near the level of the renal hilum . the collecting system was divided just cranial to the narrowest point , and the ureter was then spatulated 2 cm along its lateral aspect . the capacious pelvis was minimally spatulated along its medial aspect and reduced appropriately if excessive tissue was present . the anastomosis was performed using 4 0 absorbable vicryl suture on an sh - needle beginning with the posterior layer with a free hand - tied knot and proceeding with a continuous suture in a lateral - to - medial direction . the suture line was performed by placing the needle from outside - to - in on the renal pelvis and inside - to - out on the ureter side . a lapra - ty clip ( ethicon endosurgery , cincinnati , oh ) was used to complete the suture line at the medial aspect of the renal pelvis . the anterior layer was completed in a similar fashion by using a second 4 0 vicryl suture secured with a lapra - ty clip . forced diuresis along with intravenous indigo - carmine was provided by the anesthesia team to visually inspect the anastamotic closure . on the morning of postoperative day ( pod ) 1 , the bladder catheter was removed , and the drain was removed 12 hours later before hospital discharge , after the patient had voided without any increase in drain output . all patients had a follow - up tc-99 m mag3 renal scintigraphy at 4 weeks and 6 months following surgery . total analgesia was reported in equivalent milligrams of injectable morphine and was calculated for duration of in - patient stay only . data were maintained and analyzed with file maker pro ( file maker inc , santa clara , ca ) . five patients ( 3 female , 2 male ) underwent lsp with a mean follow - up of 15.7 months ( range , 7 to 24 ) . average age of the patients was 42.8 years ( range , 33 to 64 ) with an average body mass index of 29.3 kg / m ( range , 19.3 to 54.9 ) . mean split renal function for the obstructed kidney by tc-99 m mag3 scan was 36% ( range , 15 to 52 ) ( table 1 ) . three patients were noted to have a crossing vessel , while the other 2 patients had dense , fibromuscular adhesions surrounding the upj . mean operative time and estimated blood loss were 196 minutes ( range , 145 to 284 ) and 50 ml ( range , 10 to 150 ) , respectively . average time to regular diet was 13.3 hours ( range , 5 to 35 ) . mean durations of postoperative urinary catheter and abdominal drain were 0.9 days ( range , 0.5 to 1.5 ) and 1.6 days ( range , 1 to 2.5 ) , respectively . one patient was kept in the hospital one extra day due to difficulty voiding after catheter removal . mean analgesia consumption was 42.1 mg ( range , 21 to 65 ) of equivalent morphine . summary of preoperative and postoperative tc-99 m mag3 renal scans upjo = ureteropelvic junction obstruction ; cv = crossing vessel ; da = dense adhesion . this involved patient # 5 in our series who was a 42-year - old male with a left - sided upjo . the night of the surgery , the patient complained of colic - type flank pain with intractable nausea and vomiting . a retrograde pyelogram was performed on the morning of pod 1 , which demonstrated a normal caliber ureter with mild hydronephrosis . although there was no obvious evidence of obstruction , a 6f ureteral stent was placed without difficulty . multiplanar fluoroscopic views ensured proper placement of the guide - wire and stent in the collecting system . the patient noted complete resolution of symptoms , however , did note moderate irritatitve voiding symptoms requiring anticholinergic medications and flank discomfort while voiding during his recovery period . follow - up split function with mag-3 renal scans at both 4 weeks and 6 months following lsp were not significantly different from preoperative values ( table 1 ) . otherwise , all patients remained asymptomatic throughout their follow - up period with no evidence of obstruction . abdominal ultrasound at that time revealed no hydronephrosis , however , did demonstrate several gallbladder stones . follow - up scan at that time noted drainage with t- of 26 minutes , which had improved from the patient 's preoperative state of complete obstruction . a more recent renal scan 18 months postoperatively demonstrated a further improvement in t of 16 minutes and a stable split renal function , with no recurrence of flank discomfort . in addition , this patient , the only one with preoperative baseline renal insufficiency , had an improvement in serum creatinine from 2.5 mg / dl to 2.0 mg / dl . ureteral stenting in children typically requires a second procedure with the patient under anesthesia for stent removal . the case for nonstented pyeloplasty has been addressed in a review study of the pediatric literature where it has been shown to be safe and accepted as a standard of care . in general , the indications for ureteral stenting are many , and their use has become common in a urologic practice , especially after a pyeloplasty where a ureteral stent typically will be placed for 4 weeks to 6 weeks postop . the most recent and largest published series to date , from johns hopkins , documents 4-week routine stenting following laparoscopic pyeloplasty . unfortunately , stent use is not without potential complication . in one study evaluating the morbidity of stents , a complication rate of 94% ( 103/110 patients ) was reported , which included infection , flank pain with voiding , stent migration , and stent fragmentation . similarly , using validated questionnaires , joshi et al reported that 78% of the 62 respondents noted bothersome urinary symptoms that encompassed urinary urgency , frequency , incontinence , and hematuria . in addition , 80% of respondents experienced stent - related pain affecting daily activities , as well as some respondents noting sexual dysfunction ( 32% ) and reduced work capacity ( 58% ) . in the senior surgeon 's prior experience with 17 stented laparoscopic pyeloplasties , nine ( 53% ) of these individuals required anticholinergic medication to treat these symptoms and 2 ( 12% ) required early stent removal 3 weeks postoperatively ( unpublished data ) . in our initial lsp experience , we found no increased morbidity from performing this technique . the risks of stent migration , encrustation , stent syndrome ( defined as dysuria , frequency , flank pain , and hematuria commonly seen with short - term placement ) , increased risk of infection , and stent encrustation were replaced by the risk of possible postop obstruction and/or urinoma formation that would require retrograde stent placement . such a maneuver is potentially dangerous in that wire placement may injure the anastamosis or be mistakenly placed outside the collecting system . proper endoscopic equipment ( eg , hydrophilic coated guidewire ) and skill are required to ensure safe placement . in our study , only one patient experienced symptoms suggestive of postoperative obstruction . nevertheless , a ureteral stent was placed for 4 weeks , during which time , the patient complained of irritative voiding symptoms and renal pain during voiding . furthermore , the oldest patient in this cohort ( patient # 2 ) with a bmi of 54.9 kg / m who had recurrent bouts of pyelonephritis and flank pain at the time of presentation had dense peri - ureteral adhesions noted at the time of surgery . this patient with a preoperative split function of 35% on the right side , and complete upjo was noted to have a partial obstruction on follow - up renal scan . at 10-months postoperatively , the concept of cholecystitis was considered by the presence of gallbladder stones and sludge coupled with moderate gallbladder wall thickening . this patient has otherwise remained asymptomatic with improved serum creatinine and no recurrent bouts of pyelonephritis despite moderate objective improvement . no infectious or obstructive complications related to the use of lapra - ty clips were encountered . although postoperative ct - abdominal scans were not routinely obtained , none of the patients exhibited signs or symptoms consistent with urinoma formation , which compares favorably with a previous report that noted a 5% prolonged urinary leak and 5% rate of urinoma formation in a nonstented pediatric cohort . the benefits of a stentless procedure , if proven as efficacious and safe as a stented pyeloplasty , include reducing the risk for infection , eliminating the risk of developing stent syndrome and the need for follow - up cystoscopy 4 weeks to 6 weeks postoperatively for stent removal . the technical advantage of using the lsp technique is the avoidance of stent interference during suture - related reconstruction . lsp allows for optimal visualization and direct access to the spatulated edges for anastamosis , eliminating the risk of entangling the suture around the stent and stent migration . as such , these factors help optimize the likelihood of a watertight anastamosis . comparative results were noted to our previously published stented laparoscopic pyeloplasty series . comparing our operative times to times of previously published series shows that our mean time of 196 minutes is comparable to that of other contemporary large series , which range between 123 minutes and 252 minutes . when assessing hospitalization , we noted a shorter mean stay of approximately 1.6 days versus that of other series , which are reported to be between 2.6 days to 4.5 days . overall , there was 100% subjective success in our 5 patients with a mean follow - up of 15.7 months . with regards to objective success , all patients demonstrated stabilized or improved split renal function on mag-3 renal scan and improved drainage t- times . several limitations of this study warrant discussion . the small patient number and short follow - up limit the power to perform statistical analysis . although some critics may argue that extended follow - up beyond 24 months would be optimal , inagaki et al from johns hopkins evaluated 147 lps with a mean follow - up of 24 months and demonstrated that most failures occur within 6 months . due to small numbers in a retrospective review , we were not able to assess the benefit of lsp for primary versus secondary upjo . a prospective , randomized study comparing stented with nonstented methods is needed to address the dogma of ureteral stent placement and evaluate the postoperative complication parameters . as such , it will take a significantly larger cohort of patients with a longer follow - up to assess whether adult lsp is comparable to published success rates of stented laparoscopic pyeloplasty of 81 to 99 . we still advocate the use of a stent in all patients with a solitary kidney , patients with a difficult ureteral anastomosis , significant bleeding during upjo repair , and those individuals with a thick , noncompliant ureter due to chronic inflammation . utilizing a stentless technique has proved to be efficacious in a small cohort of patients with limited follow - up . our minimal incidence of postoperative complications is likely related to meticulous surgical technique and ability to minimize laxity on our suture line with the aid of additional lapra - ty clips as needed . certainly , a larger cohort with longer follow - up will be required to prove the durability and safety of a stentless laparoscopic pyeloplasty , which may ultimately revolutionize previous dogmatic assertions .

background and objectives : pyeloplasty , whether open or laparoscopic , has been the mainstay of treatment for ureteropelvic junction obstruction ( upjo ) . a nonstented pyeloplasty has only been reported in the pediatric literature . herein , to the best of our knowledge , we report the first published experience with laparoscopic stentless pyeloplasty ( lsp ) in the adult population.methods:patients with a normal contralateral kidney who underwent a laparoscopic pyeloplasty were included in this study . a dismembered pyeloplasty was performed without the placement of a ureteral stent . functional tc-99 m mag3 renal - scan data were compared with results at 4 weeks and 6 months postoperatively . perioperative complications and long - term follow - up were prospectively gathered.results:to date , 5 patients have undergone lsp with a mean follow - up of 15.7 months . mean age and body mass index of this group were 42.8 years and 29.3 kg / m2 , respectively . mean operative time , estimated blood loss , and hospital stay were 196 minutes , 58 ml , 1.6 days , respectively . three patients had right - sided upjo , and 2 patients had left upjo . no patient had undergone previous surgery for upjo . all patients had a ureteral stent in place at the time of surgery . no intraoperative complications occurred . only one patient complained of flank pain on pod1 . no obstruction or urinary extravasation was seen on retrograde pyelography , but a ureteral stent was placed . during our follow - up , all patients had complete resolution of their symptoms . postoperative renal scans demonstrated improved urinary drainage in all patients.conclusion:our initial experience suggests that in experienced hands , lsp may be an effective method for treating upjo .

acquired chloridorrhea ( acd ) is a rare disorder presenting with chloride - rich diarrhea , severe volume depletion , metabolic alkalosis , hypochloremia , hypokalemia , hyponatremia , acute renal failure , and low urinary chloride excretion . this array of derangements is also seen in congenital chloridorrhea ( ccd ) , a neonatal disorder that generally incurs death by the third decade of life . although ccd has been described 100 times in the literature [ 2 , 3 ] , the acquired form is still elusive and may be seen with severe intestinal stress or surgery . a 58-year - old african american male presented with profuse diarrhea and severe dehydration after right hemicolectomy , extensive distal ileal resection , ileostomy , and colostomy placement post - small bowel incarceration 9 weeks before . ileosteal output approximated 3 l of watery stool per day despite diphenoxylate with atropine and loperamide therapy . he reported salt craving but denied vomiting , diuretic use , pain , or persistent childhood diarrhea . his vital signs were temperature 98.8 f , respiratory rate 20 , pulse 89 , and blood pressure 97/71 mmhg . on examination as shown in table 1 , initial laboratory values demonstrated hyponatremia , hypochloremia , hypocalcemia , hyperphosphatemia , hypomagnesemia , acute renal failure , and surprisingly , metabolic alkalosis . urine studies revealed low sodium and chloride excretion in urine suggesting gastrointestinal origin of the metabolic alkalosis . stool studies for clostridium difficile toxin , fecal leukocytes , fecal ova , and parasites were negative . hiv elisa ( human immunodeficiency virus enzyme linked immunosorbent assay ) for 1 and 2 were nonreactive . after initial fluid replacement with normal saline , he developed hypokalemia ( potassium 2.8 meq / l ) . over several days , he received 24 l of fluid and nutrient and electrolyte replenishment , but continued to have high ostomy output . short bowel syndrome likely exacerbated nutritional deficiencies and increased gastric motility , thereby promoting high ostomy output . table 1.relevant laboratory studieslabpresentation ( some values are after hydration)2 months post - operationunitsserum studiessodium129140meq / lpotassium3.74.0meq / lchloride68105meq / lbicarbonate4128meq / lcalcium6.69.8mg / dlblood urea nitrogen10311mg / dlcreatinine10.61.2mg / dlphosphorus6.2mg / dlmagnesium1.31.4mg / dlhemoglobin10.410.6g / dlph7.545po279mmhgpco252.9mmhgbase excess21.8mmol / lalkaline phosphatase298122u / last4528u / lalt8437u / ltotal protein7.66.5albumin4.13.3ferritinx352b12150.6spot urine studiescreatinine122.9mg / dlsodium<20meq / lpotassium87.6meq / lchloride<20meq / lfecal studies ( ileostomy source)consistencywateryvolume34 lph6.0 to 6.5 -sodium11964meq / lpotassium910meq / lchloride10073meq / losmolarity307346mosm / kgpotassium decreased to 2.8 meq / l after initial fluids.our lab does not report urinary sodium < 20.our lab does not report urinary chloride < 20.the ph ( 6.06.5 ) in the fecal ( and ileal collections ) represents a low bicarbonate concentration , where naturally a higher hco3 concentration would be expected in fecal fluids . the ph ( 6.06.5 ) in the fecal ( and ileal collections ) represents a low bicarbonate concentration , where naturally a higher hco3 concentration would be expected in fecal fluids . , he improved with intravenous sandostatin ( octreotide ) and omeprazole which reduce gut motility and gastric hydrochloric acid losses . at discharge , hydration status , electrolyte abnormalities , and pre - renal failure normalized ( table 1 ) , but he required two readmissions for similar , albeit less severe relapses . after surgical re - anastomosis , pathology reported only mild ileal and colonic mucosal inflammation and hyperemia . two months post - operatively , the diarrhea resolved completely , electrolytes normalized , he was gaining weight , and octreotide and omeprazole were successfully discontinued . ccd was first recognized in 1945 , but only recently have the causative cld gene and its defective gene product , dra ( down - regulated in adenoma ) , been characterized [ 5 , 6 ] . dra is a chloride bicarbonate ( or hydroxide ) ( cl / hco3 ) membrane transporter anion exchanger found in distal ileal and colonic brush borders . all reported cases of ccd demonstrate fecal chloride > 90 meq / l with half of the cases exhibiting fecal chloride concentrations greater than the sum of fecal potassium and sodium [ 3 , 7 ] . 1.(a ) normal jejunal effluent enters the ileum and hydrogen - sodium transporters secrete hydrogen ions and reabsorb sodium and water . this promotes luminal water formation leaving luminal chloride concentrations unchanged ( hence relatively concentrated ) . normal dra transporters ( 1 ) absorb chloride , and ( 2 ) secrete hco3 ( or oh ) ( kere ) which reacts with protons , thereby neutralizing ph and forming water to be absorbed distally . dysfunctional dra can not absorb chloride or secrete bicarbonate resulting in loss of chloride- , acid- , and water - rich fluid to the environment . additionally , hyponatremia and hypochloremia develop despite avid yet insufficient renal retention , contraction alkalosis , and elevated aldosterone secretion promoting renal potassium losses . ( a ) normal jejunal effluent enters the ileum and hydrogen - sodium transporters secrete hydrogen ions and reabsorb sodium and water . this promotes luminal water formation leaving luminal chloride concentrations unchanged ( hence relatively concentrated ) . normal dra transporters ( 1 ) absorb chloride , and ( 2 ) secrete hco3 ( or oh ) ( kere ) which reacts with protons , thereby neutralizing ph and forming water to be absorbed distally . dysfunctional dra can not absorb chloride or secrete bicarbonate resulting in loss of chloride- , acid- , and water - rich fluid to the environment . additionally , hyponatremia and hypochloremia develop despite avid yet insufficient renal retention , contraction alkalosis , and elevated aldosterone secretion promoting renal potassium losses . renal insufficiency due to volume contraction is common [ 7 , 8 ] though other associated renal lesions have been described . although ccd is a disease of the young , diagnosis may be delayed if the patient copes by increasing salty food consumption . however , severe disease states are unlikely to be assuaged by diet alone , making milder or clinically silent mutations in otherwise healthy persons likely . unfortunately , neither heterozygous phenotypes nor mutations manifesting with bowel stress ( i.e. bowel resection ) are known . in contrast to ccd , our previously healthy adult patient presented with acquired chloridorrhea . acquired and congenital cases differ in that acd ( i ) responds partially to potassium replacement , ( ii ) may have small quantities of chloride in the urine rather than zero often reported in ccd , ( iii ) appears to be associated with bowel resections and ostomies , and ( iv ) can be transient and reversible with surgical correction of the bowel abnormality . in 1954 , ariel reported the first case of acd in a 66-year - old male post - colectomy with colostomy presenting with copious , chloride - rich diarrhea and hypochloremic , hypokalemic alkalosis , hypokalemia , and metabolic alkalosis . twenty years later , bieberdorf et al . demonstrated that perfusing normal colons with a chloride - poor solution rich in non - absorbable ions reproduced diarrhea and electrolyte changes found in chloridorrhea , demonstrating an inducible , complex defect requiring more than potassium repletion alone . other reports of acd include that of a 39-year - old crohn 's patient with intestinal transplantation and ileostomy and three infants with intestinal obstruction and ostomy placements [ 8 , 14 ] . ostomies are not uncommon , but the rarity of chloridorrhea suggests an underlying genetic predisposition evident only with significant bowel stress . yang et al . demonstrated in animal models that chronic intestinal inflammation promotes extensive cytokine release resulting in reduced dra expression , altered epithelial permeability and electrolyte transport , and induction of intestinal chloride loss and associated electrolyte abnormalities . our tissue sample was processed in columbus , ohio , usa . reverse transcription polymerase chain reaction ( rt pcr ) for dra expression was not available at the facility . to continue to produce large volume ( diarrheal ) fluid , there is a need for high deliveries of na and cl to the ileum . this , in turn , requires good splanchnic blood flow and adequate extracellular fluid ( ecf ) . much like a cholera patient , ecf expansion during treatment paradoxically can worsen the volume of diarrhea . the treatment of ccd and acd has been generally disappointing , though acd can be transient . marx et al . reported that addition of proton - pump inhibitor ( ppi ) therapy improved the symptoms in a crohn 's patient with acd . the ppi blocks gastric hydrochloric acid and chloride secretion , thereby reducing luminal losses to the environment when dra is dysfunctional . octreotide ( sandostatin ) is useful in nonspecific treatment of different forms of secretory diarrhea ( including verner morrison syndrome and ccd ) because of its antimotility and inhibitor effects on small intestinal secretion . while ppi ( omeprazole ) and octreotide therapy improved our patient 's clinical status until he received corrective surgery , after surgery his disease resolved and further medication was no longer necessary in summary , acd presents with profuse , chloride - rich diarrhea and a surprising contraction metabolic alkalosis rather than metabolic acidosis often associated with typical diarrhea . acd and cld may represent a disease spectrum , resulting from insult to the same gene product , dra . the available literature suggests that chronic intestinal inflammation and reduction of dra expression in genetically susceptible persons post - bowel resection and ostomy placement may result in acd . although acd is rare , it is likely under - recognized in post - bowel resection patients , and requires accurate identification in order to implement appropriate treatment

a 58-year old male with a history of small bowel resection and ileostomy presented with severe dehydration and high ostomy output . laboratory investigation indicated hypochloremia , hypokalemia , hyponatremia , metabolic alkalosis , chloride - rich diarrhea , acute renal failure , and low urinary chloride excretion . due to striking similarities to congenital chloridorrhea ( ccd ) reported in neonates , we empirically diagnosed acquired chloridorrhea ( acd , chloride diarrhea ) . this is a rare disorder resulting in profuse chloride - rich diarrhea and classic metabolic derangements affecting adults with chronic intestinal inflammation , often in association with bowel surgery . in this report , we review the relevant literature and discuss the genetic defects likely contributing to both the congenital and acquired forms of chloridorrhea .

cyclodextrin glycosyltransferases ( cgtases ; e.c.2.4.1.19 ) are starch degrading enzymes which generate cyclic oligosaccharides termed cyclodextrins ( cds ) . these oligosaccharides exist as three major types commonly known as -cd , -cd , and -cd having 6 , 7 , and 8 glucose residues , respectively . they have torus - shaped structures able to encapsulate a wide range of molecules , thereby modifying their physical and chemical properties . consequently , cds are extensively used in pharmaceutical , chemical , agricultural , cosmetic , and food industries [ 1 , 2 ] . cgtases belong to the glycoside hydrolase family 13 ( -amylase family ) . members of this family contain a catalytic ( /)8 barrel and use an -retaining mechanism but they display a variety of reaction specificities . whereas -amylases typically catalyze starch hydrolysis reaction , cgtases catalyze mainly three transglycosylation reactions ( cyclization , coupling , and disproportionation ) besides a weak hydrolytic activity . both -amylases and cgtases share three domains ( a , b , and c ) while cgtases possess two additional domains ( d and e ) . domains a and b form the catalytic core , domains c and e are involved in substrate binding whereas domain d has unknown precise function [ 6 , 7 ] . understanding the enzymatic features has been explored by cloning , sequencing , and comparing various cgtase genes [ 811 ] . the sequence data analysis has revealed the key amino acids residues that determine the reaction mechanism and product specificity , thereby enhancing genetic engineering techniques to provide modified cgtases [ 7 , 12 , 13 ] . e. coli is frequently adopted as host strain for heterologous protein expression since it is well characterised with various expression systems . however , the formation of inclusion bodies remains a significant barrier for expression of heterologous proteins in e. coli . refolding of inclusion bodies into soluble and active form needs high cost and tedious jobs . hence , maximizing the yield of soluble and active recombinant proteins in vivo by altering the culture conditions is an attractive alternative . however , no universal approach has been established to minimize the formation of inclusion bodies and some empirical conditions must be screened on an individual basis . to date , there are only a few reports that succeeded the overproduction of cgtase in e. coli by varying fermentation conditions [ 1517 ] . in a previous study , the cgtase of paenibacillus pabuli us132 was reported as good candidate for cyclodextrins production and efficient antistaling agent . in this work , we described the molecular cloning of the us132 cgtase gene as well as the amino acid sequence inspection and the comparison with other related cgtases . we also reported the production enhancement of the recombinant active enzyme in e. coli by varying mainly the cultivation conditions . paenibacillus pabuli us132 strain , previously isolated , was used as source of chromosomal dna . e. coli dh5 ( fsupe44 80 lacz m15 (laczya - argf ) u169 enda1 reca1 hsdr17 ( rk , mk ) deor thi-1 gyra96 rela1 ) was used for all plasmid constructions . e. coli er2566 ( f fhu a2 [ lon ] ompt lacz : t7 gene1 gal sula11 (mcrc - mrr)114:is10 r(mcr-73:minitn10-tets)2 r(zgb-210:tn10 ) ( tets ) enda1 [ dcm ] ) and e. coli jm109 ( ftrad36 proab laciz m15/(lac - proab e14 ( mcra ) reca1 enda1 gyra96 thi-1 hsdr17 ( rkmk ) supe44 rela1 ) were used for protein expression . the plasmids psj8 and psj9 ( this work ) carrying the cgtase us132 gene , derived from pcr2.1 ( invitrogen ) and ptrc99a vector , respectively . e. coli recombinant strains were grown on lb , m9 , 2ty , or m9zb medium containing 100 g / ml ampicillin . paenibacillus pabuli us132 strain was grown as previously described . to amplify an internal cgtase gene fragment , we used the highly conserved aa regions previously reported for cgtase enzymes as members of the -amylase family [ 4 , 21 ] . furthermore , we aligned several cgtases dna sequences and we located the corresponding cgtases conserved regions . hence , a degenerate pair of primers , s209 ( 5-gaytttgcrcccaaycat-3 ) and s211 ( 5-atcatgattgtcratraa-3 ) , corresponding respectively to the conserved regions i ( dfapnh ) and iv ( fidnhd ) , were designed . the chromosomal dna isolated from paenibacillus pabuli us132 strain was used as template for gene amplification by pfu dna polymerase ( fermentas ) . the pcr parameters were 94c for 120 seconds followed by 35 cycles of 94c for 30 seconds , 50c for 45 seconds , and 72c for 120 seconds . the pcr product was purified using wizard sv gel and pcr clean - up system ( promega ) . to amplify the entire us132 cgtase gene , we have conceived two primers , s260 ( 5-tccaatatttcttacgat-3 ) and s262 ( 5-gttacgttactccggg-3 ) , flanking the closely matching cgtase gene ( accession no . the resulting pcr fragment was cloned into the pcr2.1 vector to give psj8 plasmid harboring the us132 cgtase gene under the control of the iptg inducible t7 promoter . the psj8 was digested with hindiii and ecorv and the resultant cgtase gene was subcloned in the ptrc99a , under the control of the trc promoter ( iptg inducible ) , to give psj9 plasmid . nucleotide sequences of the us132 cgtase gene carried by three independent psj8 plasmids obtained from different pcr reactions were determined . dna sequencing was carried out using the bigdye terminator v3.1 cycle sequencing kit and the automated abi prism3100-avant genetic analyser ( applied biosystems ) . homology search was performed using blast search algorithm . for the promoter determination , the us132 cgtase sequence was submitted to the embl data bank under accession no . the heterologous expression of the us132 cgtase was investigated using different recombinant strains ( dh5/psj8 , er2566/psj8 , dh5/psj9 , er2566/psj9 or jm109/psj9 ) , various temperature cultivation ( 37c or 19c during all operating time and 37c followed by a shift to 19c after iptg induction ) and different medium composition ( lb , m9 , 2ty or m9zb ) . an overnight culture of the studied strain , cultivated at 37c , was used to inoculate 50 ml of basal medium ( in 250 ml shake flask ) with an initial od600 nm of 0.1 . protein expression was induced by addition of different iptg concentration ( 16240 g / ml ) at od600 nm between 0.8 and 1 . batch fermentation was carried out in a 7 l fermentor infors ag ch-4103 ( bottmingen , switzerland ) containing 4.5 l of 2ty medium . the fermentor was inoculated , with an initial od600 nm of 0.1 , by an overnight culture of the er2566/psj8 strain grown at 37c in 2ty broth . the ph , aeration and agitation were maintained constant at 7.4 , 1.5 vvm and 500 rpm , respectively , during all cultivation . the temperature was shifted from 37 to 19c after induction with 16 mg / l of iptg when od600 nm reached 0.81 . the cgtase extract was prepared from the periplasm by the modified osmotic - shock procedure of ausubel et al . . cells were harvested by centrifugation at 8000 g for 10 minutes and the pellets were suspended in 30 mm tris - hcl buffer ( ph 8) containing 20% sucrose and 1 mm edta . after agitation for 10 minutes at 25c , cells removed by centrifugation were rapidly suspended in ice - cold water , incubated for 10 minutes at 0c and clarified by centrifugation at 12000 g for 30 minutes . residual cells were sonicated at 4c for 6 minutes ( pulsations 3 s , amplify 90 ) using a vibra - cell 72405 sonicator and insoluble fraction ( cells debris ) was recuperated by centrifugation at 12000 g for 30 minutes . for the purification of the recombinant us132 cgtase , the er2566/psj8 periplasmic fraction was heat - treated at 60c for 15 minutes in presence of 10 mm calcium followed by centrifugation at 16000 g for 30 minutes at 4c . the supernatant was then purified using hydrophobic interaction chromatography and starch adsorption as previously described for the native enzyme . the cgtase activity , determined as dextrinisation activity , was monitored at 60c for 10 min as described in a previous work . the kinetic parameters ( km and vmax ) were determined by incubating the purified cgtase at 60c in 50 mm sodium acetate buffer ( ph 6.5 ) and using various concentrations of soluble starch ranging from 0.4 to 1.6 samples were taken at regular time intervals and the reaction was stopped by boiling for 5 minutes . the initial speed was calculated for each substrate concentration and then represented according to the lineweaver - burk method . the km and vmax were determined graphically using hyper32 program ( http://homepage.ntlworld.com/john.easterby/hyper32.html ) . to clone the us132 cgtase gene , an internal pcr fragment of about 600 bp was firstly amplified using us132 genomic dna and two primers corresponding to the conserved regions i and iv in cgtases as described in materials and methods section . the nucleotide sequence of this internal fragment ( 582 bp ) showed the highest homology ( 95% identity ) with homologous region of the putative bacillus q cgtase gene ( accession no . this result incited us to conceive two primers from the 5 and 3 flanking regions of the bacillus q cgtase gene in order to amplify the entire us132 cgtase gene using the chromosomal dna of p. pabuli us132 as template . the pcr amplification , using pfu dna polymerase , gave a nucleotide fragment of approximately 2400 bp . the cloning of this fragment in pcr2.1 vector , under the control of the t7 promoter , provided psj8 plasmid and conferred starch degrading activity for e. coli host strains . the nucleotide sequence analysis of the fragment containing the us132 cgtase gene revealed the presence of a single open reading frame ( orf ) with two potential initiation atg codons ( figure 1 ) . a putative shine - dalgarno site ( 5-agaagggtgg-3 ) , exhibiting a good rationally complementarity with the 3 end of the us132 16s rrna gene , was 7 bases upstream the first atg codon . thus , the us132 cgtase orf , consisted of 2157 bp , encoded a protein having 718 amino acids . by using the promoter prediction program , a potential 10 region ( 5-tctgca-3 ) and 35 region ( 5-ttggcg-3 ) the first 34 amino acids are considered as signal peptide according to the signalp server ( http:/www.cbs.dtu.dk / services / signalp ) . therefore , the mature enzyme was consisted of 684 amino acids with an estimated molecular mass of about 74.3 kda . the alignment of the mature us132 cgtase sequence to the data bank showed 93 , 92 , 74 , and 73% identity with the cgtases of bacillus sp . ( accession no . the highest homology ( 97% similarity and 94% identity ) was found with the cgtase of bacillus circulans strain no . 8 ( accession no . the alignment showed a difference of 39/684 amino acids which does not radically affect the product specificity . indeed , both enzymes are specific for the -cd production and are classified as -cgtases since they generated a mixture of cds composed of - , - , and -cd 20 : 58 : 13 and 10 : 64 : 20 ratios for the cgtase of strain us132 and b. circulans strain no . however , as far as we know , no data was available concerning biochemical properties of the cgtase of b. circulans strain no . 8 ( ph , thermoactivity , thermostability ) allowing the comparison of these two enzymes . from the multiple sequence alignment ( figure 2 ) , the five structural domains ( a , b , c , d , and e ) [ 11 , 26 ] could be identified in the us132 cgtase . furthermore , four highly conserved regions labelled i iv and located in the catalytic domain were found . these conserved regions would constitute the active center of the enzyme since it contained the three catalytic residues asp229 , glu257 , and asp328 referring to the cgtase of b. circulans strain no . 8 the conserved amino acids in the acceptor binding site , namely lys47 , tyr89 , asn94 , phe183 , asn193 , leu194 , tyr195 , asp196 , phe259 , phe283 , and asp371 , reported as the main determinants for cyclization reaction [ 12 , 27 ] , were also identified . the purification of the us132 cgtase from the native strain ( paenibacillus pabuli ) was extremely hampered by the viscosity of the crude supernatant , probably due to the presence of polysaccharides of unknown nature ( data not shown ) . in the aim to overcome this problem and also to overproduce the us132 cgtase , we have tested the enzyme production from the initial recombinant e. coli strain ( dh5/psj8 ) . this study showed that this latter strain , cultivated in lb medium at 37c and induced by 16 g / ml iptg for 18 hours , exhibited a very low activity of about only 0.3 u / ml . for this reason , we have studied the effect of several parameters on the production enhancement of the active recombinant cgtase . using the same culture conditions ( lb medium , 37c and induction by 16 g / ml iptg for 18 hours ) , the association e. coli er2566/psj8 increased the production of the active recombinant enzyme by 3-fold ( table 1 ) since the er2566 strain contains a chromosomal iptg inducible copy of the t7 rna polymerase gene . despite this improvement , the production of the active us132 cgtase remained negligible ( 0.92 u / ml ) compared to that obtained by the native strain ( 18 u / ml ) . to further enhance this production , the gene was placed downstream the trc promoter in psj9 and the enzyme production was investigated in various strains ( dh5 , er2566 and jm109 ) . as shown in table 1 , the association e. coli er2566/psj9 gave the highest activity of only about 1 u / ml which is nearly the same with that obtained by er2566/psj8 strain . this study showed that the use of different promoters and e. coli host strains did not significantly enhance the production of the active recombinant cgtase , which is probably due to the formation of inclusion bodies often reported for e. coli heterologous expression [ 14 , 28 ] . a well - known technique to prevent aggregation of recombinant proteins in vivo consists to reduce the operating temperature [ 15 , 16 , 29 ] . therefore , in order to improve the recombinant us132 cgtase production , er2566/psj9 and er2566/psj8 strains were cultivated at different temperatures and were induced by 16 g / ml of iptg for 18 hours : ( a ) 37c during all operating time , ( b ) 37c followed by a temperature shift to 19c after iptg induction , and ( c ) 19c during all operating time . this study showed that the conditions ( b ) and ( c ) allowed the enhancement of the active enzyme production in the periplasm for the two constructions psj8 and psj9 ( table 2 ) . the highest production ( 15 u / ml ) was reached by e. coli er2566/psj8 when the culture was performed at 37c followed by temperature shift to 19c after iptg induction . sds - page analysis of periplasmic and insoluble fractions , obtained from e. coli er2566/psj8 , showed that the cultivation of the strain at 37c expressed most the us132 cgtase as insoluble inclusion bodies with a small amount of soluble protein ( figure 3 ) explaining the low activity detected . however , the decrease of the operating temperature ( 3719c or 19c ) strongly increased the expression of the us132 cgtase as periplasmic soluble form ( figure 3 ) . the enhancement of the production of soluble and active enzyme at low cultivation temperature could be explained by : ( i ) the reduction of the high rate of protein synthesis , which prevents misfolding , ( ii ) the enhancement of chaperones expression , which allows correct protein folding . based on all obtained data , we retained for further investigation the strain e. coli er2566/psj8 and a culture temperature at 37c followed by a shift to 19c after induction . the composition of growth medium could have significant metabolic effects on both cells growth and protein production . to investigate the effect of this parameter , the strain e. coli er2566/psj8 was cultivated in 2ty , m9zb , and m9 besides lb medium used in all previous experiences . the us132 cgtase production monitored after 18 hours of induction showed that the use of m9zb and 2ty medium increased the production by about 1.1-fold ( 16.5 u / ml ) and 1.3-fold ( 20 u / ml ) , respectively , in comparison to that obtained by lb broth . however , the use of m9 medium decreased the production to attain only 8 u / ml . the enhancement of the us132 cgtase production was likely correlated with cell density since the od600 nm reached about 3.7 , 4.8 , 6 , and 2 for the lb , m9zb , 2ty , and m9 medium , respectively ( figure 4 ) . moreover , this difference in the production level of the recombinant active enzyme , using different medium , could be also influenced by the growth phase at which induction took place on . otherwise , extensive attempts to improve the enzyme production using different iptg concentrations and stressing additives ( sorbitol , mannitol , and ethanol ) did not increase the us132 cgtase production ( data not shown ) as reported for other recombinant proteins [ 16 , 28 , 32 ] . the effect of postinduction time on the us132 cgtase production was examined by analysis of samples taken , from er2566/psj8 culture performed in 2ty medium , at different times after iptg addition . following the iptg induction , the evolution of both cell growth and active enzyme production ( figure 5 ) showed that the cgtase production reached its maximum ( 22 u / ml ) after 22 hours of induction when cells are at full stationary phase . when culture attained the lysis phase the production of the active enzyme decreased by about 27% probably due to the proteases release . the fermentation , using the er2566/psj8 , was scaled up in 7 l fermentor strain under the conditions previously optimised : 2ty medium and temperature shift from 37 to 19c after iptg induction . interestingly , the production of the us132 cgtase in fermentor reached a maximum ( 35 u / ml ) after only 18 hours of induction while this maximum ( 22 u / ml ) was obtained after 22 hours in erlenmeyer . it should be mentioned that the cell density obtained in the two cases was almost the same ( about od600 nm = 6 ) . this finding suggested that the improvement of the active enzyme production was not only related to the cell growth but it could also depend on the stability of fermentation parameters ( ph and po2 ) , which probably affect translation and correct folding of recombinant proteins as well as proteolysis according to makrides . for the purification of the recombinant us132 cgtase , we have used the periplasmic fraction of the er2566/psj8 strain cultivated in the fermentor in the cultivation conditions described above . this periplasmic fraction was firstly heat - treated at 60c in order to remove e. coli thermolabile proteins . the extract was then purified using hydrophobic interaction chromatography ( hic ) and starch adsorption as described for the native enzyme . the recombinant enzyme was purified to homogeneity 25-fold with a yield of 20% ( table 3 , figure 6 ) . this recombinant us132 cgtase retained the same biochemical properties ( data not shown ) as the native enzyme ( thermoactivity , thermostability , ph stability , cds production ) . the kinetic parameters were determined by incubating the purified recombinant us132 cgtase in presence of various soluble starch concentrations and using the lineweaver - burk method . the vmax and km values were evaluated to be 253 36 mol of -cyclodextrin / mg / min and 0.36 0.18 km values determined , using also soluble starch as substrate , for cgtase from bacillus agaradhaerens , bacillus circulans e192 , bacillus firmus , and bacillus sp . ts1 - 1 were 21.2 , 5.7 , 1.21 , and 0.52 g / l , respectively . since a small value of km exhibited a high affinity for the substrate , the values shown above suggested that us132 cgtase was more specific towards starch than the other reported cgtases . the molecular characterization of the gene encoding the us132 cgtase showed that the enzyme is a novel -cgtase exhibiting 95% of identity with the -cgtase from bacillus circulans strain no . the production of the active us132 cgtase by e. coli dh5 under usual culture conditions ( 37c and lb ) was very low ( 0.3 u / ml ) suggesting the formation of inclusion bodies . the investigation of different culture parameters showed that mainly the shift of the operating temperature from 37 to 19c increased the production of the active cgtase to reach 22 u / ml . this production was further enhanced to reach 35 u / ml by using batch fermentation while the native strain produced only 18 u / ml . the recombinant us132 cgtase , purified to homogeneity , shared the same biochemical properties with the native enzyme . the determination of the kinetic parameters ( vmax and km ) of this recombinant enzyme showed that the us132 cgtase had the highest affinity towards soluble starch in comparison to other reported cgtases .

the gene encoding the cyclodextrin glycosyltransferase ( cgtase ) of paenibacillus pabuli us132 , previously described as efficient antistaling agent and good candidate for cyclodextrins production , was cloned , sequenced , and expressed in escherichia coli . sequence analysis showed that the mature enzyme ( 684 amino acids ) was preceded by a signal peptide of 34 residues . the enzyme exhibited the highest identity ( 94% ) to the -cgtase of bacillus circulans no . 8 . the production of the recombinant cgtase , as active form , was very low ( about 1 u / ml ) in shake flasks at 37c . this production reached 22 u / ml after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19c and using 2ty instead of lb medium . high enzyme production ( 35 u / ml ) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask . the recombinant enzyme showed vmax and km values of 253 36 mol of -cyclodextrin / mg / min and 0.36 0.18 g / l , respectively .

typical clinical presentations include nausea , vomiting , diarrhea , abdominal pain , weight loss , malnutrition , and peripheral edema due to hypoalbuminemia . md usually shows an insidious onset with progressive features and is associated with an increased risk of gastric cancer . these variants are associated with cytomegalovirus ( cmv ) or helicobacter pylori infection [ 4 - 6 ] . cmv - associated cases usually occur in children ; however , cases in adults have also been reported [ 7 - 10 ] . md has also been reported in patients with autoimmune diseases such as inflammatory bowel disease , sclerosing cholangitis , and ankylosing spondylitis , suggesting an immunological component to its pathogenesis . endoscopically , the markedly thickened gastric mucosal folds resemble cerebral convolutions and primarily affect the body and fundus but spare the antrum ( fig . gastric ph is increased due to loss of parietal cells , and copious thick mucus production is seen secondary to foveolar hyperplasia . foveolar hyperplasia that often results in mucosal thickness 1 cm or greater is the most striking feature of md . the foveolar epithelium shows a corkscrew morphology due to massive foveolar hyperplasia , but the overall linear architecture is maintained ( fig . oxyntic glands are atrophic with reduced or absent parietal cells , and deep glands can be cystically dilated . the lamina propria shows a variable amount of predominantly chronic inflammatory cell infiltration with scattered eosinophils . prominent vertical strands of smooth muscle in the lamina propria are also identifiable ( fig . 2d ) . these conditions range from foveolar hyperplasia in reactive conditions to malignancy ( table 1 ) . because some of these diseases share histologic characteristics with md , correlation of microscopic findings with endoscopic and clinical features is important in order to establish the correct diagnosis . because accurate diagnosis of some of these diseases requires examination of very thick gastric mucosa , large snare biopsies that capture the entire thickness of the mucosa disease entities with diffuse hypertrophic gastropathy include hypertrophic lymphocytic gastritis , hypertrophic hypersecretory gastropathy , and zollinger - ellison syndrome ( zes ) . hypertrophic lymphocytic gastritis often presents as giant fundic mucosal folds with relative sparing of the antrum similar to md . in contrast to md , the gastric mucosa in this condition shows diffuse and severe inflammation with prominent intraepithelial lymphocytes . hypertrophic hypersecretory gastropathy is a rare acquired gastropathy that involves hypersecretion of acid , pepsin , and mucin . endoscopically , it is characterized by hypertrophic gastric folds and cobblestone gastric body mucosa with atrophic antral mucosa . histologically , it is differentiated from md in that hyperplasia is seen in both the foveolar epithelium and oxyntic glands . gastric glands with cystic dilatation zes is characterized by ectopic gastrin secretion ( gastrinoma ) , increased gastric acid secretion , and intractable peptic ulcer disease , and it shows diffusely thickened gastric folds , especially in the body and fundus . nodular and linear enterochromaffin - like cell hyperplasia can frequently be observed in the disease and is associated with multiple endocrine neoplasia type 1 . gastric polyps can manifest as focal hypertrophic gastropathy and can be numerous and diffusely distributed in polyposis syndromes , resulting in a hypertrophic appearance . gastric hyperplastic polyps are the most common , accounting for 70% of gastric epithelial polyps . the antrum is the most common site for hyperplastic polyps ; however , they can develop in other areas of the stomach and can be solitary or multiple . hyperplastic polyps usually develop in the background of other gastric pathology such as atrophic gastritis , reactive gastropathy , or any form of acute or chronic gastritis . juvenile polyposis syndrome ( jps ) is characterized by hamartomatous polyps in the gastrointestinal tract , most commonly in the colon . most colonic juvenile polyps are sporadic , but germline mutations in either bmpr1a or smad4 genes have been detected in 50% of cases of jps , which is inherited in an autosomal dominant pattern . a severe gastric phenotype has been shown in smad4 mutations , particularly nonsense mutations in exon 11 and a deletion of four base pairs in exon 9 at codon 415 . histologically , gastric polyps in jps are similar to other hyperplastic or hamartomatous polyps and are characterized by foveolar hyperplasia . compared to md , gastric juvenile polyps show distorted glandular architecture , edematous stroma , lower gland to stroma ratio , and less conspicuous eosinophils and smooth muscle fibers in the lamina propria ( table 2 , fig . 2b , e ) . among 48 patients referred to vanderbilt university medical center with a diagnosis of md for a clinical trial of cetuximab conducted in individuals with advanced md , 25 ( 52% ) were confirmed with the diagnosis of md . the most common entities mistaken for md were gastric hyperplastic polyps or jps , found in seven of the total 23 non - md patients ( 30% ) . gastric polyps in syndromes with both gastrointestinal and extra - intestinal manifestations can also mimic md ; therefore , clinical information is essential for a correct diagnosis . cronkhite - canada syndrome ( ccs ) is a rare nonhereditary syndrome characterized by diffuse polyposis in the gastrointestinal tract , alopecia , cutaneous hyperpigmentation , and onychodystrophy . mixed inflammation in the lamina propria with prominent eosinophils and eosinophilic infiltration of glandular epithelium with crypt abscesses is a helpful upper gastrointestinal feature of ccs . peutz - jeghers syndrome ( pjs ) is characterized by hamartomatous gastrointestinal polyps with increased skin and mucosal pigmentation . pjs is associated with mutations in the serine / threonine kinase 11 ( stk11 ) gene and shows an autosomal dominant inheritance pattern . pjs polyps are usually characterized by hyperplastic epithelium divided by arborizing bands of smooth muscle . however , pjs polyps in the stomach show dilated hyperplastic glands and less prominent smooth muscle strands compared to pjs polyps in other gastrointestinal tract ( fig . cowden syndrome is characterized by numerous hamartomas in the gastrointestinal tract and extraintestinal sites such as skin , breast , thyroid , gynecologic , oral cavity , and central nervous system . mutations in the phosphatase and tensin homolog ( pten ) gene are detected in up to 80% of cowden syndrome cases and show an autosomal dominant inheritance pattern . a diffuse distribution of white mucosal plaques in the esophagus is a characteristic endoscopic finding , which microscopically shows thickened squamous epithelium with glycogen accumulation in the cytoplasm ( glycogenic acanthosis ) . gastric adenocarcinoma and proximal polyposis of the stomach is a recently described entity with an autosomal dominant inheritance with incomplete penetrance . numerous gastric polyps carpeting the proximal stomach are observed endoscopically with sparing of the esophagus , antrum , duodenum , and colon . these polyps histologically overlap with fundic gland polyps with multiple foci of low- and high - grade dysplasia . this syndrome is associated with a high risk of intestinal - type gastric adenocarcinoma , and 41 cases in five families have been reported to date . other entities that can mimic hypertrophic gastropathy include diffuse gastric carcinoma , gastric lymphoma , and amyloidosis . the pathophysiology of md has not been fully elucidated . however , the observation that transgenic mice overexpressing transforming growth factor alpha ( tgf- ) in the stomach develop gastric changes that resemble md and increased tgf- expression in the gastric mucosa of md patients suggest an important role of this growth factor in its pathogenesis [ 24 - 31 ] . tgf- is one of seven mammalian ligands that bind epidermal growth factor receptor ( egfr ) . egfr is a receptor tyrosine kinase that mediates signal transduction in many cell types . a common action of tgf- is increased cellular proliferation , and elevated tgf- expression has been associated with neoplastic transformation . transgenic mouse models that overexpress tgf- showed epithelial hyperplasia of liver , pancreas , stomach , small intestine , colon , mammary glands , and coagulation glands . acinoductular metaplasia was also found in the pancreas , and neoplasia was identified in the mammary glands and liver . interestingly , the stomachs of mice that overexpressed tgf- showed characteristic md features , such as foveolar hyperplasia , glandular cystic dilatation , oxyntic atrophy , increased mucin production , and reduced acid secretion . moreover , it has been shown that tgf- expression in both rna and protein is increased in the gastric mucosa of md patients . functionally , tgf- overexpression appears to direct gastric stem / progenitor cells to surface mucous cell differentiation at the expense of parietal and chief cell differentiation [ 35 - 37 ] . additionally , tgf- overexpression repatterns gastric fundic - type epithelium into antral - type epithelium , as evidenced by aberrant expression of the gastric antral markers , pdx1 and gastrin , in the gastric body of both tgf- transgenic mice and md patients . involvement of egfr signaling in the pathogenesis of md is further supported by a cmv - associated variant of md . it has been shown that the cmv principal envelope glycoprotein , gb , binds egfr , induces egfr - her3 hetero - oligomers , and activates egfr signaling . in addition , disruption of the bone morphogenetic protein / tgf- signaling pathway in mice can lead to increased levels of tgf- and amphiregulin , another egfr ligand [ 40 - 42 ] . we also found that phosphorylated egfr is increased in the involved gastric mucosa of jps patients ( fig . 3 ) . these findings could contribute to the histolopathologic similarities between gastric jps and md . it is advised to rule out spontaneous remission variants associated with cmv or helicobacter pylori infection by appropriate clinical testing . reports on efficacy of steroids , anticholinergics , acid suppression , and octreotide are inconsistent . partial or total gastrectomy is reserved for patients with intractable or debilitating disease and for cases with a high risk of developing gastric cancer . the link to tgf- and egfr signaling in the pathogenesis of md led us to design a clinical trial using a neutralizing monoclonal antibody ( cetuximab ) against egfr for md patients . all seven patients who completed a month of cetuximab treatment showed statistically significant improvement in quality - of - life indices , increased parietal cell mass and gastric acidity . in addition , four patients showed near - complete histological resolution . to our surprise , improvement in symptoms usually started within one to two days after the first cetuximab infusion , and parietal cell mass was significantly increased within 24 hours . therefore , based on the results of this study , cetuximab should be considered a therapeutic option for md patients .

mntrier s disease is a rare protein - losing hypertrophic gastropathy . histologically , it can be mistaken for other disorders showing hypertrophic gastropathy . the pathogenesis of mntrier s disease is not fully understood ; however , it appears that the epidermal growth factor receptor ( egfr ) ligand , transforming growth factor alpha , contributes to the pathogenesis of this disorder . in this review , we will discuss disease entities that can mimic mntrier s disease and the role of egfr signaling in mntrier s disease .

daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of complicated skin and skin structure infections , as well as for staphylococcus aureus bloodstream infections , including those occurring with right - sided infective endocarditis [ 1 , 2 ] . data analyses from the eu - core registry consisting of 6075 patients indicate that daptomycin is relatively safe and well tolerated , even at high doses ( > 6 mg / kg / day ) . serious adverse events possibly related to daptomycin occurred in 2% of patients , but hypersensitivity reactions were rare . these registry data are important , in particular when facing a previously undescribed adverse event to a compound . here a 60-year - old woman was admitted to our hospital because of immobilizing lumbar back pain and elevated inflammatory markers in blood tests . spine infection was suspected , and after obtaining blood cultures , the patient was treated empirical therapy with ceftriaxone ( 2 g once daily ) . antimicrobial treatment was switched to amoxicillin / clavulanate ( 2.2 g every 4 h on days 3 and 4 of hospitalization ) , and then streamlined to flucloxacillin ( 2 g every 4 h ) after identification of methicillin - susceptible s. aureus . despite 2 mri scans , a pet - ct scan , an anti - granulocyte scintigraphy and several transesophageal echocardiographies , the source of bacteremia was not identified . on day 6 , blood cultures were still positive for s. aureus . thus , persistent s. aureus bacteremia without identified source was postulated . in parallel , acute kidney injury developed . because of persistent bacteremia and renal impairment , antimicrobial treatment was switched to a combination therapy with daptomycin ( 10 mg / kg body weight ) and ceftriaxone ( 2 g once daily ) . shortly after completion of the first daptomycin administration ( 60 min infusion ) , the patient developed lip and tongue swelling and dyspnea . after adrenaline inhalation and intravenous administration of clemastine and methylprednisolone , symptoms and findings resolved . no skin eruption or diffuse lymphadenopathy was noted in clinical examinations , and results of laboratory investigations showed no elevated eosinophil counts , atypical lymphocytosis or increased serum alanine aminotransferase . thus , the differential diagnosis of drug reaction with eosinophilia and systemic symptoms ( dress ) appeared unlikely , and a diagnosis of acute angioedema was made . doses were adapted according to the gradually recovering renal function , and monitored by vancomycin trough levels in serum . blood cultures showed no growth on day 9 and results remained negative on day 12 . the workup of the case included investigations into the cause of angioedema and renal failure . the timely occurrence of the latter and the histopathological characteristics of the kidney biopsy result were consistent with acute interstitial nephritis . high - dose beta - lactam exposure during the first 46 days of hospitalization was considered the most likely cause . after intermittent hemodialysis ( days 1019 ) and corticosteroid treatment , renal function values returned to normal on the day of discharge ( day 32 ) . a detailed review of the drug charts strongly suggested a timely association between angioedema and daptomycin . the role of other compounds ( nonsteroidal anti - inflammatory drugs , opioids ) in causing direct histamine release or immunoglobulin e ( ige)-mediated reaction was also reviewed . these compounds were repeatedly challenged without an obvious clinical reaction . because no similar case was previously described in the literature , the differential diagnosis included a more commonly known reaction to ceftriaxone , although exposure to ceftriaxone in the preceding week spoke against such a reaction . also , the workup of the patient s history revealed that she was exposed to 1st and 2nd generation cephalosporins in the previous years without any notable reaction . daptomycin re - exposure was considered in the intensive care unit ( icu ) . an intradermal test ( idt ) the results were positive for daptomycin ( 5 mg / ml , wheal diameter 5 mm after 20 min ) , but negative for ceftriaxone ( 2 mg / ml ) . acute angioedema shortly after daptomycin infusion and the positive idt result suggested an immediate hypersensitivity reaction to daptomycin . to the best of our knowledge , this is the first report of angioedema after daptomycin exposure . a literature review identified only three published cases of immediate - type hypersensitivity reactions to daptomycin . bagwell et al . reported a 24-year - old woman with multiple antibiotic allergies . she had experienced hypersensitivity reactions to daptomycin previously , and after re - exposure , pruritus and shortness of breath developed . metz and thyagarajan described a 33-year - old man who did not tolerate vancomycin because of a diffuse urticarial eruption . after the third daptomycin administration , he developed a generalized urticarial rash ( no lip or tongue swelling or shortness of breath ) . his symptoms improved with diphenhydramine , but the urticarial rash returned after each subsequent dose . on the basis of the reported patient history , it is unclear whether this reaction was ige mediated or nonspecific ( e.g. , mast cell activation ) . the third case illustrates desensitization to ceftaroline in a 32-year - old woman with multiple allergies , including anaphylaxis to daptomycin ( not further specified ) . in contrast to these cases , our patient had no known allergy , and angioedema developed acutely after the first dose . daptomycin was first approved by the us food and drug administration ( fda ) in september 2003 . after the hallmark study on daptomycin for s. aureus bacteremia and endocarditis was published in 2006 , numerous safety data on this agent became available [ 3 , 1013 ] . in addition to known adverse events ( e.g. , myotoxicity ) , an association of daptomycin with eosinophilic pneumonia has been raised through an fda drug safety communication . moreover , rare clinical manifestations such as acute generalized exanthematous pustulosis and drug rash with eosinophilia and systemic symptoms have been reported . anaphylaxis and angioedema are labeled as reactions of unknown frequency and causality [ 1 , 2 ] . worldwide , 72 immediate - type hypersensitivity reactions to daptomycin have been reported to who pharmacovigilance centers . in 20 of these cases , the reaction occurred on the first day of daptomycin treatment . in the present case , the more frequently reported association of beta - lactams with hypersensitivity reactions and the lack of a similar case report associated with daptomycin were misleading . when performing an idt , a procedure with sequent dilutions we performed the idt in the icu with a 10 dilution of the original vial concentration ( 50 mg / ml ) and all necessary precautions were taken in case emergency treatment was needed . as done in our investigations , healthy volunteers with or without previous exposure to the drug can be used as negative controls to evaluate nonspecific skin reaction . although one healthy control may be insufficient to draw firm conclusions , the idt result and a negative control was a helpful tool together with the clinical observation and detailed time analyses of drug application in pointing towards a daptomycin - associated hypersensitivity reaction . because of the clinical circumstances , no in vitro testing with specific ige level measurements to daptomycin could be performed , and no tryptase was measured . although , in our view , ige - mediated reaction is likely , it can not be ascertained . nevertheless , clinicians should be aware that daptomycin can cause immediate - type hypersensitivity reactions . our case report indicates that acute angioedema can be triggered by daptomycin , even upon first administration . v. gisler , s. mller , l. mller , l. jrg - walther and p. sendi have nothing to disclose . this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made .

introductiondaptomycin is a cyclic lipopeptide antibiotic , frequently administered for staphylococcus aureus bloodstream infections . numerous studies have shown that daptomycin is relatively safe and well tolerated . serious adverse events possibly related to this antimicrobial compound are rare . we report a case of acute angioedema triggered by daptomycin.case reporta 60-year - old woman with s. aureus bacteremia without identified source was treated intravenously with high - dose beta - lactams at our institution . because s. aureus bacteremia persisted on day 6 , and in parallel , acute kidney injury developed , antimicrobial treatment was switched to a combination therapy with daptomycin and ceftriaxone . shortly after completion of the first daptomycin administration , the patient developed lip and tongue swelling and dyspnea . acute angioedema was clinically evident . antibiotic therapy was switched to vancomycin , and the further clinical course was favorable . an intradermal test showed a significant wheal diameter for daptomycin , but negative results for ceftriaxone.conclusionthe association with daptomycin in this case is either probable or certain . clinicians should be aware that daptomycin can cause immediate - type hypersensitivity reactions , including acute angioedema , even upon first administration .

the evolution of the amniotic egg was one of the great evolutionary innovations in the history of life , freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments1 . among amniotes , genome sequences are available for mammals2 and birds35 , but not for non - avian reptiles . here we report the genome sequence of the north american green anole lizard , anolis carolinensis . we find that a. carolinensis microchromosomes are highly syntenic with chicken microchromosomes , yet do not exhibit the high gc and low repeat content that are characteristic of avian microchromosomes3 . also , a. carolinensis mobile elements are very young and diverse more so than in any other sequenced amniote genome . this lizard genome s gc content is also unusual in its homogeneity , unlike the regionally variable gc content found in mammals and birds6 . we describe and assign sequence to the previously unknown a. carolinensis x chromosome . comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins . an anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations .

the study design is cross - sectional and includes adult diabetic patients and control subjects ( aged 3070 years ) from five european cities ( london , belfast , lyon , barcelona , and rome ) examined between 2004 and 2007 . all patients came from five european hospital - based centers involved in action lada , a european union funded multicenter european study with the aim of identifying immune and clinical risk factors for adult - onset autoimmune diabetes ( http://www.actionlada.org ) . diabetes was designated according to standard criteria , and lada was defined as patients aged 3070 years with gadas who did not require insulin treatment for at least 6 months after diagnosis ( 7,8 ) . type 1 diabetic patients and normal subjects fulfilling the inclusion criteria were ascertained consecutively from three of these five european centers ( london , barcelona , and rome ) . inclusion criteria were diagnosis of diabetes ( with at least two fasting blood glucose measurements 7 mmol / l ) , time from diagnosis < 5 years for all non insulin - requiring diabetic patients , and age 3070 years at examination . patients came from europe but with different ethnicity ( 91.7% caucasian , 4.6% middle eastern , 1.4% asian , 1.3% african , and 1.0% mixed race ) . control subjects were individuals from local communities attending primary care centers for routine examination with age range and sex ratio similar to those of the patients and were all caucasian . exclusion criteria for both patients and control subjects were incomplete data set , current pregnancy , renal disease with a raised creatinine level or proteinuria , or acute illness at the time of testing ; in addition , patients with non insulin - requiring diabetes with duration of > 5 years and control subjects with any clinical disease , therapy , or family history of autoimmune disease were excluded . data on medication and risk factors were registered by the attending physician on the basis of the medical files . serum and plasma samples were collected according to standard procedures and were stored at 80c . lipids and lipoproteins ( serum total and hdl cholesterol and triglycerides ) were determined by standardized assays at each center . all initially non insulin - requiring patients were tested for gada in a central laboratory ( london ) as part of the european union action lada program . metabolic syndrome was assessed according to the ncep criteria ( 6 ) as follows : waist circumference > 102 cm in men and > 88 cm in women , triglycerides > 1.70 mmol / l , hdl cholesterol < 1.00 mmol / l in men and < 1.30 mmol / l in women , blood pressure 130/85 mmhg or taking antihypertensive medication , and fasting glucose 6.1 mmol / l . we chose to identify all diabetic patients in this study as fulfilling the criteria for hyperglycemia . three of five criteria were required for the diagnosis of metabolic syndrome . because raised glucose is one criterion for metabolic syndrome and all diabetic patients , by definition , the radioimmunoprecipitation assay for gadas uses human islet gad65 cdna with in vitro transcription and translation systems as described previously ( 12 ) . all samples were centrally tested in london in duplicate , including positive and negative control standard sera . each assay for gada included in - house standard serum from a pre - diabetic individual serially diluted to an end point equivalent at 70 world health organization ( who ) units above which samples were scored positive . a separate positive serum sample ( equivalent to the who standard of 200 who iu ) was used as an in - house control to standardize each assay for unit calculation . in the diabetes antibody standardization program antibody workshop our assay had a sensitivity of 74% and specificity of 98% for gadas ( 13 ) . in the latest diabetes antibody standardization program workshop ( 2007 ) our assay had a sensitivity of 80% and specificity of 98% for gadas ( m.i.h . positive samples were retested to confirm gada positivity and reduce the false - positive rate . to compare the prevalence of metabolic syndrome with respect to the gada titer in patients with lada , we sought to divide them into two subgroups using a q - q plot of gada - positive patients . the differences between groups were analyzed with a test or fisher 's exact test when appropriate . quantitative variables were analyzed with a general linear model univariate , and a post hoc analysis was performed with a bonferroni test ; data are presented as means sd . a logistic regression analysis was performed to evaluate confounding by covariables , with adjustment for sex , age of onset , disease duration , and ethnicity to calculate the odds ratio ( or ) for metabolic syndrome ; three dummy variables were created to include three groups of patients with control subjects as a reference group . values for gada levels , triglycerides , and hdl cholesterol were log - transformed to normalize distributions . all analyses were performed using spss for windows ( spss , chicago , il ) . q - q probability plots were used to analyze the distribution of gada measurements for normality . observed antibody values were plotted along the horizontal axis against expected normal values under normality on the vertical axis using blom 's proportion estimation formula . the study protocol is in accordance with the declaration of helsinki and was approved by local ethics committees in each study center . metabolic syndrome was assessed according to the ncep criteria ( 6 ) as follows : waist circumference > 102 cm in men and > 88 cm in women , triglycerides > 1.70 mmol / l , hdl cholesterol < 1.00 mmol / l in men and < 1.30 mmol / l in women , blood pressure 130/85 mmhg or taking antihypertensive medication , and fasting glucose 6.1 mmol / l . we chose to identify all diabetic patients in this study as fulfilling the criteria for hyperglycemia . three of five criteria were required for the diagnosis of metabolic syndrome . because raised glucose is one criterion for metabolic syndrome and all diabetic patients , by definition , the radioimmunoprecipitation assay for gadas uses human islet gad65 cdna with in vitro transcription and translation systems as described previously ( 12 ) . all samples were centrally tested in london in duplicate , including positive and negative control standard sera . each assay for gada included in - house standard serum from a pre - diabetic individual serially diluted to an end point equivalent at 70 world health organization ( who ) units above which samples were scored positive . a separate positive serum sample ( equivalent to the who standard of 200 who iu ) was used as an in - house control to standardize each assay for unit calculation . in the diabetes antibody standardization program antibody workshop our assay had a sensitivity of 74% and specificity of 98% for gadas ( 13 ) . in the latest diabetes antibody standardization program workshop ( 2007 ) our assay had a sensitivity of 80% and specificity of 98% for gadas ( m.i.h . positive samples were retested to confirm gada positivity and reduce the false - positive rate . to compare the prevalence of metabolic syndrome with respect to the gada titer in patients with lada , we sought to divide them into two subgroups using a q - q plot of gada - positive patients . the differences between groups were analyzed with a test or fisher 's exact test when appropriate . quantitative variables were analyzed with a general linear model univariate , and a post hoc analysis was performed with a bonferroni test ; data are presented as means sd . a logistic regression analysis was performed to evaluate confounding by covariables , with adjustment for sex , age of onset , disease duration , and ethnicity to calculate the odds ratio ( or ) for metabolic syndrome ; three dummy variables were created to include three groups of patients with control subjects as a reference group . values for gada levels , triglycerides , and hdl cholesterol were log - transformed to normalize distributions . all analyses were performed using spss for windows ( spss , chicago , il ) . q - q probability plots were used to analyze the distribution of gada measurements for normality . observed antibody values were plotted along the horizontal axis against expected normal values under normality on the vertical axis using blom 's proportion estimation formula . the study protocol is in accordance with the declaration of helsinki and was approved by local ethics committees in each study center . of the 2,011 subjects studied , for diabetic patients ( n = 1,652 ) , mean sd age was 52.7 10.3 years , duration of diabetes was 5.2 6.7 years , and 51.8% were men . for control subjects ( n = 359 ) , of note , age was significantly lower in patients with type 1 diabetes ( 43.8 9.8 years ) than in those with lada ( 49.7 10.4 years ) ( p < 0.0001 ) and in both was lower than for type 2 diabetic patients ( 55.1 10.1 years ) ( p < 0.0001 ) . the prevalence of metabolic syndrome , including hyperglycemia as a component , was 75.5% in all diabetic patients and 26.5% in control subjects ( p < 0.0001 ) . metabolic syndrome was detected in significantly more patients with type 1 diabetes ( 31.9% ) and lada ( 41.9% ) than in control subjects ( p = 0.006 ) . referring to control subjects , the ors ( 95% ci ) for metabolic syndrome are as follows : for type 2 diabetes 22.5 ( 1533.7 ) ( p < 0.0001 ) ; for lada 2.2 ( 1.23.6 ) ( p < 0.004 ) ; and for type 1 diabetes 1.1 ( 0.71.9 ) ( p = 0.6 ) . there was a significant difference in the or between type 1 diabetes and lada for metabolic syndrome , even after correction for age , duration of disease , sex , and ethnicity : 3.2 ( 1.28.3 ) ( p = 0.015 ) . the prevalence of metabolic syndrome was higher in type 2 diabetic patients ( 88.8% ) than in patients with either lada ( 41.9% ) or type 1 diabetes ( 31.6% ) ( p < 0.0001 for both comparisons ) . no differences were seen in sex for metabolic syndrome ( 50% men vs. 50% women ) . the risk of metabolic syndrome increases with age : 1.05 ( 1.041.06 ) per year ( p < 0.0001 ) . when glucose was excluded as a variable , metabolic syndrome was not more prevalent in patients with autoimmune diabetes ( type 1 diabetes and lada ) ( 17.3% ) than in control subjects ( 23.7% ) but remained more prevalent in type 2 diabetic patients ( 47.8% ) ( p = 0.001 for all groups ) . metabolic syndrome was less prevalent in patients with type 1 diabetes ( 16.9% ) than in those with lada ( 25.0% ) or control subjects ( 23.7% ) ( p = 0.04 ) , and the prevalence was similar in the two latter groups ( p = 0.7 ) . for control subjects the ors ( 95% ci ) were as follows : for type 2 diabetes 2.4 ( 1.63.6 ) ( p < 0.0001 ) ; for lada 0.9 ( 0.501.60 ) ( p = 0.69 ) ; and for type 1 diabetes 1.1 ( 0.391.12 ) ( p = 0.60 ) . the frequency of metabolic syndrome for all groups was still similar in men ( 53.1% ) and women ( 46.9% ) ( p = 0.45 ) . the risk of metabolic syndrome still increased with age : 1.02 ( 1.011.03 ) per year ( p < 0.0001 ) . it follows that factors associated with metabolic syndrome include diabetes and older age and , when glucose is excluded as a trait , type 2 diabetes and older age . of the individual components of the metabolic syndrome , after adjustment for age of onset , duration of disease , sex , and ethnicity , waist circumference was similar in patients with type 1 diabetes and lada ( p = 0.44 ) but was lower in each of these groups than in patients with type 2 diabetes ( p < 0.0001 for both comparisons ) , systolic and diastolic blood pressures were similar in patients with type 1 diabetes and lada ( p = 0.28 and p = 0.49 , respectively ) but was lower in each of these groups than in patients with type 2 diabetes ( p < 0.0001 ) , triglycerides were similar in patients with type 1 diabetes and lada ( p = 0.63 ) but lower in each of these groups than in patients with type 2 diabetes ( p < 0.0001 ) , and hdl cholesterol was similar in patients with type 1 diabetes and lada ( p = 0.40 ) and in both groups was higher than in patients with type 2 diabetes ( p < 0.0001 ) . waist circumference in the combined diabetes groups ( n = 1,652 ) was directly associated with age at sampling ( r = 0.28 ; p < 0.0001 ) and inversely related with disease duration ( r = 0.28 ; p < 0.0001 ) . of the individual components , increases in waist circumference ( r = 0.16 ; p < 0.0001 ) and systolic blood pressure ( r = 0.29 ; p < 0.0001 ) and elevated triglycerides ( r = 0.13 ; p < 0.0001 ) but not diastolic blood pressure ( r = 0.03 ; p = 0.26 ) were more common with increasing age , whereas hdl cholesterol ( r = 0.09 ; p < 0.008 ) decreased with age . a q - q plot of gada - positive patients was performed to seek distinct populations , and we identified an inflection point corresponding to 200 who iu consistent with two modes ( data not shown ) . further , a plot of the gada titer according to patient frequency revealed a possible bimodality , and the lowest value between the two modes was at a gada titer of 200 who iu ( data not shown ) . therefore , we arbitrarily analyzed the metabolic syndrome according to gada positivity in those with a gada titer of > 200 or < 200 who iu and found that it was similar in lada patients with high ( > 200 iu ) ( n = 39 ) and low ( 70200 iu ) ( n = 78 ) gada titers ( 47.3 and 40.3% , respectively ) even after correction for age , sex , duration of disease , and ethnicity ( p = 0.37 , 95% ci 0.291.6 ) . the frequency of features of metabolic syndrome formed a hierarchy that was similar in all the groups , irrespective of the presence or type of diabetes , such that high blood pressure > elevated waist circumference > high triglycerides > low hdl cholesterol . among those patients with the metabolic syndrome , the cluster of hyperglycemia , increased waist circumference , and high blood pressure was seen in 62.1% of those with the metabolic syndrome , whereas hyperglycemia , hypertension , high triglycerides , and low hdl cholesterol were seen in 17.9% . these observations indicate that the metabolic syndrome is a frequent finding in autoimmune diabetes but is not more frequent in autoimmune diabetes than in normal subjects when glucose is excluded as a risk factor . in contrast , metabolic syndrome is far more prevalent in type 2 diabetes , even when glucose is excluded as a variable . whether glucose was or was not used as a variable , we found that individual components of the metabolic syndrome in both patients with type 1 diabetes and those with lada were similar but in each group were less than those seen in type 2 diabetic patients . it follows that there is no evidence from this data set that autoimmune diabetes is distinct in terms of prevalence of metabolic syndrome from that in normal subjects and the hypothesis that it would be distinct is rejected . nevertheless , metabolic syndrome was more prevalent in patients with lada than in those with type 1 diabetes when glucose was included as a variable . glucose is a debatable component of the metabolic syndrome and was introduced for assessing type 2 diabetes and not type 1 autoimmune diabetes ( 14 ) . when glucose was excluded , the prevalence of metabolic syndrome in patients with autoimmune diabetes was not greater than that in normal subjects . therefore , there is no evidence to suggest that autoimmune diabetes is due to decreased insulin sensitivity ; instead , decreased insulin sensitivity might predispose to an earlier time of diagnosis . such decreased insulin sensitivity could explain why age , overweight , and physical inactivity are as strongly predictive of lada as they are of type 2 diabetes ( 15,16 ) . these present observations indicate that metabolic syndrome is as common in adults with autoimmune diabetes as it is in normal subjects . thus , agents such as metformin and other insulin sensitizers may be beneficial in autoimmune diabetes , as they are in type 2 diabetes , in line with recent studies indicating that metformin is efficacious in type 1 diabetes ( 17 ) . of the three current criteria for metabolic syndrome ( idf , ncep , and who ) , we used the ncep criteria because they are more appropriate to apply here to populations of variable geographical origin rather than the idf criteria , for simplicity , and the who criteria , which include insulin resistance and microalbuminuria ( 6 ) . although it is possible that metabolic syndrome should be defined differently in autoimmune diabetes , including type 1 diabetes and lada , the hierarchy of features associated with metabolic syndrome was similar to that found in type 2 diabetic patients and control subjects . nevertheless , the metabolic syndrome was not originally introduced to identify a feature of autoimmune diabetes but to capture the clustering of a group of continuous variables associated with cardiovascular risk . that cluster has subsequently been extended to include measures of endothelial dysfunction and low - grade inflammation , but it remains unclear whether these new parameters are also features of autoimmune diabetes ; for example , type 2 diabetes is associated with increased levels of proinflammatory serum cytokines and acute - phase proteins , especially in association with obesity , whereas in type 1 diabetes such inflammatory changes are mild or nonexistent ( 18 ) . patients with lada have not been consistently found to exhibit the systemic low - grade inflammation previously identified in type 2 diabetic patients ( 18 ) . thus , it is reasonable to conclude that autoimmune diabetes , whether type 1 diabetes or lada , differs from type 2 diabetes with respect to systemic low - grade inflammation , as it does with metabolic syndrome . previous studies of metabolic syndrome in lada have confirmed that , although it is prevalent , it is less prevalent than in type 2 diabetes ( 19,20 ) . there are only two previous studies comparing lada with type 1 diabetes , each suggesting a tendency for lada to have more features of the metabolic syndrome , but control subjects were not included ; each study was very small with a combined total of 94 patients with lada and 112 type 1 diabetic patients , and one of them used highly selected patients with lada ( 9,21 ) . large studies of type 1 diabetes have identified a prevalence of metabolic syndrome of 39% in finnish patients , which was approximately three times the separately observed prevalence in nondiabetic finnish subjects ( 22 ) and higher than the frequency in american patients with type 1 diabetes ( 22% ) ( 23 ) . however , in the latter study frequency of metabolic syndrome increased from 14.6 to 36.1% after a duration of diabetes of 9 years ( 23 ) . it follows that there is a dynamic in the frequency of metabolic syndrome in autoimmune diabetes , whether type 1 diabetes or lada , which must be considered in comparing the groups . therefore , the disparity in the prevalence of metabolic syndrome between the finnish and american studies and our own european study probably reflects differences in population characteristics including duration of diabetes , renal function , intensive insulin therapy , male sex , and age , all of which have been independently associated with metabolic syndrome . there is some limited evidence , other than the clinical phenotype , to support the contention that lada is an intermediate form of diabetes between type 1 diabetes and type 2 diabetes . lada , albeit defined as gada positivity irrespective of therapy , was reported to be associated with the ct and tt genotypes of the tcfl2 gene and , therefore , apparently shares genetic features with type 2 diabetes ( 24 ) . however , the high false - positive rate of the gada assay used in that study raises issues regarding the validity of the observation . we limited this potential error by repeating gada assays in gada - positive subjects , thereby reducing the risk of false positivity to < 0.2% , much less than the proportion of patients with lada . the error from not testing other diabetes - associated autoantibodies is probably small , given that 94% of patients with lada sampled from this cohort were detected by gada testing alone ( m.i.h . . the proposal that the metabolic syndrome is more prevalent in patients with lada who also have a low gada titer compared with those with an arbitrarily selected high titer of gada was not confirmed in this present study using a q - q plot to identify two apparently distinct gada - positive distributions ( 20 ) . however , there is substantial variation between laboratories with respect to threshold gada titers defining a positive result , and caution should be exercised in using such thresholds . thus , metabolic differences between lada and type 1 diabetes may not be categorical but part of a continuum , implying that lada is one end of a rainbow of a pathophysiological variation encompassing autoimmune diabetes ( 1 ) . if we accept the fact that metabolic syndrome is a surrogate marker for insulin resistance , there is now evidence that patients with lada are more insulin resistant than adult type 1 diabetic patients but not when glucose is excluded as a variable , when insulin resistance in both is similar to that in normal subjects . this conclusion is important , as it implies that autoimmune diabetes , whether type 1 diabetes or lada , can be identified by diabetes - associated autoantibodies , e.g. , gadas , as a categorical trait , irrespective of individual components of the metabolic syndrome or the need for insulin treatment . indeed , we have previously suggested that insulin treatment is an insubstantial feature to diagnose lada because it is subject to local issues such as medical practice and context , including availability of gada results ( 25 ) . thus , the role of insulin resistance in autoimmune diabetes may be limited , and there is no characteristic clinical phenotype of adult - onset autoimmune diabetes ; specifically , neither forms of autoimmune diabetes showed an increased frequency of metabolic syndrome in striking contrast with type 2 diabetes .

objective the purpose of this study was to estimate whether prevalence of metabolic syndrome in adult european diabetic patients is associated with type of diabetes.research design and methods a consecutive series of patients attending hospital - based diabetes clinics were assessed for the frequency of metabolic syndrome and compared with population - based control subjects as part of the action lada study . in total , 2,011 subjects ( aged 3070 years ) were studied , including 1,247 patients with recent - onset type 2 diabetes without glutamic acid decarboxylase autoantibodies ( gadas ) , 117 non insulin - requiring patients with gadas who had not received insulin therapy for at least 6 months after diagnosis ( designated latent autoimmune diabetes of adults [ lada ] ) , 288 type 1 diabetic patients , and 359 normal subjects.resultsfrequency of metabolic syndrome was significantly different in patients with type 1 diabetes ( 31.9% ) and lada ( 41.9% ) ( p = 0.015 ) and in both conditions was less frequent than in type 2 diabetic patients ( 88.8% ) ( p < 0.0001 for each ) . eliminating glucose as a variable , the prevalence of metabolic syndrome was similar in patients with autoimmune diabetes ( type 1 diabetes and/or lada ) ( 17.3% ) and control subjects ( 23.7% ) but remained more common in type 2 diabetic patients ( 47.8% ) ( p = 0.001 for all groups ) . in both type 1 diabetic patients and those with lada , individual components of metabolic syndrome were similar but less common than in type 2 diabetic patients ( p < 0.0001 for each).conclusions the prevalence of metabolic syndrome is significantly higher in type 2 diabetic patients than in patients with lada or adults with type 1 diabetes . excluding glucose as a variable , metabolic syndrome is not more prevalent in patients with autoimmune diabetes than in control subjects . metabolic syndrome is not a characteristic of autoimmune diabetes .

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 .

leukemias strike males and females in all ages and represent about one - third of childhood cancers . the most common type is childhood acute lymphoblastic leukemia ( also called acute lymphocytic leukemia or all ) . all is a fast growing cancer with an overproduction of abnormal immature white blood cells , called lymphoblasts or leukemic blasts . this type of leukemia usually affects children ages 1 10 years ( 1 ) . relapsed , recurrence leukemia can occur during therapy or after completion of treatment and represents the main cause for treatment failure ( 2 ) . in egypt about four new cases of all per 100.000 children are diagnosed each year in the national cancer institute ( nci ) , cairo university . it comprises about 30% of all pediatric malignancies and 70% of pediatric leukemia . in children between the ages of 2 and 10 all is the predominant form of leukemia with higher rates in males than females ( 3 , 4 ) . several studies confirm that altered secretion of the adipocytokines from adipose tissue is believed to increase the risk of various types of cancer . leptin and adiponectin , adipocyte secreted hormones , are well - studied factors relation to malignancies . leptin was found to be inversely associated with risk of chronic lymphocytic leukemia ( cll ) , however , no significant association between cll and adiponectin ( 5 ) . among the pathophysiological mechanisms underlying the association between obesity and cancers are insulin resistance ( 6 ) , low - grade systemic inflammation ( 7 ) , and altered secretion of adipokines ( 8) , though the mechanisms may differ between different types of cancer . high concentration of circulating insulin may signal cells to proliferate through different mechanisms either by directly signaling growth , or by increasing the levels of other more potent growth factors . insulin - like growth factors ( igf ) is one of the growth factors that have an important pathogenic role in cancers through promoting cell proliferation and decreasing apoptosis ( 6 ) . adiponectin , a hormone exclusively derived from adipocyte and secreted in high concentration into the blood ( 9 ) . previous studies postulated an inverse relation between serum levels of adiponectin and the risk of breast , endometrial , prostate , colorectal , kidney cancer and acute myeloid leukemia ( aml ) in children ( 1015 ) . accumulating evidence suggests that adiponectin may have an important protective role in carcinogenesis through exerting an insulin - sensitizing ( 16 ) , anti - inflammatory ( 17 ) , and antiangiogenic effect ( 18 ) . moreover , it exerts direct anti - carcinogenic effects through the amp - activated protein kinase ( ampk ) system . the mechanism seems to involve cell cycle arrest through the up - regulation of p53 and p21 ( 19 ) . independent of ampk activation , adiponectin decreases the production of reactive oxygen species ( ros ) ( 20 ) , which appears to cause decreased ampk activation ( 21 ) leading to inhibition of cell proliferation . it is produced only in small amount by white adipose tissue ( 22 ) while relatively high levels of resistin mrna are detectable in circulating mononuclear cells ( 23 ) . resistin may have an adiposity - independent role in breast carcinogenesis ( 24 ) and may be a good biomarker of gastric cancer ( 25 ) . it also has a role in the relation between inflammation and insulin resistance ( 26 ) . although , it is well documented that adipose tissue dysfunction increases the risk of some types of cancer such as that of the colon , breast and prostate , only few studies have elucidated whether disturbances of adipocytokines are linked to all . therefore , in our present study we evaluated the disturbance of adipose tissue hormonal secretion ( adiponectin and resistin ) in de novo and relapsed acute lymphoblastic leukemia among egyptian children , determined whether this disturbance is implicated in recurrence of acute lymphoblastic leukemia , and investigated the correlation between adiponectin and resistin in both groups . this study was conducted on 51 children patients suffering from all ( 29 males and 22 females ) ; they were selected from medical oncology center , mansoura university . all subjects provided informed consent and the study protocol was approved by the ethics committee of the mansoura university hospitals . patients were classified into : de novo acute lymphoblastic leukemia group ( de novo all ) : included 32 patients ( 18 male and 14 female ) with age range ( 3 18 years).relapsed acute lymphoblastic leukemia group ( relapsed all ) : included 19 patients ( 11male and 8 female ) with age range ( 5 17 years ) . besides 10 healthy children ( 6 male and 4 female ) with age range ( 5 18 years ) , served as a healthy control group . de novo acute lymphoblastic leukemia group ( de novo all ) : included 32 patients ( 18 male and 14 female ) with age range ( 3 18 years ) . relapsed acute lymphoblastic leukemia group ( relapsed all ) : included 19 patients ( 11male and 8 female ) with age range ( 5 17 years ) . besides 10 healthy children ( 6 male and 4 female ) with age range ( 5 18 years ) , served as a healthy control group . samples were divided into two aliquots , the first contained ; whole blood ( with anticoagulant ) for complete blood count and the other aliquot was allowed to clot at room temperature and the sera were separated by centrifugation at 3000 r.p.m . for 10 min . all patients and control were subjected to : complete blood count was done by coulter counter ( cell dyn 3500 ) and examination of lishman or wright - stained peripheral blood smears.determination of serum adiponectin level by human adiponectin / acrp30 quantikine elisa kit , which is quantikine colorimetric sandwich elisas , catalog no . 614mckinley place ne minneapolis , mn55413 usa.determination of serum resistin level by the quantikine human resistin kit which is a quantitative sandwich enzyme immunoassay technique purchased from r&d systems , inc . complete blood count was done by coulter counter ( cell dyn 3500 ) and examination of lishman or wright - stained peripheral blood smears . determination of serum adiponectin level by human adiponectin / acrp30 quantikine elisa kit , which is quantikine colorimetric sandwich elisas , catalog no . determination of serum resistin level by the quantikine human resistin kit which is a quantitative sandwich enzyme immunoassay technique purchased from r&d systems , inc . samples were divided into two aliquots , the first contained ; whole blood ( with anticoagulant ) for complete blood count and the other aliquot was allowed to clot at room temperature and the sera were separated by centrifugation at 3000 r.p.m . for 10 min . all patients and control were subjected to : complete blood count was done by coulter counter ( cell dyn 3500 ) and examination of lishman or wright - stained peripheral blood smears.determination of serum adiponectin level by human adiponectin / acrp30 quantikine elisa kit , which is quantikine colorimetric sandwich elisas , catalog no . 614mckinley place ne minneapolis , mn55413 usa.determination of serum resistin level by the quantikine human resistin kit which is a quantitative sandwich enzyme immunoassay technique purchased from r&d systems , inc . complete blood count was done by coulter counter ( cell dyn 3500 ) and examination of lishman or wright - stained peripheral blood smears . determination of serum adiponectin level by human adiponectin / acrp30 quantikine elisa kit , which is quantikine colorimetric sandwich elisas , catalog no . 614mckinley place ne minneapolis , mn55413 usa . determination of serum resistin level by the quantikine human resistin kit which is a quantitative sandwich enzyme immunoassay technique purchased from r&d systems , inc . complete blood counts data of studied participants are summarized and compared in table 1 . in both all groups ( de novo and relapsed ) , red blood cell counts were significantly lower , whilst white blood cell counts were significantly higher compared with healthy controls ( p<0.05 ) . additionally , relapsed all group has significant increase in white blood cell counts when compared with de novo all group ( p < 0.05 ) . adiponectin and resistin levels of studied groups are expressed in mean s.d and shown in table 2 . all groups ( de novo and relapsed ) showed significantly lower adiponectin levels 10.031 1.934 and 7.731 1.083 ( ng / ml ) respectively compared with healthy controls 11.91 2.234 ng / ml ( p < 0.05 ) , while exhibited significantly higher resistin levels 7.353 1.582 and 9.784 1.656 ( ng / ml ) respectively in comparison with healthy controls 4.92 1.55 ( ng / ml ) . moreover , a significant increase in resistin levels was observed in relapsed all group when compared with de novo all group ( p < 0.05 ) . pearson coefficient correlation showed inverse correlation between adiponectin and resistin levels in all groups ( r = 0.51 , p < 0.001 , fig . the current study addressed the hypothesis that dysregulation in adipocytokines has a potential role in carcinogenesis and cancer progression focusing on leukemia . the aim of this study was to evaluate adiponectin and resistin concentration in newly diagnosed and relapsed all children and whether the disturbance in those two adipokines is implicated in all relapse . we have identified that the level of adiponectin is decreased in de novo all children compared to healthy controls , its level also decreased in relapsed all compared to both healthy controls and de novo all patients . ( 28 ) who confirmed the low plasma level of adiponectin at all diagnosis compared with controls . in an in vitro study ( 29 ) the authors have investigated the functions of adiponectin in haematopoiesis and found that adiponectin predominantly inhibits proliferation of myeloid cell lines , and induces apoptosis in myelomonocytic leukemia lines , but did not suppress proliferation of erythroid or lymphoid cell lines . this hormone has also been inversely associated with both adult forms of cancer that have been epidemiologically investigated , namely breast cancer ( 11 , 30 ) and endometrial cancer ( 12 , 31 ) . we also reported a decreased level of adiponectin in relapsed all compared to both healthy controls and de novo all patients . a possible explanation could be due to a direct effect of adipose tissue dysfunction on the leukemia itself , perhaps mediated by adiponectin and other adipocyte - derived hormones . alternatively ; it may be an adipocyte interaction with leukemia cells to impair chemotherapy of all as suggested earlier ( 32 ) . adiponectin is a direct angiogenesis inhibitor that induces apoptosis in activated endothelial cells ( 18 , 33 ) . similarly , decreased adiponectin level facilitates the development of cancer by preventing pathologic cell mitosis ( 34 , 35 ) . additionally , adiponectin level is attributed to the further increase in production of inflammatory cytokines in the cachectic stage by cancer cell itself ( 36 , 37 ) . leukemia per se causes a more intensive inflammatory process than malignancies of solid organs , with proinflammatory cytokines further suppressing adiponectin ( 28 ) . our study also demonstrated that resistin level in egyptian children with all was high in de novo compared to healthy controls , also in relapsed all resistin level was high compared to controls and de novo all . resistin in all subjects was inversely correlated with adiponectin level ( r = 0.51 , p<0.001 ) . ( 28 ) who showed that in children with all , resistin levels are high at diagnosis compared with controls . moreover , correlations from their study suggested that leukemia related to inflammatory cytokines release serum lipids may stimulate leptin and resistin secretion and vice versa . changes in adipose tissue and metabolism occur in multiple myeloma ( mm ) , acute leukemia , and chronic lymphocytic leukemia ( cll ) ( 38 ) . they studied resistin level in serum of patients with hematologic malignancies and they found that resistin level was significantly higher in lymphoma patients than in chronic lymphocytic leukemia ( cll ) , acute leukemia and control groups . although only a few studies have analyzed resistin in patients with malignancies , the general properties of resistin could contribute to tumorigenesis ( 39 ) . resistin has been also shown to induce production of vascular endothelial growth factor receptor ( vegfr ) and the formation of endothelial cell tubes ( 40 ) . moreover , resistin is seen mainly as an inflammatory factor ( 41 ) which is associated with tnf- and il-6 , and may up regulate several adhesion molecules and cytokines ( 26 ) . we reported decreased adiponectin and elevated resistin levels in association with de novo and relapsed all in egyptian children . this finding suggests the implication of both hormones in all pathogenesis which might be useful prognostic markers in guiding the treatment of all in the future . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

backgroundadipose tissue secretes a large number of adipocytokines such as leptin , resistin , and adiponectin . many of these hormones and cytokines are altered in obese individuals and may lead to disruption of the normal balance between cell proliferation , differentiation , and apoptosis . the aim of our work was to investigate the disturbance of secretion of adiponectin and resistin in de novo and relapsed acute lymphoblastic leukemia ( all ) in egyptian children and determine whether adiponectin and resistin are implicated in increased risk relapse compared to healthy individuals.methods:measurements of adiponectin and resistin were performed at diagnosis , in 32 patients with de novo all aged 3 to 18 years ( mean 9.8 y ) and 19 children with relapsed all aged 5 to 17 ( mean 9.9 yr ) . 10 apparently healthy children with matched age and sex were used as controls.results:mean adiponectin levels were low ( p < 0.05 ) , whereas mean resistin levels were high ( p<0.05 ) at diagnosis and relapsed all ( compared to healthy controls ) . a significant decrease of adiponectin levels was observed in relapsed all compared to de novo all . in contrast resistin was significantly increased in relapsed all compared to de novo patients . adiponectin in all subjects inversely correlated with resistin level ( r = 0.51 , p < 0.001).conclusion : low adiponectin and high resistin level at diagnosis suggest their implication in all pathogenesis and may serve as potential clinically significant diagnostic markers to detect leukemic relapse .

infantile hemangiomas ( ih ) are benign tumors in the pediatric population with an incidence of up to 10% . they are usually not present at birth but undergo a rapid proliferation within the first weeks of life . this is followed by the plateau stage and then a slow involution of the lesion such that 60% of 4-year - olds and 76% of 7-year- olds will experience complete regression of their hemangiomas . a subset of ih that could be considered high risk and might require treatment would include multiple ih , ocular involvement , oropharyngeal and airway involvement , large or disfiguring lesions , and ulcerated hemangiomas . for high risk ih , corticosteroids have traditionally been the mainstay of treatment with variable response and significant side effects including hypertension , increased risk of infection , cushingoid appearance , and growth suppression . since 2008 , propranolol has become a favored first line treatment for ih . propranolol shows good efficacy , rapid improvement in the lesion and fewer severe adverse events compared to corticosteroids . nadolol is a beta - blocker that has similar mechanism of action as propanolol , but has less central nervous system penetration and is dosed twice rather than three times per day . an 11-week - old healthy baby girl presented to the dermatologist with a well - defined red plaque over her right face that had been getting progressively more visible over the last 7 weeks [ figure 1 ] . the baby 's parents had noticed a whitish - red mark over the patient 's cheek in the hours after her birth , which they attributed to trauma from vaginal delivery . the lesion developed a bruised appearance over the next 2 weeks , and then grew more noticeably red , well defined and thickened . ( b ) patient at 21 weeks of age , after 10 weeks of treatment physical exam revealed a well - defined 7 3 cm , soft , warm red plaque following a v2 distribution on the right side of the patient 's face affecting the right temple , zygoma , malar cheek and lower eyelid , compatible with an ih . due to concern over the facial v2 distribution , phaces syndrome , which can be associated with segmental ih , ophthalmology exam , ekg , abdominal ultrasound , echocardiogram , and mri of head were all normal . phaces refers to posterior fossa malformations , hemangiomas , arterial anomalies cardiac defects , eye abnormalities , sternal cleft and supraumbilical raphe syndrome . the patient was started on nadolol at 0.5 mg per kg , adding 0.5 mg per kg each week until the 4 week where a maintenance dose of 2 mg per kg was reached . there was marked decrease in the redness , vascularity , and elevation of the hemangioma , including over the lower eyelid [ figure 1 ] . ongoing improvement in the appearance of the hemangioma on nadolol was noted at age 22 weeks , with a plan to continue therapy until age 1 and then titrate slowly off the medication . many recent studies have shown propranolol to be highly effective in treating high - risk or disfiguring ih . potential advantages of nadolol over propranolol include its inability to cross the blood brain barrier , which may lead to decreased sleep disturbance and irritability , and a longer half - life with dosing twice a day . a small cohort - blinded study showed that nadolol might be somewhat more effective as a treatment for ih than propranolol . typically , few side effects are reported in patients receiving beta - blockers for ih . blood pressure , heart rate , and serum glucose should be monitored in infants receiving beta - blockers . sweating , shakiness , tachycardia , hypotonia , and hunger are early signs of hypoglycemia in infants . beta - blockers produce an immediate vasoconstrictive effect due to inhibition of adrenaline - mediated vasodilation . this leads to decreased erythema and softening of the hemangioma within a few treatments . during the proliferative stage of ih , beta - blockers are thought to decrease the expression of vascular endothelial growth factor ( vegf ) , thereby opposing aberrant angiogenesis . beta - blockers have been also theorized to decrease inhibition of apoptotic pathways in ih , which may hasten tumor involution .

infantile hemangiomas are common benign tumours of infancy affecting up to 10% of children . they are typically not present at birth but undergo a rapid proliferation stage and then plateau in growth before resolving spontaneously . recently , beta - blockers have been favoured over systemic corticosteroids for treatment of disfiguring or life - threatening infantile hemangiomas . we present a case of an 11-week - old female with a 7 week history of an evolving hemangioma along a facial v2 distribution . physical exam revealed a well - defined bright red plaque over the right zygoma and lower eyelid . mri , echocardiograph , and liver ultrasound were normal . patient was treated with nadolol and had a rapid and substantial regression of the hemangioma . nadolol is an effective treatment option for disfiguring facial infantile hemangioma . the use of beta - blockers as treatment offers clues into the pathogenesis of infantile hemangioma , which is not yet completely understood

anti - mllerian hormone ( amh ) is secreted into the circulation by small growing follicles in the ovary , until they have reached the size at which they may be selected for dominance ( 68 mm ) . since the cohort of small growing follicles is in equilibrium with the total number primordial follicles , serum amh levels reflect the ovarian follicular pool . amh is therefore considered an accurate marker of ovarian reserve [ 3 , 4 ] . moreover amh levels vary less across different menstrual cycles as well as within one menstrual cycle as compared to other biomarkers of ovarian activity , such as fsh , which has a number of obvious clinical advantages [ 47 ] . indeed , according to different studies , the measurement of amh on a random basis throughout the menstrual cycle is associated with a very good accuracy when predicting ovarian response [ 810 ] . however while first studies reported a very low variability throughout the menstrual cycle [ 1114 ] , a number of more recent studies [ 1517 ] indicate a reduction of circulating amh in the luteal phase , hence raising the question if amh should better be measured on a fixed day of the menstrual cycles to foster standardization and to allow better cross comparison between individual assessments . in this review , we shed light on the partly controversial issue of amh variability , with particular attention on amh intracycle variability , that has been recently widely debated . moreover we evaluate the impact of ethnicity , bmi , and smoking behaviour on amh interindividual variability . finally we discussed changes in amh serum levels in two conditions of ovarian suppression , namely contraceptives use and pregnancy . when talking about hormonal stability , two different types of variability should be considered : the interindividual and the intraindividual variability . the interindividual variability of amh refers to variations in amh levels between different subjects and is first of all secondary to a very high variability in the number of growing follicles within groups of women of similar age [ 1820 ] . the high interindividual variability in amh is not surprising , given the wide variability of ovarian reserve in women . generally , high interindividual variability is a good characteristic for a hormone when used as discriminatory biomarker in a clinical setting ( figure 1 ) . indeed the high interindividual variability of amh makes it an ideal candidate biomarker with which to discriminate patients for diagnostic , prognostic , and other clinical purposes . in uni- and multi - variate analyses , black [ 21 , 22 ] and hispanic women exhibit serum amh levels 25% lower than those found in caucasian women of similar age . furthermore , an unexpectedly high number of black women has undetectable amh levels despite relatively young age and regular menstrual cycles , hence indicating a potential discrepancy between actual ovarian reserve and what is indicated by amh measurement in this population ( figure 2 ) . more research on the underlying biological phenomena and consequences of this finding however , this finding indicates that care should be taken when using amh reference values across different ethnicities . some papers , even if limited to small numbers of patients , indicated a negative relationship between bmi and serum amh levels [ 23 , 24 ] . in a recent large study performed in a healthy general female population , amh was negatively related to bmi , but the relationship was age dependent . in other words , in women , amh levels decreased and bmi increased with age ; hence , the relationship between amh and bmi was only secondary to the stronger relationship of the two variables with age . there is clear evidence that smoking may directly accelerate ovarian follicular depletion , thereby reducing the age at menopause [ 29 , 30 ] . moreover , smoking has been shown to alter metabolic path for several hormones including estradiol . however contradictory results have been reported on the relationship between smoking and amh , with some authors [ 3133 ] reporting reduced amh levels in smokers versus nonsmokers and others [ 18 , 27 , 3436 ] reporting similar values in both groups of women . in a more recent study , amh levels of 416 healthy women , including 99 smokers and 317 nonsmokers , were analyzed . as shown in figure 3 , at any age , the distribution of smokers was uniform in all quartiles of amh distribution ( figure 3 ) . in other words , in reference to a given age , a similar number of smoking women had high or low amh levels , respectively . accordingly , the debate on the impact of smoking on the follicular pool and the circulating amh levels has not yet been settled . in conclusion , according to the published studies , it seems that the variability in ovarian reserve and secondly ethnicity may largely explain the high degree of interindividual variability in amh levels . the intraindividual variability is indicative of variations in amh levels in one single subject and may be secondary to true biological variations in levels of circulating amh in women . we propose to distinguish among a long - term variability , a short - term variability and an ultra - short - term variability . the first refers to variations in amh levels that occur year after year and are indicative of the decline in the ovarian reserve of a single woman . the second depends on the monthly physiologic variability in ovarian function ; hence , the short - term variability may refer to intermenstrual cycle variability . the ultra - short - term variability indicates the day - by - day variability and refers to intramenstrual cycle variability . in a recent prospective longitudinal study , serum amh levels have been measured in healthy young prepubertal girls ( 6 to 13 years of age ) every 6 months for 3 years and the mean intraindividual coefficient of variation ( cv ) for amh was reported as 22% . this indicates that circulating amh shows on average only minor fluctuations within a limited time span ; thus , a random amh measurement is likely to be representative indeed for a given girl . the long , term variability in adult women has been mainly studied in cross - sectional studies , with some of them including as many as 1015 thousand patients [ 18 , 27 , 3841 ] . overall , the studies are in good agreement that amh declines with advancing age with a pattern that recalls the exponential decay of the primordial follicular pool [ 2 , 42 ] , which is best described by a quadratic equation . the intermenstrual cycle variability has been analyzed in two well conducted prospective studies [ 14 , 43 ] . both studies concluded that 89% of the variation in amh was due to between - subject variation , while only 11% of variability was secondary to individual fluctuation in amh levels ( figure 4 ) . furthermore , a recent prospective study reported a correlation of 0.88 between amh measurements performed on cycle day 2 or 3 in two subsequent cycles in women with regular spontaneous cycles . amh showed the highest between - cycle - correlation within an array of hormones assessed , including testosterone , fsh , e2 , inhibin b , and lh . a highly debated issue relates to whether amh significantly varies or not throughout the menstrual cycle . several studies have suggested that serum amh levels fluctuate little during the menstrual cycle , as would be expected from the evidence that amh is not secreted by the dominant follicle or corpus luteum [ 1114 ] ( figure 5 ) . amh is unique among the known hormones produced by antral follicles , because its secretion seems to be only marginally influenced by gonadotropins and it is dramatically reduced as follicles reach the full gonadotropin sensitivity . as a consequence , amh levels during the follicular phase do not reflect the activity of the developing large dominant follicle of the month , and conversely on any time point of the menstrual cycle amh levels provide information on the number of small antral follicle present in the ovary which are available for cyclic follicular recruitment . to study the intraindividual variability of amh , van disseldorp et al . the authors reported that the intraindividual variability of amh was only 13% and , most importantly , when dividing patients into quintiles according to basal amh levels , the intraindividual fluctuations were shown to fall in the same quintile in 72% of the cases and to cross two quintiles in only 1% of the cases . in contrast , some authors have noted significant fluctuations within one menstrual cycle [ 1517 ] . a very recent study found serum amh levels significantly lower in the luteal than follicular phase with a variation pattern similar to pituitary fsh , and the intraindividual variance of amh was as high as 80% . however the study was based on a very small group of subjects ( n = 12 ) , and some of them had as few as five blood samples throughout an entire menstrual cycle . moreover when analyzing values for single patients , the proposed decline of amh in the luteal phase was not evident in 25% of patients ( figure 6 ) , hence raising the questions if the observed reduction of amh in the luteal phase might be simply casual instead due to a biological reason indeed . in another prospective study including 20 women , serum amh levels were shown to fluctuate throughout the menstrual cycle . in this case , the observed fluctuations were absolutely random throughout the cycle and not associated to typical gonadotropin or ovarian steroid patterns . moreover , the amplitude was proportional to basal amh levels : women with low amh levels exhibited only minor fluctuations , whereas women with high basal amh levels showed relatively higher fluctuations . the author speculated to categorize amh pattern in the ageing ovary pattern and the younger ovary pattern . in spite of the good quality of the study , where blood samples were collected from each woman daily along a whole menstrual cycle , some criticisms have been put on roberts paper for the lack of the calculation of the intraindividual coefficient of variation , which is considered the optimal analysis for hormonal variability . however , at bottom line , sowers et al . 's study indicates in a clear and convincing way that serum amh levels vary throughout the menstrual cycle , that fluctuations may be relevant in those women with high basal levels , and most importantly that fluctuations are randomly distributed during the cycle . the random and noncyclic fluctuations in amh indicate that measuring the hormone on a fixed day of the menstrual cycle would not yield any advantage of a random assessment , for example , on any day of the menstrual cycle . in order to verify the effect of female age on the degree of amh fluctuations , a recent study re - evaluated for the third time the data previously described by hehenkamp et al . and van disseldorp et al . , the absolute intraindividual variation of amh ( deltaamh ) , that is , the difference between maximum and minimum serum level throughout one cycle , was found to be significantly and negatively associated with age . in other words , younger women had significantly larger fluctuations in amh levels than older women . it may be concluded that in patients with low ovarian reserve ( usually aged women ) , amh fluctuations have little clinical relevance , while in young patients with usually high ovarian reserve , fluctuations of amh might indeed impact on the discriminatory capability of diagnostic and predictive tests , respectively . the observed variability in amh levels may have a limiting effect on the main current application of amh as a predictive test in ivf practice . amh is widely used to predict the ovarian response and to individualize the treatment according to this prediction [ 5 , 9 , 46 , 47 ] . if amh values cross the cut - off values proposed for the ovarian response categories because of true biological variability in amh , this might lead to misclassification and erroneous treatment of patients . hence the impact of the documented amh variability needs to be tested in a clinical setting on a typical target population undergoing a clinically relevant predictive testing scenario . as reported in detail in several reviews and metanalysis [ 5 , 4850 ] , amh is the best hormonal marker for the prediction of ovarian response in ivf . when using a random amh measurement in order to prospectively predict ovarian response to exogenous fsh , correct categorization of 75% of patients in the three categories poor , normal , or hyper - responder can potentially be obtained . accordingly , although not as stable as thought before , amh still remains the most reliable ovarian hormone and the best hormonal predictor of ovarian response to stimulation in ivf , with the important advantage of being randomly measurable . since amh production by antral follicles has been considered to be largely gonadotropin independent , a logical consequence is that pituitary suppression , as occurring with oral contraceptives ( oc ) or physiologically during pregnancy , should not be associated with relevant changes in serum levels . a number of studies have been published on the effect of oc on amh levels [ 24 , 44 , 5157 ] and most of the available evaluations are not prospective . the very few prospective studies unfortunately limited the analysis to a few observational months ( 1 to 4 months ) of oc treatment and are thus potentially and insufficiently informative . oc use has been reported either to insignificantly influence amh concentration [ 44 , 51 , 52 , 54 , 55 ] or to reduce it significantly [ 53 , 5658 ] . a large cross - sectional study compared 180 and 76 twenty - year old oc users and nonusers , respectively , and found that long - term oc use was associated with a significant mean reduction in amh levels by 13% . recently a cohort study based on 863 healthy women ( 228 oc users and 504 nonusers ) reported that amh serum levels were 29.8% lower in oc users than those in nonusers . . however no dose - response relation was found between the dose of ethinyl estradiol and the impact on serum amh concentration . in a well - conducted prospective study , amh levels during oc pill intake in long - term oc users ( n = 25 ) and 2 months after stopping the oc were assessed . interestingly , a mean increase in amh levels by 30% after cessation of the oc was found ( from a mean of 2.0 ng / ml during oc to a mean of 2.6 ng / ml two months after the last pill , p = 0.001 ) . the study population consisted of 42 healthy women randomized to use for 9 weeks an oc in the form of either an oral pill ( n = 13 ) , a transdermal patch ( n = 15 ) , or a vaginal ring ( n = 14 ) . after 9 weeks of contraceptive use , serum amh levels decreased significantly by almost 50% as compared to baseline in all treatment groups ( figure 7 ) . this evidence is indicative of a suppressive effect of hormonal contraception on circulating amh levels , at least when considering long - term use . thus , serum amh concentration may not retain its accuracy as predictors of the ovarian reserve in women using hormonal contraceptives for long time . pregnancy is a physiological condition associated with ovarian suppression because of suppressed endogenous gonadotropin release . according to the concept that amh reflects the continuous fsh - independent noncyclic growth of small follicles in the ovary , it would be expected to find nonrelevant alteration in its levels during pregnancy . indeed , an early small cross - sectional study reported unmodified amh levels throughout pregnancy . subsequent studies reported contradictory results , with some confirming this finding [ 60 , 61 ] , while others describing a decrease in amh levels during pregnancy [ 62 , 63 ] . it has also been reported that the decline in amh during pregnancy is evident when using the beckman coulter but not the dsl assay . however , in the only longitudinal study available ( n = 60 ) , authors found a significant decrease in amh levels in the 2nd and 3rd trimesters compared to the 1st trimester and the mean reduction at the end of pregnancy was of about 50% . this study indicated that during pregnancy , there is a relative ovarian quiescence and reduced follicular maturation with a consequent decrease in the population of follicles secreting amh . at the same time , at least part of the observed reduction in amh levels during pregnancy could also be explained by the pregnancy - associated hemodilution and increased plasma - protein binding . in conclusion , on top of the age related decline in amh , significant fluctuations have been reported for a number of conditions and this has to be taken into account when interpreting values in clinical practice . this suggests that in clinical practice , amh can be measured independently of the cycle phase . prolonged ovarian suppression as induced by physiological or pharmacological interventions may reduce amh levels , since the long and profound pituitary gonadotropin suppression is associated with a reduced number of antral follicles . the exact role of patients ' characteristics , as ethnicity , and some habits , as smoking , on intra- and interindividual variability of amh need to be investigated further .

anti - mullerian hormone ( amh ) is an ovarian hormone expressed in growing follicles that have undergone recruitment from the primordial follicle pool but have not yet been selected for dominance . it is considered an accurate marker of ovarian reserve , able to reflect the size of the ovarian follicular pool of a woman of reproductive age . in comparison to other hormonal biomarkers such as serum fsh , low intra- and intermenstrual cycle variability have been proposed for amh . this review summarizes the knowledge regarding within - subject variability , with particular attention on amh intracycle variability . moreover the impact of ethnicity , body mass index , and smoking behaviour on amh interindividual variability will be reviewed . finally changes in amh serum levels in two conditions of ovarian quiescence , namely contraceptives use and pregnancy , will be discussed . the present review aims at guiding researchers and clinicians in interpreting amh values and fluctuations in various research and clinical scenarios .

discoidin domain receptors ( i.e. , ddr1 and ddr2 ) are transmembrane receptor tyrosine kinases ( rtks ) that specifically recognize fibrillar collagens as extracellular ligands . ddr1 and ddr2 are highly involved in fundamental cellular processes , including cell proliferation , migration , adhesion , and matrix remodeling . the dysregulation of ddr1 has been linked to a variety of human cancers and inflammatory conditions such as fibrotic disorders and atherosclerosis . collective evidence indicates a critical link between ddr1 and pulmonary fibrosis , a lethal disease with few therapeutic options . for instance , a ddr1 deletion has been reported to alleviate bleomycin ( blm)-induced lung inflammation and pulmonary fibrosis by blocking p38 mitogen - activated protein kinase ( p38 mapk ) activation . we and others have identified several classes of ddr1 inhibitors with different selectivity profiles and have demonstrated their therapeutic potential for various human cancers ( figure 1 ) . however , these small molecules still have relatively poor target specificity and there are limited reports on the efficacy of the pharmacologic inhibition of ddr1 in models of pulmonary fibrosis . in this article , we report the structure - based design of tetrahydroisoquinoline derivatives as new highly selective ddr1 inhibitors with promising therapeutic effects in a blm - induced pulmonary fibrosis mouse model . selective ddr1/ddr2 kinase inhibitors . ddr1 shares approximately 61% sequence identity with abelson ( abl ) kinase in its adenosine triphosphate ( atp ) binding domain , and most reported selective ddr1 inhibitors are derivatized from abl antagonists . previous investigations revealed that a stacking interaction between the chemical molecule and tyr of abl is critical for most of the reported abl inhibitors ( supporting information ( si ) , figure s1a , b , c ) , but the corresponding interaction is unnecessary for ddr1 binding . indeed , ddr1-in-1 achieved selective ddr1 inhibition because its hinge binding moiety is oriented away from the p - loop , avoiding the potential interactions with tyr in abl . however , the molecule could form an additional interaction with phe in abl , which contributed greatly to its relatively low selectivity between ddr1 and abl ( si , figure s1d ) . diminishing this interaction a series of 1 , 2 , 3 , 4-tetrahydroisoquinoline derivatives were designed as novel , selective ddr1 inhibitors in which a pyrimidinyl group was utilized as the potential hinge binding moiety . a n-(3-((4-methylpiperazin-1-yl)methyl)-5-(trifluoromethyl ) phenyl)carboxamide group was also introduced based on our previous investigation ( figure 2a ) . ( b ) molecular docking of 6a ( cyan ) into the ddr1-ponatinib ( yellow ) costructure ( pdb i d : 3zos ) . ( c ) co - crystal structure of 6c with ddr1 ( pdb i d : 5fdp ) . ( e ) superposition of 6c with abl ( pdb i d : 3ik3 ) . our preliminary modeling suggested that the initial lead , 6a , could fit nicely into the ddr1 binding pocket and maintain the key interactions with ddr1 . the pyrimidinyl moiety of 6a could form an essential hydrogen bond with the nh of met in the hinge region of ddr1 . two additional hydrogen bonds have also formed between the linker amide and glu in the c - helix and asp in the asp - phe - gly ( dfg ) motif , respectively ( figure 2b ) . encouragingly , compound 6a failed to dock into the abl binding pocket , suggesting its potential selectivity among kinases . briefly , the substituted methyl 4-(2-(2,2,2-trifluoroacetamido)ethyl)benzoate ( 8) was prepared by the trifluoroacetylation of methyl 4-(2-aminoethyl)benzoate ( 7 ) and then underwent a classical pictet spengler reaction to yield the protected tetrahydroisoquinoline derivatives ( 9 ) , which were deprotected and reacted with a hydrochloric acid solution in meoh to form the key intermediate ( 10 ) . compound 10 was coupled with 5-bromopyrimidine , 3-bromopyridine , or bromobenzene through a buchwald the final products were obtained by treating intermediate 11 with different anilines under basic conditions . reagents and conditions : ( a ) trifluoroacetic anhydride , 0 c to rt , 5863% ; ( b ) ( hcho)n , conc h2so4 , 0 c to rt , 5189% ; ( c ) ( i ) k2co3 , meoh / h2o ( 2:1 ) , rt , ( ii ) hclmeoh , meoh , rt , 8695% ( two steps ) ; ( d ) 5-bromopyrimidine or 3-bromopyridine or bromobenzene , pd(dba)2 , ruphos , cs2co3 , toluene , 80 c , 4289% ; ( e ) substituted aniline , t - buok , thf , 20 c to rt , 6986% . ddr1 inhibition of the compounds was determined using a well - established lance ultra kinase assay . the potential target selectivity was additionally evaluated by monitoring their inhibition against abl . compound 1 was included as a positive control , which displayed similar ic50 values to the previously reported data . it was shown that 6a exhibited modest ddr1 inhibitory activity , with an ic50 value of 442 nm , while its potency against abl was strikingly inferior ( ic50 > 10.0 m ) . further computational investigation suggested that a small hydrophobic recess formed by val , ala , and met was available in the atp binding pocket of ddr1 ( figure 2b ) . a lipophilic substituent at r1 may occupy this pocket to achieve improved potency . the ( r)-methyl ( 6b ) and ( r)-ethyl derivatives ( 6d ) displayed a 20-fold and 12-fold potency improvement , respectively . the ( s)-methyl compound ( 6c ) , in which the methyl moiety was oriented away from the pocket , as confirmed by a 2.3 cocrystal structure with ddr1 ( figure 2c ) , displayed a similar inhibitory potency to that of the r1 unsubstituted 6a . a large group at the r1 position was predicted to be detrimental to the binding of ddr1 . as expected , r1-(r)-isopropyl ( 6f ) caused a substantial potency loss for ddr1 . additionally , compounds 6e and 6 g , which featured ( s)-ethyl and ( s)-isopropyl , respectively , displayed almost no ddr1 inhibition . the x - ray crystal structure also confirmed the presence of a strong hydrogen bonding network between the new inhibitor and ddr1 ( figure 2c ) . the deletion of a hydrogen bond by eliminating the n atoms in the pyrimidinyl group ( 6i ) totally abolished ddr1 inhibitory potency . not surprisingly , when the pyrimidinyl group was replaced by a pyridinyl moiety , the resulting compound , 6h , exhibited an almost identical ic50 value to that of 6a . it was also noteworthy that all the new ddr1 inhibitors exhibited excellent ddr1 selectivity over the structurally related abl kinase ( table 1 ) . to rationalize this target selectivity , the inhibitor 6c was superimposed into the abl structure ( pdb i d : 3ik3 ) ( figure 2e ) . it was shown that the 1 , 2 , 3 , 4-tetrahydroisoquinoline scaffold in 6c forced the pyrimidinyl moiety to adopt a different dihedral angle that prevented the formation of critical interactions with tyr and phe in abl . moreover , the distance between the n atom in the pyrimidinyl group and met in abl was predicted to be 4.09 , which exceeds the limit to form a potential hydrogen bond . ddr1 experiments were performed using the lance ultra kinase assay , according to the manufacturer s instructions . abl1 activity experiments were performed using the fret - based z - lyte assay , according to the manufacturer s instructions . further structural optimization of the inhibitor 6b yielded 6j as a promising candidate , with an ic50 value of 9.4 nm against ddr1 ( table 1 ) . the compound also exhibited reasonable pharmacokinetic ( pk ) properties , with an oral bioavailability of 66.8% and a t1/2 value of 1.25 h at an oral dose of 20 mg / kg ( table 2 ) in rats . the pk profile of compound 6j was also investigated in icr mice , which showed that the compound had a similar oral bioavailability to that of rats . however , the area under concentration time curve ( auc ) value of the compound in mice was obviously higher than that in rats , suggesting its good absorption property in mice . icr mice ( male , 24 animals per group ) weighing 1830 g were used for the study . sd rats ( male , 3 animals per group ) weighing 180220 g were used for the study . the ddr1 inhibition of 6j was further validated by determining its binding affinity with the ddr1 protein ( conducted by discoverx , san diego , ca ) . it was shown that 6j bound tightly to ddr1 , with a binding constant ( kd ) value of 4.7 nm . the target specificity of 6j was also investigated by conducting a kinase selectivity profiling study against a panel of 468 kinases ( including 403 nonmutated kinases ) at 1.0 m , which is approximately 210-fold above its kd value against ddr1 using the discoverx screening platform . it was shown that 6j displayed excellent target selectivity , with s(10 ) and s(1 ) scores of 0.022 and 0.012 , respectively ( table s5 ) . the potential off - target kinases tested included cyclin - dependent kinase 11 ( cdk 11 ) , ddr2 , ephrin type - b receptor 8 ( ephb8 ) , muscle - specific receptor tyrosine kinase ( musk ) , nerve growth factor receptor a ( trka ) , trkb , and trkc . however , the ic50 value of 6j against ddr2 was determined to be 188 nm in our lance ultra kinase assay , indicating that 6j was 20-fold less potent against ddr2 . further determination of the binding affinities ( kd values ) revealed that 6j exhibited an approximately 21120-fold less potency against the majority of the other off target kinases , with the exception of trkb and trkc , which displayed kd values of 22 and 18 nm , respectively ( figure 3 ) . compound 6j was profiled at a concentration of 1.0 m against a diverse panel of 468 kinases by discoverx . ( b ) binding constants ( kd values ) of compound 6j against the top hits . further investigation revealed that the activation of ddr1 as well as its downstream signaling intermediate , p38 , were both dose - dependently suppressed by 6j in primary human lung fibroblasts ( figure 4 ) , suggesting the efficacy of this compound against ddr1-induced signaling . in light of the critical role of ddr1 in blm - induced pulmonary fibrosis , we treated mice with compound 6j after the onset of a blm challenge . inhibitor 6j was orally administered at 10 and 50 mg / kg twice daily ( bid ) for 2 weeks based on its pk properties ( table 2 ) . unlike those from the phosphate buffered saline ( pbs ) treated mice , which had large alveolar spaces and were weakly stained by masson s trichrome , the lungs from blm - challenged animals exhibited a reduction in alveolar spaces and were stained blue by masson s trichrome , demonstrating typical fibrotic features . the compound prevented these blm - induced pathological changes in a dose - dependent manner ( figure 5a ) . these results agreed with the expression levels of fibrotic markers in lung tissue lysates , including fibronectin and -smooth muscle actin ( sma ) ( figure 5b ) . further analyses also revealed that the administration of compound 6j caused a dose - dependent suppression in the content of hydroxyproline ( figure 5c ) , a unique amino acid found in collagen . the above data collectively indicate the promising therapeutic potential of 6j against the blm - induced pulmonary fibrosis . 6j inhibited ddr1-mediated signaling in a concentration - dependent manner in primary human lung fibroblasts ( 24 h treatment ) . fourteen days after the onset of blm injury , c57bl/6 mice ( five animals each group ) received an oral gavage of 6j twice daily at the indicated dosages , and the lungs were collected on day 28 . the upper panels in ( a ) display the hematoxylin and eosin ( h&e ) staining images of the dissected lungs . the images in ( b ) show the results of the immunoblotting with the indicated antibodies . the histogram in ( c ) shows the determined hydroxyproline content . * * * p < 0.001 . in summary , a series of 1 , 2 , 3 , 4-tetrahydroisoquinoline derivatives were designed as novel highly selective ddr1 inhibitors . compound 6j strongly suppressed ddr1 , with a single digital nm ic50 value , but it is significantly less potent in a panel of 400 nonmutated kinases . thus , to the best of our knowledge , this compound represents one of the most selective ddr1 inhibitors to date . the compound also demonstrated reasonable pk properties and a promising oral therapeutic effect in a blm - induced mouse pulmonary fibrosis model . its strong ddr1 inhibitory potency and extraordinary target specificity make compound 6j not only a promising lead compound for new drug discovery but also a valuable research probe for further biological investigation of its target . reagents and solvents were obtained from commercial suppliers and used without further purification . h and c nmr spectra were recorded on a bruker av-400 spectrometer at 400 mhz and bruker av-500 spectrometer at 125 mhz . the low or high resolution of esi - ms was recorded on an agilent 1200 hplc - msd mass spectrometer or applied biosystems q - star elite esi - lc - ms / ms mass spectrometer , respectively . the purity of compounds was determined to be over 95% ( > 95% ) by reverse - phase high performance liquid chromatography ( hplc ) analysis . hplc instrument : dionex summit hplc ( column , diamonsil c18 , 5.0 m , 4.6 mm 250 mm ( dikma technologies ) ; detector , pda-100 photodiode array ; injector , asi-100 autoinjector ; pump , p-680a ) . elution : 85% meoh in water with 0.1% modifier ( ammonia , v / v ) ; flow rate , 1.0 ml / min .

the structure - based design of 1 , 2 , 3 , 4-tetrahydroisoquinoline derivatives as selective ddr1 inhibitors is reported . one of the representative compounds , 6j , binds to ddr1 with a kd value of 4.7 nm and suppresses its kinase activity with an ic50 value of 9.4 nm , but it is significantly less potent for a panel of 400 nonmutated kinases . 6j also demonstrated reasonable pharmacokinetic properties and a promising oral therapeutic effect in a bleomycin - induced mouse pulmonary fibrosis model .

the nasal cavity and paranasal sinuses - including the maxillary , ethmoid , sphenoid , and frontal sinuses are collectively referred to as the sinonasal tract . although the sinonasal tract and nasopharynx have identical - appearing ciliated respiratory epithelium , the epithelium of the sinonasal tract is ectodermally derived , while that of nasopharynx is endodermally derived . this embryologic difference may be a factor in the development of certain epithelial lesions unique to this surface . carcinomas of the nasal cavity and paranasal sinuses account for 0.2 - 0.8% of all malignant neoplasms and 3% of those occurring in the head and neck . fine needle aspiration ( fna ) of paranasal sinus lesions is difficult due to closed architecture , and only one study has been documented in the literature . the present study was taken to find the efficacy of intra - operative cytology and frozen section taking paraffin - embedded histology as gold standard for frequently encountered tumor and tumor - like lesions of the nose and paranasal sinuses . thirty cases of tumor and tumor - like lesions of nose and paranasal sinuses were included in the study . of these,20 were non - neoplastic and 10 were neoplastic . the most common age group both in males and females involved was 20 - 30 years with the mean age of presentation being 30.5 years . cytological smears were prepared by imprint / squash methods from the representative tissue pieces and stained with giemsa and papanicolaou stain . a rapid opinion regarding the benign or malignant nature of the lesion and the type of tumor was given . imprint smears were prepared from the material obtained by blotting the specimen by a filter paper and lightly touching and rolling it on a clean , grease - free glass slide . for squash smear , small fragments of tissue were kept between two slides , which were then pulled apart gently . in nose and paranasal sinuses lesion , squash smear preparation was little difficult because of presence of mucus , which made the tissue slippery . the smears thus prepared were immediately fixed in 95% ethyl alcohol for 30 seconds and stained with papanicolaou stain . thirty cases of tumor and tumor - like lesions of nose and paranasal sinuses were included in the study . of these , most common site of involvement was left nasal cavity ( 50% ) followed by right nasal cavity ( 40% ) and paranasal sinuses ( 10% ) . benign lesions included nasopharyngeal angiofibroma , and malignant lesions included 4 cases of squamous cell carcinoma . non - neoplastic lesions were seen in 12 males and 8 females in age group of 11 - 50 years . all cases were described as non - neoplastic lesion on imprint [ figure 1a ] and squash cytological smear technique using giemsa stain . in imprint smears , one case had markedly increased cellularity of pseudo stratified epithelial cells suggestive of epithelial hyperplasia and in 4 cases along with columnar cells , clusters of squamous epithelial cells were also seen , which was suggestive of squamous metaplasia . one case showed few large hyperchromatic atypical cells , leading to suspicion of neoplastic lesion in squash smear . ( a ) imprint from a nasal polyp showing clusters of columnar epithelial cells ( giemsa , 400 ) , ( b ) polyp with pseudo stratified squamous epithelial lining with edematous stroma ( paraffin block ; h and e , 100 ) ; ( c ) imprint from angiofibroma showing columnar epithelial cells , endothelial cells and fibroblasts ( giemsa , 400 ) ; ( d ) numerous thin walled blood vessels with fibrous stroma ( frozen section ; h and e , 100 ) on frozen section , in 2 cases , morphological details of cells were not clear and one case showed only degenerative tissue . this may be due to improper technique . in one case , underlying glands showed multilayered glandular lining with cellular change that gave rise to suspicion of neoplastic lesion . one case showed focal area of epithelial hyperplasia , which corresponded to increased cellularity seen in imprint smear . cases with degenerative tissue on frozen section showed areas of squamous metaplasia with focal areas of degeneration on histological examination . case with increased cellularity on imprint smear and focal areas of hyperplasia on frozen section showed no area of hyperplasia on histological examination [ figure 1b ] . nasopharyngeal angiofibroma was seen in 6 cases in males of age group of 11 - 20 years . all cases were described as benign lesions on imprint [ figure 1c ] and squash cytological smear technique . on frozen section [ figure 1d ] , all the cases were described as benign lesions that correlated well with diagnosis by h and e section . on histological examination , all cases were diagnosed as nasopharyngeal angiofibroma . squamous cell carcinoma was seen in 4 cases ; 2 in male and 2 in female of age group of 40 - 50 years . all cases were diagnosed by imprint and squash cytological smear technique as malignant lesions . on frozen section , all cases were described as malignant lesions correlated well with histological diagnosis of non - keratinizing squamous cell carcinoma on paraffin - embedded blocks [ figure 2a - d ] . ( a ) clusters of pleomorphic hyperchromatic squamous cells ( imprint ; giemsa , 400 ) ; ( b ) clusters of pleomorphic hyperchromatic cells with high nuclear : cytoplasmic ratio . ( squash smear ; giemsa , 400 ) ; ( c ) frozen section suggestive of malignant lesion probably squamous cell carcinoma ( h and e , 100 ) ; ( d ) section from paraffin block suggestive of non keratinizing squamous cell carcinoma ( h and e , 100 ) sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy of imprint smears , squash smears , and frozen section were calculated taking histology as gold standard . the sensitivity , specificity , positive predictive value , and negative predictive value , of imprint cytology , squash cytology , and frozen section were 100% , 100% , 100% , and 100% ; 100% , 96.15% , 80.0% , and 100% ; and 100% , 96.15% , 80.0% , and 100% , respectively . overall diagnostic accuracy of imprint smears , squash smears , and frozen section were 100% , 96.7% , and 96.7% , respectively . out of the two cytological technique imprint smears had better specificity as cells morphology could be more accurately appreciated as compared to squash smears [ table 1 ] . comparison of sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy on imprint , squash smears , and frozen section non - neoplastic lesions were seen in 12 males and 8 females in age group of 11 - 50 years . all cases were described as non - neoplastic lesion on imprint [ figure 1a ] and squash cytological smear technique using giemsa stain . in imprint smears , one case had markedly increased cellularity of pseudo stratified epithelial cells suggestive of epithelial hyperplasia and in 4 cases along with columnar cells , clusters of squamous epithelial cells were also seen , which was suggestive of squamous metaplasia . one case showed few large hyperchromatic atypical cells , leading to suspicion of neoplastic lesion in squash smear . ( a ) imprint from a nasal polyp showing clusters of columnar epithelial cells ( giemsa , 400 ) , ( b ) polyp with pseudo stratified squamous epithelial lining with edematous stroma ( paraffin block ; h and e , 100 ) ; ( c ) imprint from angiofibroma showing columnar epithelial cells , endothelial cells and fibroblasts ( giemsa , 400 ) ; ( d ) numerous thin walled blood vessels with fibrous stroma ( frozen section ; h and e , 100 ) on frozen section , in 2 cases , morphological details of cells were not clear and one case showed only degenerative tissue . this may be due to improper technique . in one case , underlying glands showed multilayered glandular lining with cellular change that gave rise to suspicion of neoplastic lesion . one case showed focal area of epithelial hyperplasia , which corresponded to increased cellularity seen in imprint smear . cases with degenerative tissue on frozen section showed areas of squamous metaplasia with focal areas of degeneration on histological examination . case with increased cellularity on imprint smear and focal areas of hyperplasia on frozen section showed no area of hyperplasia on histological examination [ figure 1b ] . non - neoplastic lesions were seen in 12 males and 8 females in age group of 11 - 50 years . all cases were described as non - neoplastic lesion on imprint [ figure 1a ] and squash cytological smear technique using giemsa stain . in imprint smears , one case had markedly increased cellularity of pseudo stratified epithelial cells suggestive of epithelial hyperplasia and in 4 cases along with columnar cells , clusters of squamous epithelial cells were also seen , which was suggestive of squamous metaplasia . one case showed few large hyperchromatic atypical cells , leading to suspicion of neoplastic lesion in squash smear . ( a ) imprint from a nasal polyp showing clusters of columnar epithelial cells ( giemsa , 400 ) , ( b ) polyp with pseudo stratified squamous epithelial lining with edematous stroma ( paraffin block ; h and e , 100 ) ; ( c ) imprint from angiofibroma showing columnar epithelial cells , endothelial cells and fibroblasts ( giemsa , 400 ) ; ( d ) numerous thin walled blood vessels with fibrous stroma ( frozen section ; h and e , 100 ) on frozen section , in 2 cases , morphological details of cells were not clear and one case showed only degenerative tissue . this may be due to improper technique . in one case , underlying glands showed multilayered glandular lining with cellular change that gave rise to suspicion of neoplastic lesion . one case showed focal area of epithelial hyperplasia , which corresponded to increased cellularity seen in imprint smear . cases with degenerative tissue on frozen section showed areas of squamous metaplasia with focal areas of degeneration on histological examination . case with increased cellularity on imprint smear and focal areas of hyperplasia on frozen section showed no area of hyperplasia on histological examination [ figure 1b ] . nasopharyngeal angiofibroma was seen in 6 cases in males of age group of 11 - 20 years . all cases were described as benign lesions on imprint [ figure 1c ] and squash cytological smear technique . on frozen section [ figure 1d ] , all the cases were described as benign lesions that correlated well with diagnosis by h and e section . nasopharyngeal angiofibroma was seen in 6 cases in males of age group of 11 - 20 years . all cases were described as benign lesions on imprint [ figure 1c ] and squash cytological smear technique . on frozen section [ figure 1d ] , all the cases were described as benign lesions that correlated well with diagnosis by h and e section . squamous cell carcinoma was seen in 4 cases ; 2 in male and 2 in female of age group of 40 - 50 years . all cases were diagnosed by imprint and squash cytological smear technique as malignant lesions . on frozen section , all cases were described as malignant lesions correlated well with histological diagnosis of non - keratinizing squamous cell carcinoma on paraffin - embedded blocks [ figure 2a - d ] . ( a ) clusters of pleomorphic hyperchromatic squamous cells ( imprint ; giemsa , 400 ) ; ( b ) clusters of pleomorphic hyperchromatic cells with high nuclear : cytoplasmic ratio . ( squash smear ; giemsa , 400 ) ; ( c ) frozen section suggestive of malignant lesion probably squamous cell carcinoma ( h and e , 100 ) ; ( d ) section from paraffin block suggestive of non keratinizing squamous cell carcinoma ( h and e , 100 ) sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy of imprint smears , squash smears , and frozen section were calculated taking histology as gold standard . the sensitivity , specificity , positive predictive value , and negative predictive value , of imprint cytology , squash cytology , and frozen section were 100% , 100% , 100% , and 100% ; 100% , 96.15% , 80.0% , and 100% ; and 100% , 96.15% , 80.0% , and 100% , respectively . overall diagnostic accuracy of imprint smears , squash smears , and frozen section were 100% , 96.7% , and 96.7% , respectively . out of the two cytological technique imprint smears had better specificity as cells morphology could be more accurately appreciated as compared to squash smears [ table 1 ] . comparison of sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy on imprint , squash smears , and frozen section squamous cell carcinoma was seen in 4 cases ; 2 in male and 2 in female of age group of 40 - 50 years . all cases were diagnosed by imprint and squash cytological smear technique as malignant lesions . on frozen section , all cases were described as malignant lesions correlated well with histological diagnosis of non - keratinizing squamous cell carcinoma on paraffin - embedded blocks [ figure 2a - d ] . ( a ) clusters of pleomorphic hyperchromatic squamous cells ( imprint ; giemsa , 400 ) ; ( b ) clusters of pleomorphic hyperchromatic cells with high nuclear : cytoplasmic ratio . ( squash smear ; giemsa , 400 ) ; ( c ) frozen section suggestive of malignant lesion probably squamous cell carcinoma ( h and e , 100 ) ; ( d ) section from paraffin block suggestive of non keratinizing squamous cell carcinoma ( h and e , 100 ) sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy of imprint smears , squash smears , and frozen section were calculated taking histology as gold standard . the sensitivity , specificity , positive predictive value , and negative predictive value , of imprint cytology , squash cytology , and frozen section were 100% , 100% , 100% , and 100% ; 100% , 96.15% , 80.0% , and 100% ; and 100% , 96.15% , 80.0% , and 100% , respectively . overall diagnostic accuracy of imprint smears , squash smears , and frozen section were 100% , 96.7% , and 96.7% , respectively . out of the two cytological technique imprint smears had better specificity as cells morphology could be more accurately appreciated as compared to squash smears [ table 1 ] . comparison of sensitivity , specificity , positive predictive value , negative predictive value , and diagnostic accuracy on imprint , squash smears , and frozen section exfoliative cytology is based on spontaneous shedding of cells derived from the lining of an organ into a body cavity ; hence , they can be removed by non - abrasive means . organs usually applied for exfoliative cytology are female genital tract , respiratory tract , urinary tract , effusions . abrasive cytology is based on purpose to enrich the sample with cells obtained directly from the surface of the target organ . various instruments like cervical scraper or spatula , gastric balloon , esophageal balloons , and number of brushing instruments with or without fiberoptic guidance are used in abrasive cytology . fine needle aspiration cytology is a method where a fine needle is used to remove a sample of cells from a suspicious mass for diagnostic purposes . fnac is regarded as minimally invasive , cost - effective technique with diagnostic accuracy in the range of 90 - 99% . because of early and rapid diagnosis , high sensitivity , specificity , and cost - effectiveness , the use of intra - operative cytology and frozen section is rapidly increasing . these are , to provide rapid gross or microscopic diagnosis for intra- or peri - operative patient management , to optimally process tissue for special studies to be used for diagnosis , treatment , or research , and to confirm lesional tissue is present for diagnosis . combinations of frozen sections and cytological imprint preparation , squash , or crush preparation are often used in intra - operative diagnosis . the nasal cavity and paranasal sinuses are exposed to a variety of infections , chemically irritating and antigenically stimulating , mechanical , and traumatic influences . as a consequence of these exposures , the nose and paranasal sinuses accounts for less than 1% of all malignant tumors in the general and not more than 3% of the head and neck region malignancies . initially , most of the cytological studies in nose and paranasal sinuses were done by collection of secretion / exfoliated cells . belic et al . , evaluated the cytologic examination of secretions of nose and maxillary sinuses in patients with naso - sinusal disorders . they concluded that cytological examination of secretions of the nose and maxillary sinuses can diagnose chronic bacterial inflammation with great accuracy as compared to the allergic process , but for documentation of malignancy , cytology is not a reliable diagnostic method . they further observed that cytologic examination of secretions of the nose and maxillary sinuses may be used as an additional method to indicate possible etiologic factors and to direct further diagnostic steps . bogaerts et al . and voss et al . , studied patients of chronic sinusitis and the patients who had an increase d incidence of carcinoma , respectively , by exfoliative cytology . they concluded that exfoliative cytology was found to be a reliable means of detecting the differential diagnosis of exudative rhinopathy and preneoplastic changes in the nasal mucosa of individuals in selected risk groups . maru et al . , studied the 151 patients of various inflammatory , benign , and malignant conditions in rhinological problems and compared brush cytology with histo - pathological examination . they found that brush cytology method can be used as an adjuvant to clinical diagnosis and histo - pathological diagnosis . they concluded that variety of non - neoplastic and neoplastic conditions can involve the sinonasal region . fnac is a reliable diagnostic procedure in a good number of cases . to the best of our knowledge , there is no well - documented study in literature searched , describing the efficacy of intra - operative cytology in the lesions of nose and paranasal sinuses . the utility of intra - operative cytology and frozen section of various neoplastic and non - neoplastic lesion of the body for assisting the operating surgeon by providing an accurate and rapid diagnosis and intra - operative cytology has gained popularity in the last two decades . intra - operative cytologic evaluation has emerged as an accurate , simple , and rapid diagnostic tool in the interpretation of neoplastic and non - neoplastic conditions . well - prepared cytologic preparation may eliminate the need for frozen section or at least influence its interpretation . kim et al.,10 ] evaluated 664 specimens retrospectively by intra - operative imprint cytology to find its significance as a diagnostic adjunct . they concluded that the use of intra - operative cytology demonstrated the technique to be of value in providing abbreviated preparation time ( 3 - 5 min ) , supportive diagnostic information when frozen section was equivocal or when frozen - section evaluation could not be done ( e.g. , excessively small sample ) . it further adds contributory information for final diagnosis on difficult cases and was found to be excellent teaching material for cytopathology . a frozen section is an important intra - operative consultation for rapid diagnosis of specimens while a patient is undergoing surgery , usually under general anesthesia . liu et al . , had investigated the utility of intra - operative touch preparation with comparison of frozen section in 122 cases taken from various sites . the rate of correct diagnosis for touch preparation was 88.5% as compared to 86.1% for frozen section . scucchi et al . , studied the 2250 intra - operative cytology with frozen section with the final diagnosis on paraffin - embedded sections . the sensitivity and specificity combined intra - operative cytology and frozen sections were respectively 98.2% and 100% . for frozen section , the sensitivity was 89.9% and specificity was 97.9% as compared to the touch cytology , which had a sensitivity of 94.9% and specificity of 96.8% . the combined diagnostic accuracy of intra - operative cytology and frozen section to distinguish benign from malignant lesions was 99.2% . guarda la also compared the results of intra - operative cytology and frozen section taken from various organs of body and found that the accuracy of cytology and frozen section were 98.4% and 99.2% , respectively . in some of the earlier studies from our center , the diagnostic accuracy of imprint cytology was found to be 98% , 98.5% , and 83.3% for neoplastic lesions of esophagus , colo - rectum , and ovary , respectively . in another study , diagnostic accuracy of imprint and squash cytology for diagnosing cns malignancy was found to be 92.0% and 89.3% , respectively . the combined efficacy was 94.6% . in our study , the sensitivity , specificity , and positive predictive value of imprint cytology , squash cytology , and frozen section were 100% , 100% , and 100% ; 100% , 96.15% , and 80.0% ; and 100% , 96.15% , and 80.0% , respectively . overall diagnostic accuracy of imprint smears , squash smears , and frozen section were 100% , 96.7% , and 96.7% , respectively . imprint smears had better specificity as cells morphology could be more accurately appreciated and provide better cellular details and fewer artefacts in comparisons of squash smears and frozen section . the main disadvantage of squash cytology was loss of cellular and architectural details . in nose and paranasal sinuses lesion , squash smear preparation are little difficult because of presence of mucus , which make the tissue slippery . the main drawback of this study was smaller number of cases , but still by this preliminary study , it can be concluded that intra - operative cytology and frozen section examinations of lesions of nose and paranasal sinuses are useful , quick , and reliable diagnostic technique for rapid and early diagnosis in the operation theatre and can be used as an adjunct to histopathology for better management of patients . another study with larger number of cases may further help to deciding its efficacy and use in day - to - day practice .

background : the nasal cavity and paranasal sinuses - including the maxillary , ethmoid , sphenoid , and frontal sinuses are collectively referred to as the sinonasal tract . fine needle aspiration ( fna ) of paranasal sinus lesions for early diagnosis of neoplastic lesions is difficult due to closed architecture , and only one study has been documented in the literature.aims:to find the efficacy of intra - operative cytology and frozen section for frequently encountered tumor and tumor - like lesions of the nose and paranasal sinuses.materials and methods : thirty cases were included in this study . the material for cytologic examination and frozen sections were taken per - operatively . cytological smears were prepared by imprint / squash methods from the representative tissue pieces and stained with leishman - giemsa and papanicolaou . frozen section and paraffin - embedded section were stained with hematoxylin and eosin stain . a rapid opinion regarding the benign or malignant nature of the lesion and the type of tumor was given.results:the sensitivity , specificity , and positive predictive value of imprint cytology , squash cytology , and frozen section were 100% , 100% , and 100% ; 100% , 96.15% , and 80.0% ; and 100% , 96.15% , and 80.0% , respectively . overall diagnostic accuracy of imprint smears , squash smears , and frozen section was100% , 96.7% , and 96.7% , respectively . imprint smears had better specificity as cells morphology could be more accurately appreciated.conclusions:intra-operative cytology and frozen section examinations of lesions of nose and paranasal sinuses are useful , quick , and reliable diagnostic technique for rapid diagnosis in the operation theatre and can be used as an adjunct to histopathology for better management of patients .

a 64-year - old man , weighing 47 kg and having a height of 154 cm , was scheduled for a pericardiectomy for treatment of constrictive pericarditis . when he was hospitalized with a two - week history of aggravating dyspnea , he had been placed on outpatient follow - up for pulmonary tuberculosis , previously diagnosed with dyspnea on exertion and pleural effusion . his past medical history included diabetes mellitus diagnosed 20 years ago and stent insertion in the left anterior descending ( lad ) coronary artery eight years ago . preoperative ecg revealed left atrial enlargement , and st depression and flat t - wave on lead ii , iii and v4 - 6 . coronary computerized tomography demonstrated that the lad stent was intact , but there was > 50% narrowing of the left circumflex coronary artery , and pericardial thickening with adhesion to the right atrial and left ventricular lateral walls . echocardiography showed a left ventricular ejection fraction of 73% , and mild pulmonary hypertension with a systolic right ventricular pressure of 38 mmhg . an hour before surgery , premedication was done with i m morphine 3 mg . on arrival , continuous monitoring of ecg lead ii and v5 was initiated , and the radial artery cannulation was done under local anesthesia for pressure monitoring . vital signs before induction of anesthesia were hr 85/min , abp 108/68 mmhg , and spo2 98% . rocuronium 50 mg was administered to facilitate endobronchial intubation with a 35 fr left - sided double - lumen tube . after intubation , lungs were ventilated with a tidal volume of 8 - 10 ml / kg , adjusted to maintain end - tidal co2 within 33 - 38 mmhg . anesthesia was maintained with 1.0 - 1.5% sevoflurane in 60% oxygen in air and continuous infusion of remifentanil at 0.1 - 0.3g / kg / min . after induction of anesthesia , the patient was turned to the head - down position with the head rotated leftward by approximately 60 to facilitate internal jugular catheterization . an 18-g puncture needle was inserted at the conjoined site of both bellies of the scm , and was advanced toward the ipsilateral nipple until free regurgitation of venous , non - pulsatile blood flow was observed . during the first attempt , the puncture needle had to be withdrawn , as the j - tipped guide wire could be advanced < 10 cm . after 5 min of manual compression , jugular venous puncture was retried in the same manner , and non - pulsatile , free - regurgitant blood flow was again obtained . in the second attempt , the j - tipped guide wire was inserted into the vessel without significant resistance , even though it was initially difficult to advance the wire and the puncture needle had to be withdrawn a few millimeters to obtain free regurgitant blood flow . then , a 7-fr double - lumen central venous catheter ( arrow gard blue , arrow international , inc . , usa ) was inserted using the seldinger method and was suture - fixed at 16 cm in the skin after confirming free inflow of fluid . at the end of procedure , the guide wire withdrawn showed no deformations . central venous pressure ( cvp ) was measured to be 20 mmhg with the patient in a supine position and thereafter was maintained within 30 - 40 mmhg in the right semi - lateral decubitus position at approximately 30 during one - lung ventilation for surgery . such a high cvp was considered to be related to the constrictive pericarditis , as the pressure measurement was less than the mean arterial pressure of the radial artery ( 80 - 85 mmhg ) and the waveform was flat without pulsations , being affected by right - side one - ling ventilation and surgical manipulation of the heart . during the catheterization , vital signs were stable with hr 83/min , bp 90 - 100/65 - 68 mmhg , and spo2 99 - 100% . arterial blood gas analysis was done after induction of anesthesia and revealed ph 7.486 , pao2 331 mmhg , and paco2 36.2 mmhg with fio2 0.6 . approximately 50 minutes after the surgery began , the pressure waveform from the central venous catheter changed abruptly into what was seemingly an arterial waveform , with a mean pressure of 61 mmhg , almost same to the mean arterial pressure of 62 mmhg ( fig . 1 ) . simultaneously , blood regurgitated through the fluid line connected to the central venous catheter . even though about 300 ml of fluid had already been administered , arterial blood gas measurement was done on blood withdrawn from the central venous catheter , indicating a pao2 of 438.7 mmhg with a fio2 of 0.6 , confirming that it was arterial blood . 2 ) . fluid infusion was immediately stopped , and the catheter was removed when the surgery was completed . after confirming that there was no bleeding or developing hematoma in the puncture site with > 15 min of compression , the double - lumen tube was replaced with a single - lumen endotracheal tube . thereafter , the patient was transferred to the intensive care unit with the endotracheal tube in situ . extubation was done after seven days of mechanical ventilatory care for tachypnea and co2 retention , and the patient was transferred to the general ward without central venous catheter - related complications on the 10th postoperative day . arterial puncture is most frequently encountered , but rarely results in more serious complications , such as hematoma development compromising airway , thromboembolism causing cerebrovascular accident , arteriovenous fistula or pseudoaneurysm . arterial catheterization can happen after arterial puncture , but the incidence is very low ( < 0.1% ) . obesity , hypotension , reduced hemoglobin , low oxygen saturation or unavailability of ultrasonic guidance are risk factors for arterial catheterization . in healthy patients , arterial puncture is easily recognized by pulsatile regurgitation of bright red blood ; however , in patients with hypoxemia or hypotension , those visual parameters would be unreliable due to a weak pulsation of dark red blood . in contrast , patients with increased cvp , including those with congestive heart failure , tricuspid regurgitation , or constrictive pericarditis , may present with forced venous regurgitation , which can be misinterpreted as arterial puncture . in this case , clinically significant pitfalls on differentiation of arterial puncture are the features of the regurgitant blood flow , including color , pulsation , force , or velocity . as fio2 had been maintained at 0.6 after induction of anesthesia , it was difficult to macroscopically discriminate between arterial and venous blood . also , we already anticipated increased blood regurgitation due to the elevated diastolic pressure caused by restrictive physiology of pericarditis . it would be highly suspicious of an arterial puncture if massive regurgitation of bright red blood with pulsation or large hematoma was observed ; however , in this case , those findings were not definitive . first , overlap between the carotid artery and jugular vein could be increased with leftward head rotation . together with a relatively deep insertion of the puncture needle , such an increased overlapping could facilitate a penetration of the anterior wall of the carotid artery through the jugular vein . . demonstrated that when the head was maximally rotated leftward without discomfort , an overlap of > 75% was observed in 54% of patients , especially in those with advanced age ( > 60 yr ) and venous dilation . kim et al . also found that the degree of overlap between the carotid artery and jugular vein increased with leftward head rotation in korean patients . considering the small build of our patient , puncture needle could be inserted relatively deep , and eventually penetrate the anterior wall of the carotid artery after advanced through the jugular vein . and non - deformed guide wire withdrawn could be another evidence of carotid puncture , not subclavian artery . second , multiple attempts at venous puncture are known to increase the incidence of mechanical complications of cvc , reportedly increasing by about 10-fold after at least two unsuccessful attempts even with an experienced physician . the ultrasound - guided method is reported to be helpful to reduce the complications . it is unclear why the pressure waveform abruptly changed 1 hr after the arterial catheterization . non - pulsatile , plain waveform , mimicking venous property despite arterial insertion , might be attributed to kinking of the catheter or partial obstruction of the catheter tip against the vessel wall . some measurement errors in pressure transducer system , such as excessive damping , could result in such a plain arterial waveform . abrupt change in pressure waveform and measurement would be attributed to a sudden release of previously kinked catheter or position shift of catheter due to surgical manipulation of the heart . hwang and kim reported a case of gradual increase in cvp from 34 - 37 mmhg when the catheter was indwelled , to almost the same to the mean arterial pressure . similar to our case , they confirmed arterial insertion via arterial blood gas measurement because the pressure waveform did not appear to be arterial and they assumed damping to be a primary cause . they accomplished the catheterization at the first attempt , and presented no difficulties during catheterization . proper discrimination between the unmasked arterial and central venous waveforms could be difficult due to atrial fibrillation , which presumably resulted from hypotension during induction in their patient with severe coronary artery disease . unlike their case , we observed the abrupt change of previously plain waveform to arterial waveform ( fig . 3 ) , requiring no comparison of arterial gas measurement with venous blood . recently , percutaneous / intravascular intervention with a hemostatic device was shown to be safe and applicable treatment for arterial trauma . in this case , however , the catheter was removed and then manually compressed . this can sometimes cause bleeding , cerebrovascular accident or pseudoaneurysm , the risk of which , however , is dependent on the catheter size , elapsed time from catheterization , and the site of catheterization . because the arterial catheterization was maintained briefly , no hematoma formation in the aorta was confirmed with tee , and the internal jugular vein could be compressed more easily than the subclavian vein , the removal and compression were judged to be appropriate . the concern was the possibility of surgical field contamination ; the removal and compression were done after the surgery was finished . when visual discrimination between arterial and venous blood regurgitation is unreliable , anesthesiologists should confirm where one punctures using all the available methods one has on the scene , especially after at least two unsuccessful attempts or in patients with advanced age or clinical conditions resulting in jugular venous dilation . to prevent arterial catheterization , one should limit the leftward rotation of the head by < 40 and consider using ultrasound - guided method after more than two unsuccessful attempts .

we present a case of inadvertent arterial insertion of a central venous catheter , identified during a pericardiectomy procedure after observing abrupt changes in pressure waveform and confirmed via arterial blood gas analysis and transesophageal echocardiography . central venous pressure measurement was initially 20 mmhg in supine , and then elevated to 30 - 40 mmhg in right lateral decubitus , presumably resulting from constrictive physiology of pericarditis . the pressure waveforms , however , abruptly changed from a venous to an arterial waveform during surgery . when visual discrimination between arterial and venous blood regurgitation is unreliable , anesthesiologists should confirm that using all the available methods one has on the scene , especially after at least two unsuccessful attempts or in patients with advanced age or clinical conditions resulting in jugular venous dilation . to prevent arterial catheterization , one should limit the leftward rotation of the head by < 40 and consider using ultrasound - guided method after more than two unsuccessful attempts .

in recent years , three major alterations in the perception of gluten toxicity and cd have occurred . firstly , the definition of cd has evolved with the advent of new testing strategies , and a broader view of cd has been brought forward , encompassing non - classic cd presentations increasingly seen both in adults and in children . secondly , the diagnostic approach to cd has changed , with the appearance of new diagnostic guidelines , which in selected cases of children and adolescents give the opportunity to make the diagnosis of cd without a duodenal biopsy . other new guidelines directed particularly at adults keep the duodenal biopsy as an essential part of the diagnosis . an increasing percentage of otherwise - healthy individuals in europe , the us , and australasia have adopted a gluten - free diet as part of their lifestyle , suggesting but in no way proving that ncgs is a real entity . this article will discuss the features that separate ncgs from cd and investigate the evidence base for the presence of ncgs , including studies that support ncgs as well as studies that stress the limitations of ncgs ( table 1 ) . abbreviations : dbpcfc , double - blind placebo - controlled food challenge ; ig , immunoglobulin ; ncgs , non - celiac gluten sensitivity ; tg2 , transglutaminase . cd may be defined as a chronic small intestinal immune - mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals . the old definitions included the term typical versus atypical , which now is discouraged , and the term latent cd , which also is discouraged . cd is characterized by positivity of immunoglobulin a ( iga ) tissue transglutaminase 2 ( tg2 ) antibodies in serum , which is currently the most powerful screening test for cd . the presence of hla - dq2 ( dqa1 * 0501 , * 0505 ) or hla - dq8 ( dqb1 * 0201 , * 0202 ) may support the cd diagnosis , but in most western populations these hla types exist in 25% to 30% of healthy subjects . conversely , the absence of these hla types is a very strong negative predictor for cd , particularly useful in those already on a gluten - free diet . still , the characteristic findings in the duodenal mucosa with crypt hypertrophy and villous atrophy form a mainstay in the diagnosis . in the spectrum of histological changes , it is the presence of villous atrophy that is most indicative of cd , whereas crypt hypertrophy alone or intraepithelial lymphocytosis is much less predictive of cd . indeed , intraepithelial lymphocytosis is more common in other non - gluten - related conditions and in clinically normal subjects . wheat allergy is a distinct entity , characterized by an ige - mediated response to wheat . ncgs should largely be considered only when tests for cd are negative , absent tg2-iga on a normal diet , absence of the dependence of hla dq2 or dq8 , which may be slightly increased in patients with ncgs . also , the characteristic histological findings in the duodenal mucosa are absent . as to the distinction from wheat allergy , ige - wheat antibodies are absent in ncgs . the symptoms of ncgs most resemble those of irritable bowel syndrome ( ibs ) with bowel movement and abdominal pain ( figure 1 ) . ncgs has been claimed to be a cause of psychiatric conditions , including autism and schizophrenia [ 10 - 12 ] . proving the role of gluten or even wheat in the genesis of symptoms has been challenging . dbpcfcs with gluten / wheat have been used to document ncgs ( or non - celiac wheat sensitivity ) in adult patients . their patient population was retrospectively collected within the out - patient group of ibs , and the authors performed dbpcfcs with wheat in a total of 276 patients . gluten sensitivity was documented by subject report of symptoms by using a visual analogue score ( vas ) rather than objective findings . with a similar design , an australian group performed dbpcfcs in a group of patients who claimed gluten sensitivity , again based on a vas . however , in a follow - up study , the research group was able to demonstrate that the active component of the sensitivity was not the gluten fraction of wheat but rather in the so - called fodmaps ( fermentable oligo- , di- , mono - saccharides and polyols ) . these data raise a general word of caution to the purity of the ingredients in the challenge material . furthermore , they underscore the drawbacks of relying on subjective findings rather than on objective findings , which are sadly lacking for ncgs , even when challenges are carried out in a blinded fashion . abbreviations : ibs , irritable bowel syndrome ; ncgs , non - celiac gluten sensitivity . in children , circumstantial evidence and retrospective data collected from hospital files suggest that a considerable proportion of children suspected of cd , but without serological and histological evidence of cd , may have ncgs . it may be difficult to use a vas , in particular in younger children . one thorough study has been published ; however , to the best of our knowledge , no dbpcfcs have been performed in children with gastroenterological symptoms of ncgs . one study was performed in children with autism , which suggests that the symptoms may be ameliorated by a gluten - free diet . despite the popular interest in gluten and gluten - related disorders , studies of the biological mechanisms to support ncgs are scarce . some reports have described increased cellularity in the intestinal mucosa and increased immune reactivity in the mucosa in patients with ncgs . in a norwegian study , biopsy material from patients with ncgs was compared with material from patients with cd . production of cytokines interferon - gamma ( ifn- ) and myxovirus resistance a ( mxa)was clearly increased in patients with cd , whereas a less marked change in the patients with ncgs was increased cellularity of cd3-positive t cells , with an increase in mrna for ifn- after challenge with gluten . amylase trypsin inhibitors ( atis ) have been identified in wheat as potent stimulators of innate immune reactions , notably the stimulation of toll - like receptor 4 ( tlr4 ) in monocytes , macrophages , and dendritic cells , which release a number of cytokines , including interleukin-8 ( il-8 ) and tumor necrosis factor - alpha ( tnf- ) , in blood from patients with cd as well as healthy subjects and in intestinal biopsies from patients with cd . these data suggest an activation avenue that applies both to cd as well as to ncgs or other inflammatory conditions . however , no further documentation that atis are involved in ncgs has been provided . as no consistent biomarkers are available for ncgs , the diagnosis must rely on clinical symptoms and signs ( for example , with a vas ) but preferably with objective findings . such a diagnosis should be based on dbpcfc , except in infants and small children . the caveat in this approach is that , whereas symptoms of food allergy typically occur within the first few minutes to hours to days after the specific challenge , the symptoms of ncgs may occur after a longer time period . furthermore , the dose of gluten should equal the doses ingested in the daily food . in theory , the dbpcfc should be repeated at least twice to minimize the risk of positivity based simply on chance . it must be kept in mind that , in patients who suspect themselves of food allergy , only roughly one out of three actually has a reproducible reaction toward the suspected food , and similar numbers of positivity ( or even lower ) may be found for ncgs . certainly , a way ahead is to learn from our allergology colleagues to be proficient in dbpcfc as well as develop convenient new practical designs of dbpcfc and at the same time continue the search for accurate biomarkers . steffen husby has participated in an advisory board with tillotts pharma , has acted as a speaker for thermo fisher scientific , and has received grant support from the danish strategic research council , the region of southern denmark , and odense university hospital research funds . joseph murray has received grant support from alba therapeutics , alvine pharmaceuticals , inc . , and the national institutes of health . he serves on the advisory board of alvine pharmaceuticals , inc . and is a consultant to amag pharmaceuticals , entera health , inc . , glenmark , sonomaceuticals , llc , biolinerx , glaxosmithkline , and genentech . he received payment for development of educational presentations from the national foundation for celiac awareness .

non - celiac gluten sensitivity ( ncgs ) has been introduced recently as a potentially common disease on the basis of studies of patients with claimed reactivity to gluten but without the characteristics of celiac disease ( cd ) . cd is characterized by antibody reactivity toward the autoantigen transglutaminase 2 , characteristic histological abnormalities of the small intestine , and an almost obligatory genetic haplotype ( hla - dq2 or dq8 ) . the diagnosis of ncgs is based largely on the clinical suspicion of hyper - reactivity to gluten and the absence of the characteristics of cd . few published studies have used double - blind placebo - controlled food challenges ( dbpcfcs ) for the diagnosis of ncgs , and none in children . innate immune reactivity to amylase trypsin inhibitors has been suggested as the pathogenic principle in ncgs , but confirmatory evidence is lacking . also , further clinical studies including dbpcfcs are needed .

forensic odontology has established itself as an important and often indispensible science in medicolegal matters and identification of the dead . the forensic importance of dental tissue has been well recognized because tooth is the hardest of all human tissues . they are well preserved for a long period even after death , hence dental remains are the most stable biological evidence encountered in crime cases and yield useful information . blood grouping has been one of the major factors for identification of biological materials in forensic investigations and is a widely used technique in forensic laboratories . the presence of abo blood group and rhesus factor is applied to inherited antigens detected on red cell surface by specific antibodies . once the blood group and rhesus factor are established , it remains unchanged throughout life . kind in 1960 discovered the presence of abo blood group in saliva by absorption - elution ( ae ) method . blood grouping from dried stain by elution procedure was described more than 50 years ago but not employed widely in forensic serology , until 1960 , when kind refined this technique . ae technique / procedure originally devised by siracusa is now employed in all forensic laboratories because it is proven to be most sensitive , reliable and reproducible . pulp tissue is one of the most protected tissues being surrounded from all sides by dental hard tissues . pulp contains numerous blood vessels and blood group antigens are certainly present in tooth pulp . the distribution of abo substances from the pulp cavity wall to the dentin edge and the enamel gradually reduces , because of fewer possibilities of diffusion of antigens from both blood and saliva . the existence of blood group antigens in tooth dentin and enamel and their nature has been substantiated by infusion sedimentation phenomena combined with inherently present antigens . this theory describes the infusion of water - soluble antigens from saliva into the tooth tissue . the presence of abo blood group and rhesus factor antigen in soft and hard dental tissues makes a possible contribution in human identification even in decomposed bodies . therefore , blood group and rhesus factor determination for biological evidence on tooth material is of great importance in forensic odontology . the aim of this study was to determine the abo blood grouping and rhesus factor from dentin and pulp of extracted teeth using ae technique at 0 , 3 , 6 , 9 and 12 months after extraction . a brief case history with relevant medical history was recorded from patients selected for study and consent was taken . the blood groups were determined for all the study participants using capillary blood by slide agglutination method . carious teeth and grossly decayed teeth were excluded ; and teeth extracted for periodontal , and orthodontic purposes were included in the study . the extraction procedure was carried out under local anesthesia following the aseptic protocol in the department of oral and maxillofacial surgery . the extracted teeth were dried and stored in labeled bottles for a span of 3 , 6 , 9 and 12 months . the pulp was scooped with a spoon excavator and dentin was powdered using a straight fissure bur [ figure 1 ] . the blood grouping from teeth was performed by ae technique using powdered dentin and dental pulp . the pulverized dentin powder and pulp were divided into three equal parts and were taken into six sterile test tubes containing 2 ml of saline and labeled , respectively . to each of these test tubes , three drops of antiserum a , b , d was added respectively , and the test samples were sufficiently soaked with antiserum for 2 h and left standing at room temperature . each sample was washed five times with cold saline solution by centrifuging it at 3000 rpm for 5 min and the supernatant was removed with pipette . then two drops of fresh saline were added to the sample , and the test tubes were heated in a water bath at a temperature of 5055c for 10 min to elude the antibodies . ( a ) dissected teeth samples , ( b ) pulverized dentine and ( c ) dried pulp tissue a drop of 0.5% red cell suspension of known blood group a , b and o was freshly prepared and immediately put into respective test tubes . the samples were incubated at 37c for 30 min to enhance agglutination , and then , it was centrifuged at 15002000 rpm for 1 min . by gentle shaking of the test tube , the presence or absence of red cell agglutination was ascertained macroscopically and microscopically at a magnification of 4 [ figures 2 and 3 ] . macroscopic observation of the agglutination in dentin ( l ) and pulp ( r ) samples microscopic observation of the agglutination in dentin ( l , unstained , x100 ) and pulp ( r , unstained , x400 ) samples ( 4 ) data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for small sample . two - tailed p < 0.05 was considered as statistically significant , and 0.01 was considered as highly statistically significant . data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for small sample . two - tailed p < 0.05 was considered as statistically significant , and 0.01 was considered as highly statistically significant . abo blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . in dentine , the sensitivity ranged from 100% to 73% and pulp sensitivity ranged from 100% to 80% . there was no significant difference in the sensitivities of dentin and pulp [ table 1 ] . abo blood grouping for pulp and dentine rh factor grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased up to 9 months . in dentine , the sensitivity ranged from 100% to 40% and pulp sensitivity ranged from 100% to 23% . overall pulp showed better sensitivity than dentin except at 12 months time period where dentin showed better sensitivity than pulp . moreover , in comparison with the sensitivities of dentin and pulp , the p values obtained were not significant [ table 2 ] . rh factor blood grouping for pulp and dentine when compared the sensitivity of abo and rh factor blood grouping of dentin and pulp in accordance to time period dentin and pulp showed almost similar results from 0 to 9 months . at 12 months both dentin and pulp showed vast difference in the sensitivities of abo and rh blood grouping with significant p value [ table 3 ] . comparison of abo and rh factor blood grouping for dentin and pulp the overall mean sensitivity of dentin for abo blood grouping is 88% and for rh factor , blood grouping is 79% , with a significant p = 0.03 . the overall mean sensitivity of pulp for abo blood grouping is 90% and for rh factor , blood grouping is 82% with a significant p = 0.04 . dentin and pulp showed the p = 0.58 for abo blood grouping and 0.46 for rh factor blood grouping [ table 4 and figure 4 ] . overall comparison of blood group systems and teeth components graph of comparisons of the overall sensitivity of abo and rh factor blood grouping of dentin and pulp lattes have rightly said the fact that belonging to a definite blood group is a fixed character of every human being and can be altered neither by lapse of time nor by intercurrent disease . human identification is the mainstay of civilization , and the identification of unknown individuals has always been of paramount importance to society . the use of blood group substances in medico - legal examination is grounded on the fact that once a blood group is established in an individual , it remains unchanged for a lifetime . for several decades , the technique ae is the most sensitive and the most widely employed . according to kind , nickolls and periera and outtridge , ae has proved to be markedly more sensitive than the absorption - inhibition test . ae has shown more success rate than mixed - agglutination for certain antigens . rh blood group is considered to be the most complex genetically of all the blood type systems since it involves 45 different antigens on the surface of red cell . teeth are used for blood grouping and are considered as a hallmark for identification of biological materials in forensic investigations . considering this fact in our present study , an attempt was made to detect abo and rh factor antigen from dentin and pulp over a time period of 0 , 3 , 6 , 9 and 12 months . for abo blood grouping , both dentin and pulp showed 100% sensitivity with the samples tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . pulp showed better sensitivity than dentin but the difference was statistically insignificant , suggesting both dentin and pulp have almost equal antigenic potential although pulp is better , the sensitivity weakened as the time period increased . till date , the various studies conducted by smeets et al . , shetty and premlata , ballal and david and ramnarayan et al . at different time period showed pulp to be better tool than dentin and there was a decrease in the sensitivity of the dentin and pulp as the time periods increased . the overall decrease in the sensitivity could be due to dehydration , the loss of pulp antigens , insufficient quantity of pulp , calcification of the canals , cell lysis ; contamination of the tooth or time lapse for the procedure . for rh factor blood grouping , both dentin and pulp showed 100% sensitivity when the samples were tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . pulp showed better results than dentine , but the difference was statistically insignificant , suggesting both dentin and pulp have almost equal antigenic potential which weakened as the time period increased , similar to abo antigens . this may be attributed to autolysis , dehydration and the loss of pulp antigens similar to abo antigens . is the only study available in english language literature that was conducted for determination of rh factor antigens along with abo blood group antigens in freshly obtained pulp samples . in our study , both pulp and dentin samples were used and tested at 0 month to an extensive time period of 12 months . abo and rh factor blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . at 12 months , both dentin and pulp showed a drastic decrease in the sensitivity for rh factor blood grouping than the abo blood grouping with a statistically significant difference . the sensitivity of pulp was better than dentin in both the blood grouping systems with an insignificant p value . abo blood grouping was better than rh factor grouping in both the teeth components and the p values obtained were significant . this indicates that the antigenicity of pulp is better than dentin in both abo and rh blood groups , and abo antigens were better expressed than rh factor blood group antigens . the outcome of our study showed that abo and rh blood group antigens could be detected up to 12 months , and there are no studies in the english literature to compare this study . this shows that pulp contains more amounts of antigen than the dentin and its antigenicity decreases with time . blood grouping on teeth is not a straight forward technique ; the concentrations of blood group antigens are low in the teeth when compared to other tissues and body fluids . in this study , it is assumed that the origin of blood group antigen in dental hard tissue is based on the infusion sedimentation phenomenon combined with inherently present antigens . considering all the factors that support the presence of blood group antigens in dentin and pulp and also the pitfalls of false positive result or mistyping of blood group , over a period in this study , we came to a close consequence that the results obtained with pulp were better than that of dentine . another aspect to be highlighted here is abo and rh factor antigens were detected from both dentin and pulp even up to 12 months after extraction of the teeth . blood grouping has been one of the bases for identification of biological materials in forensic investigations and abo blood grouping is a widely used technique in forensic laboratories . the presence of abo blood group antigens along with rh factor antigens in pulp and hard dental tissues makes it a potential substance in the identification of highly decomposed bodies or body part where teeth and bones are the only significant tissue remains . blood group substances in the hard dental tissues thus remain unaffected even in adverse environmental conditions . teeth were used as a mode of identification of blood group in this study because teeth are one of the most indestructible parts of the body and exhibit the least turnover of natural structure . the presence of abo blood group and rh factor antigen in soft and hard tissue makes it possible for identification of the deceased . ae test to identify blood groups in teeth may be of immense value not only in the identification of accused but also in the investigation of mass disaster and fire victims . on the basis of results obtained in our study , both dentin and pulp are reliable sources of blood group determination for upto 12 months for abo and rh factor blood grouping , especially where teeth ensues to be the only remnants existing for individual identity . although expression of abo blood groups and rh factor was seen in both dentin and pulp , intensity of abo blood groups and rh factor was higher in pulp than dentin and abo blood group antigens were better expressed than rh factor antigens in both dentin and pulp . blood group determination from teeth warrants advance exploration as the establishment of identification of a person from the skeletal remains is of paramount importance to a forensic odontologist . till date , in english language literature no studies have been conducted to detect abo and rh factor blood group antigens from tooth material for up to 12 months . this study detected blood groups antigens of abo and rh systems from tooth material over this extensive time period of 12 months . this study is thus a quantum of what has been known and learned and how much more needs to be understood in this challenging branch of forensic odontology .

objective : the aim of the study was to determine blood groups and rhesus factor from dentin and pulp using absorption - elution ( ae ) technique in different time periods at 0 , 3 , 6 , 9 and 12 months , respectively.materials and methods : a total of 150 cases , 30 patients each at 0 , 3 , 6 , 9 and 12 months were included in the study . the samples consisted of males and females with age ranging 1360 years . patient 's blood group was checked and was considered as control . the dentin and pulp of extracted teeth were tested for the presence of abo / rh antigen , at respective time periods by ae technique.statistical analysis : data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for the small sample.results:blood group antigens of abo and rh factor were detected in dentin and pulp up to 12 months . for both abo and rh factor , dentin and pulp showed 100% sensitivity for the samples tested at 0 month and showed a gradual decrease in the sensitivity as time period increased . the sensitivity of pulp was better than dentin for both the blood grouping systems and abo blood group antigens were better detected than rh antigens.conclusion:in dentin and pulp , the antigens of abo and rh factor were detected up to 12 months but showed a progressive decrease in the antigenicity as the time period increased . when compared the results obtained of dentin and pulp in abo and rh factor grouping showed similar results with no statistical significance . the sensitivity of abo blood grouping was better than rh factor blood grouping and showed a statistically significant result .

the incidence of coronary artery anomalies is approximately 1% among patients undergoing cardiac catheterization,1 - 4 ) 0.29% among autopsy specimens,5 ) and less than 0.1% among prospective echocardiographic screenings.6 ) most anomalies are incidentally detected and do not create clinical problems.1 - 4 ) however , 19 - 33% of sudden cardiac deaths in the young population are attributable to coronary artery anomalies.7 - 9 ) an anomalous origin of the right coronary artery ( rca ) from the left sinus is a very rare anomaly , and its incidence is 0.019% to 0.49% on coronary angiography.1 - 4 ) however , recent angiographic studies have reported a relatively high incidence ( 5.6% ) of coronary artery anomalies and anomalous rca origins from the left sinus ( 0.92%).10 ) anomalous origin of the rca ( 0.17% ) is more common than anomalous origin of left coronary artery ( lca , 0.047%).1 ) coronary artery anomalies are classified as benign ( 80.6% ) but potentially serious anomalies ( 19.4% ) . potentially serious anomalies include ectopic origin from the pulmonary artery , ectopic origin from the opposite aortic sinus , single coronary artery , and large coronary fistulae.1 ) these anomalies may be associated with sudden death . anomalous origin of either the right or left main coronary artery from the contralateral sinus with an interarterial course between the aorta and the pulmonary trunk may also be associated with sudden death and may cause myocardial ischemia , arrhythmia , and syncope.11 - 19 ) there have been several reports concerning these anomalies and their attendant clinical problems in korea.20 - 25 ) the incidence of anomalous rca is higher in angiographic studies and lower in autopsy studies compared to that of anomalous lca , which is also a more common cause of sudden death ( table 1).11 - 17 ) anomalous lca is more common in young male subjects,11 - 17 ) possibly because of the longer intramural course and larger area of dependent myocardium in the left ventricle.26)27 ) the incidence of anomalous rca from the left coronary sinus differs among the races : the incidence in western countries is 27% , and the incidence in japan is 79% ( 44 of 56 patients ) . death has not been reported in relation to this anomaly in any of the 44 japanese patients . an interarterial course existed in 12 anomalous lca cases.17 ) study of the proximal structures , including the takeoff portion , is important in formulating a treatment plan,3)10 - 19)26)28 - 30 ) and many imaging tools are now available to evaluate this anomaly . this review describes the pathophysiology , imaging methods , surgical methods , and treatment of anomalous rca from the left coronary sinus with interarterial course . the pathophysiology of the restricted coronary blood flow seen in this anomaly is suggested to be as follows . the acute takeoff angle , slit - like orifice , and compression of the intramural segment by the aortic valve commissure are all thought to narrow the orifice . lateral luminal compression of the intramural portion of the coronary artery and compression of the coronary artery between the aorta and the pulmonary artery are also possible mechanisms ( fig . 1).11 - 19)28)31)32 ) some autopsy - based studies have shown that slit - like orifice structure and acute angle takeoff are more common in sudden cardiac death patients.14 - 16 ) in a mdct - based study , acute angle takeoff was correlated with luminal stenosis of the orifice.25 ) however , there is still controversy concerning the mechanism by which the interarterial course is compressed between the aorta and the pulmonary artery . an intravascular ultrasound ( ivus ) study26 ) found that luminal compression of the coronary artery was totally attributable to the aorta because the pressure of the pulmonary artery was much lower than that of the aorta . another mdct study showed that the narrowing of the orifice was more severe than that of the interarterial course was.25 ) however , the sudden cardiac death associated with this anomaly is related to severe exercise , and our study was conducted at rest , so further mechanistic evaluation is necessary . some authors insist that anomalous coronary arteries increase the risk of coronary artery obstructive disease,33)34 ) but the prevailing opinion is that the anomalous portion of the coronary artery is not vulnerable to obstructive disease,4)29)35)36 ) the incidence of concomitant congenital anomalies is 4.2 - 24% ; common anomalies include bicuspid aortic valve and mitral valve prolapse.3)25 ) the methods used to evaluate anomalous rcas include echocardiography , angiography , mdct , and mri . noninvasive tools such as mdct19)37 - 41 ) and mri42 - 45 ) can provide precise information about the complex anatomy of coronary artery anomalies , though mdct is favored due to its higher spatial resolution and rapid exam time . additionally , mdct can provide numerous multiplanar image reconstructions to permit precise evaluation of the takeoff portion and course of the anomalous coronary artery ( fig . transthoracic echocardiography provides limited information in this regard , and transesophageal echocardiography provides more information,22 ) but both of these methods are invasive . evaluation is difficult in angiography because the complex three - dimensional structure of the anomalous coronary artery is displayed in a two - dimensional plane , and selective cannulation of the anomalous coronary artery is made difficult by the small , slit - like orifice . the success rate of selective cannulation is 55 - 61%.25)39)40)41 ) this success rate is not correlated with the takeoff angle or the orifice size or shape , so the relatively low figure has been attributed to limited experience on the part of physicians , who rarely see this anomaly.25 ) mdct - guided cannulation may be useful in increasing the success rate of cannulation . ivus is also a useful method for obtaining cross - sectional luminal images,21 ) but cannulation is difficult , and this method is also invasive . mdct provides excellent information concerning orifice location and the course of the anomalous coronary artery , so mdct should be performed prior to angiography or ivus . the size and shape of the slit - like orifice differ according to image projection , so multiplanar image reconstruction is necessary in precise evaluation . angiography and echocardiography are invasive , have a relatively low cannulation success rate , and are limited with respect to multiplanar image reconstruction . therefore , mdct is the best method for imaging coronary artery anomalies despite its radiation . the unroofing procedure31)32 ) manipulates the orifice , enlarges the orifice , and makes an acute angulation , which decreases the lateral compression of the intramural segment . possible serious complications of this procedure include aortic valve incompetence due to injury of the intercoronary commissure . associated findings support the suggestion that the pulmonary artery has little or no effect on the constriction of coronary blood flow . percutaneous coronary intervention ( pci)20)46)47 ) relieves systolic compression , but selective cannulation and stent insertion in the anomalous rca are difficult to perform due to the small , ectopic orifice and the long , curved intramural portion of the anomalous rca . prior to pci , the anomalous rca must be evaluated by mdct ; mdct - guided cannulation is helpful in selective cannulation and intervention.20)21 ) coronary artery bypass grafting ( cabg)32)48 ) is technically easy because it does not entail opening of the aorta or manipulation of the intercoronary commissure . however , the native anomalous rca is patent at rest , so obstruction of the cabg by competition flow is possible.32 ) ligation of the native anomalous rca proximal to the anastomosis is a feasible method for preventing competition flow.48 ) coronary reimplantation32 ) of the anomalous rca in the right coronary sinus is also useful , but the disadvantage of this method is that it carries the risk of neoostial stenosis . sudden death without symptoms occurs frequently in patients with anomalous lcas , so surgical repair is recommended.16)49 ) however , sudden death is extremely rare in asymptomatic patient with anomalous rcas , and there is no sudden death in children under 10 years of age or adults over 30 years of age.49 ) eckart et al.9 ) reported 21 coronary artery anomalies related to sudden death among 6,300,000 military recruits , and all cases were anomalous lcas from the right coronary sinus . according to the mdct - based study of lee et al.,25 ) significant stenosis ( > 50% ) of an anomalous rca occurred in only 1 of 24 patients , and this patient , whose symptoms disappeared after an unroofing procedure ( fig . other patients with more obtuse angles and mild or absent narrowing of the orifice and artery exhibit no anomaly - related problems in the absence of treatment ( fig . one report has suggested that subclinical ischemic changes in the myocardium are relatively frequent ( 8 of 16 patients ) in anomalous rca patients in the postoperative period.50 ) treatment of anomalous rca with an interarterial course from the left coronary sinus is still debated because most anomalous rcas are benign , with a small risk of sudden death and late myocardial ischemia after surgery is undertaken . young patients ( < 35 years ) with symptoms or ischemia should undergo surgery . in young patients ( < 35 years ) without symptoms or ischemia , gersony49 ) suggests that anomalous rcas should be followed without intervention and believes that the benefit of excessive exercise limitation is doubtful . i agree with this suggestion and believe most patients should be followed without aggressive treatment . if a young , symptomatic patient has significant luminal narrowing on imaging studies , surgical intervention should be considered . multiplanar mdct image reconstruction at the takeoff portion of the anomalous rca permits precise evaluation of the takeoff angle , size of the slit - like orifice , and course of the anomalous rca . precise and thorough imaging evaluation of anomalous rcas is necessary prior to selection of treatment method .

anomalous origin of the right or left coronary artery from the contralateral sinus of valsalva is often asymptomatic , but many patients , particularly young ones , present with sudden death or myocardial ischemia without symptoms . the mechanism of sudden death in this entity is unclear and has not been fully evaluated . these anomalies are rare , and many cardiologists and radiologists are unfamiliar with them . surgical repair is recommended , especially with anomalous origin of the left coronary artery ( lca ) . however , there is controversy concerning the treatment of anomalous right coronary artery ( rca ) with interarterial course due to its relatively high incidence and the fact that it leads to few , if any , clinical problems .

the face is widely regarded as a symbol of self and a smile as a window into one 's personality . in this , the teeth play important roles in the maintenance of a positive self - image , and loss of teeth may result in significant disabilities that can profoundly disrupt social activities . tooth loss may be traumatic and upsetting , and it is regarded as a serious life event that requires significant social and psychological readjustment . the nonacceptance of edentulousness and the individual 's feelings about dentures , which have been the traditional way of replacing missing teeth , are important for the acceptance of new dentures . traditionally , missing teeth are replaced by removable partial dentures , fixed partial dentures ( bridges ) and complete dentures in cases of complete edentulousness . the need to replace lost teeth with a near - natural successor has encouraged rapid research and advancement in the field of dental implants , especially in advanced economies . dental implant is an artificial root that is surgically inserted into the jawbone to support a single tooth replacement ( crown ) , fixed partial or complete denture or maxillofacial prosthesis . it is an ideal option for people with good oral health who have lost a tooth or teeth due to injuries , periodontal diseases , failure of endodontics , etc . it is also used for the treatment of edentulousness and is associated with improved denture retention , stability and functional efficiency and , thus , improving the quality of life of the patient . currently , dental implants are widely accepted as a prosthetic treatment of completely or partially edentulous patients , and studies have shown significant improvement in patients attitudes toward their dental health after treatment with implant prostheses . unfortunately , the same can not be said of developing countries with poorer access to dental care , where different authors have reported wide variability in the acceptance of this newer option of tooth replacement . in our setting , a resource - challenged environment , conservative dentistry is relatively young and there is dearth of information regarding patients knowledge of tooth replacement as a whole and the use of implant - retained prostheses as an option of replacement of missing tooth . with recent improvement in socioeconomic infrastructures in this environment and expansion of the middle class , access to oral health care hence , in order to identify goals in the promotion of oral health and improvement of the overall quality of life of patients with tooth loss , the study was conducted to evaluate the knowledge of patients about tooth replacement as a whole and assess their awareness of implant - retained prosthesis as an option for tooth replacement . a descriptive cross - sectional study of patients attending the dental centre of the university college hospital , ibadan , nigeria was conducted over a period of 6 months . the university college hospital , ibadan , is a major referral tertiary hospital in the south - western part of the country . in the conduct of this survey , the guidelines of ethical consideration were strictly adhered to and participants filled the questionnaire after signing informed consent . information was obtained with the use of structured , self - administered questionnaires given to patients aged 18 years or older , who consented to participate in the study at the dental clinics during the study period between september 2012 and february 2013 . data collected included the sociodemographic characteristics of the study participants , knowledge about implant - retained tooth replacement as an option of replacement , the cost , source of information and knowledge about other options of tooth replacement as a whole . tests of association between variables were conducted using chi square statistics , with the level of statistical significance set at p < 0.05 . a total of 220 participants took part in the study and 199 ( 90.5% ) properly filled questionnaires were analyzed . the ages of the study participants ranged from 18 to 84 years , with a mean of 37.6 ( 16.5 ) years and a male to female ratio of 1:1.1 . one hundred and two participants were married , 86 were single , 10 were widowed and one was divorced . majority of the participants ( 141 , 70.9% ) had tertiary education , while only eight ( 4.0% ) had no formal education [ table 1 ] . demographic characteristics the majority of the participants ( 184 , 92.5% ) were aware of the possibility of replacing lost teeth . a total of 102 ( 51.3% ) participants knew of different ways to replace missing teeth , while 94 ( 47.2% ) had no idea of such methods . the awareness of the different possible options of tooth replacement as perceived by the participants is shown in table 2 . a significantly higher proportion of those who were aware of the possibility of replacing missing teeth knew that removable partial dentures were an option ( p < 0.000 ) . also , a significantly higher proportion of those that were aware of the possibility of replacement did not know about implant as a replacement option for missing teeth compared with removable partial dentures ( p < 0.01 ) [ table 3 ] . knowledge on ways of replacing missing teeth awareness of tooth replacement and knowledge about options of replacement one hundred and forty ( 71.1% ) participants had not heard of dental implant while only 57 ( 28.9% ) of them had heard about it as an option for replacing missing teeth . of those who had heard about this option , 22 ( 38.6% ) believed that they had received reasonable amount of information on the option , 26 ( 45.6% ) perceived that the information received had been inadequate and nine ( 15.8% ) were unsure of the quantity and quality of information they had on it . only 21 of those participants who have heard about implant and claimed it was well explained could explain what they understood by dental implants , of whom 15 ( 71.4% ) gave the correct information about the implant - retained dental prosthesis . the majority ( 68% ) of the participants got informed about implant - retained prosthesis through the dentist , 23% got it through the media while 6% of the participants got to know through the internet [ figure 1 ] . source of information on dental implant while 45 ( 22.6% ) participants would like to have implant - retained prosthesis as an option for replacement of missing teeth , 24 ( 13.3% ) said they would not want it , while the majority , 112 ( 61.9% ) , were not sure if they would like to have it or not . of the 78 participants who gave reasons why they would not like to have their missing tooth / teeth replaced with dental implant - retained prosthesis , majority ( 46.2% ) claimed that they would not consider it because they do not have any idea about what it is , while seven ( 9.0% ) thought it was expensive [ figure 2 ] . reasons for not considering dental implant one hundred and fifty - four ( 84.6% ) of the participants would like to be educated on dental implant as an option for replacement of missing teeth , while just nine ( 4.9% ) did not want to know about it and 19 ( 10.4% ) cared less about the education on dental implant . one hundred and ten ( 61.8% ) of the participants claimed they would consider implant - retained prosthesis as an option for tooth / teeth replacement if they had good knowledge about it . while 15 ( 8.4% ) would not consider it , 53 ( 29.8% ) of the participants are not sure of considering it even if it is well explained to them . a statistically significant relationship was found when education on implant and if participant would consider implant is well explained ( p = 0.000 ) . among the 57 participants who have heard about implant , only 21 ( 36.8% ) have the knowledge about the cost implication , of which 15 ( 71.4% ) have the right information on cost implication and the remaining six ( 28.6% ) have wrong information on the cost . the study also showed a statistically significant relationship between those who have heard about implant - retained prosthesis and their knowledge of cost implication ( p = 0.000 ) . the ages of the study participants ranged from 18 to 84 years , with a mean of 37.6 ( 16.5 ) years and a male to female ratio of 1:1.1 . one hundred and two participants were married , 86 were single , 10 were widowed and one was divorced . majority of the participants ( 141 , 70.9% ) had tertiary education , while only eight ( 4.0% ) had no formal education [ table 1 ] the majority of the participants ( 184 , 92.5% ) were aware of the possibility of replacing lost teeth . a total of 102 ( 51.3% ) participants knew of different ways to replace missing teeth , while 94 ( 47.2% ) had no idea of such methods . the awareness of the different possible options of tooth replacement as perceived by the participants is shown in table 2 . a significantly higher proportion of those who were aware of the possibility of replacing missing teeth knew that removable partial dentures were an option ( p < 0.000 ) . also , a significantly higher proportion of those that were aware of the possibility of replacement did not know about implant as a replacement option for missing teeth compared with removable partial dentures ( p < 0.01 ) [ table 3 ] . knowledge on ways of replacing missing teeth awareness of tooth replacement and knowledge about options of replacement one hundred and forty ( 71.1% ) participants had not heard of dental implant while only 57 ( 28.9% ) of them had heard about it as an option for replacing missing teeth . of those who had heard about this option , 22 ( 38.6% ) believed that they had received reasonable amount of information on the option , 26 ( 45.6% ) perceived that the information received had been inadequate and nine ( 15.8% ) were unsure of the quantity and quality of information they had on it . only 21 of those participants who have heard about implant and claimed it was well explained could explain what they understood by dental implants , of whom 15 ( 71.4% ) gave the correct information about the implant - retained dental prosthesis . the majority ( 68% ) of the participants got informed about implant - retained prosthesis through the dentist , 23% got it through the media while 6% of the participants got to know through the internet [ figure 1 ] . source of information on dental implant while 45 ( 22.6% ) participants would like to have implant - retained prosthesis as an option for replacement of missing teeth , 24 ( 13.3% ) said they would not want it , while the majority , 112 ( 61.9% ) , were not sure if they would like to have it or not . of the 78 participants who gave reasons why they would not like to have their missing tooth / teeth replaced with dental implant - retained prosthesis , majority ( 46.2% ) claimed that they would not consider it because they do not have any idea about what it is , while seven ( 9.0% ) thought it was expensive [ figure 2 ] . reasons for not considering dental implant one hundred and fifty - four ( 84.6% ) of the participants would like to be educated on dental implant as an option for replacement of missing teeth , while just nine ( 4.9% ) did not want to know about it and 19 ( 10.4% ) cared less about the education on dental implant . one hundred and ten ( 61.8% ) of the participants claimed they would consider implant - retained prosthesis as an option for tooth / teeth replacement if they had good knowledge about it . while 15 ( 8.4% ) would not consider it , 53 ( 29.8% ) of the participants are not sure of considering it even if it is well explained to them . a statistically significant relationship was found when education on implant and if participant would consider implant is well explained ( p = 0.000 ) . among the 57 participants who have heard about implant , only 21 ( 36.8% ) have the knowledge about the cost implication , of which 15 ( 71.4% ) have the right information on cost implication and the remaining six ( 28.6% ) have wrong information on the cost . the study also showed a statistically significant relationship between those who have heard about implant - retained prosthesis and their knowledge of cost implication ( p = 0.000 ) . the present study showed a high rate of awareness of replacement of missing teeth in this environment , with 92.5% of the participants being aware that missing teeth can be replaced . furthermore , the option of removable partial dentures for teeth replacement was the most recognized by the participants . this may be because removable partial dentures have been the most widely available and traditional way of replacement in this country ; it is relatively cheap and affordable for most socioeconomic classes in resource - poor settings and has been found to be satisfactory to patients in terms of appearance , retention and stability . while 28.9% of the participants have heard about dental implant , only 22.6% of them were aware that it is an option of replacement of missing teeth . there was a significant relationship ( p = 0.01 ) when those who have heard of dental implant were compared with those who were aware of possible replacement of missing teeth , with a higher proportion of those who have knowledge of replacement of missing teeth showing low knowledge about dental implant as an option of tooth replacement . the level of awareness of implant as an option for replacement of missing teeth recorded in this study is higher than what was recorded by kumar et al . , who had an awareness of 4.83% . this may be due to the fact that the present study was conducted in a tertiary hospital situated in an urban environment , with the majority of the participants ( 84.8% ) having at least secondary school education as against the population studied by kumar et al . , who were people of lower socioeconomic and education levels of the area ( khammam , andhra pradesh in india ) and were also unskilled workers educated only to the secondary level or lower . however , the level of awareness recorded in this study is lower when compared with that reported by al johanny et al . berge and best , who recorded the level of awareness in their different studies to be from 66.4% to as high as 77% . this may be due to the relatively low level of practice of implant dentistry in this environment and sensitization of patients by dentists toward the use of implant - retained prosthesis as option of tooth replacement . dentists were the major source of information ( 68% ) to those who have heard about dental implants followed by the media , internet and other sources such as friends . this is similar to the study by kumar et al . , which recorded the dentist as being the main source of information ( 38.25% ) . however , this result is contrary to some other studies that have media and internet as the major source of information on dental implant . this could be due to the fact that majority of the people in this environment depend on professionals such as dentists , on their source of information and the fact that accessibility to the internet is relatively expensive and still limited to those of middle and high socioeconomic classes . however , of the 21 participants who had this option of tooth replacement explained to them by dentists , 15 ( 71.4% ) had the right information while six ( 28.6% ) were either wrongly informed or did not understand what was explained about this option of tooth replacement . although this misconception about dental implant has been recorded in a study , it was more through information from the internet and media . of the majority of the participants who claimed that they were not sure if they would like to have dental implants as an option to replace missing teeth , the major reason given by 46.2% of these participants was the fact that they do not have any idea about it , while 39.7% had no particular reason . high cost of implant as perceived by seven ( 9% ) of the participants was another reason given for not considering dental implants . this further buttresses the need for adequate education , as was confirmed in this study by the response of the participants to education on dental implants , with majority of the participants claiming that they would appreciate it if they could be better informed about the advantages and disadvantages that might make them consider it as a better option . , satpathy et al . and al johany , who also recorded a higher percentage of their participants requesting to have more knowledge on dental implants . a statistically significant relationship was also found when education on implant and consideration of dental implant for use by participant was compared ( p = 0.000 ) . thus , if the procedure of implant - retained prosthesis is well explained to patients , they will consider having it as an option of replacing missing teeth . there was misconception and low level of knowledge about the cost implication of dental implants among the 57 participants who have heard about it , with just 21 ( 36.8% ) claiming that they know the cost , of whom 15 ( 71.4% ) were rightly informed . this low awareness and misconception about the cost is similar to that reported by rustemeyer and bremerich and tepper . there was also a significant relationship about the knowledge of cost implication when those who have heard about the implant prosthesis were compared with their knowledge of the cost implication ( p = 0.000 ) . thus , there is need for patients adequate information vis a vis the cost , advantages , disadvantages and possible complications of dental implant prostheses as a better option for teeth replacement . the study has shown that there is a low level of awareness about dental implant in this environment even though people are aware of the possibility of tooth replacement . adequate awareness and rich , right and detailed information are the necessary tools that project dental implant - retained prostheses as the best option for the tooth and lost maxillofacial tissue replacement . the dentist as a professional has the major role to play in this regard , and this can be fulfilled by implementing patient education programmes and counseling centers on dental implant use , advantages and possible complications in order to prepare a patient 's mind .

aim : a survey was set out to evaluate the knowledge of patients about tooth replacement as a whole , and assess their awareness of implant - retained prosthesis as an option of tooth replacement.materials and methods : information on sociodemographic characteristics , knowledge about implant - retained tooth as an option for missing tooth replacement , cost implication , source of information and knowledge about other options of tooth replacement were obtained from patients attending the dental clinics of the university college hospital , ibadan , using structured self - administered questionnaires . data were analyzed using spss version 20.result:a total of 220 patients aged 18 - 84 years with a mean age of 37.6 ( 16.5 ) years participated in the study , with a male to female ratio of 1:1.1 . the majority ( 92.5% ) knew that missing teeth can be replaced , while a significantly lower proportion ( 28.9% ) knew about dental implants as an option ( p < 0.01 ) . dentists were the major source of information on dental implants ( 68% ) . only 21 ( 36.8% ) of those who had heard about dental implant had knowledge about the cost ( p < 0.000).conclusion : a low level of awareness about dental implant as tooth replacement option exist in this environment , although most of the study participants were aware that missing teeth can be replaced .

galactosamine ( gal ) is a 6-carbon amino sugar derivative of galactose . under physiological conditions , it is a component of specific glycoprotein hormones , such as follicle - stimulating hormone or luteinizing hormone . gal is a potent hepatotoxic substance , which can cause hepatocyte death both by necrosis and apoptosis . it inhibits the synthesis of liver rna via the production of uridine diphosphate hexosamines , which block the transcription of genetic material . according to 1 study , gal seems to sensitize liver cells to other hepatotoxic agents ( e.g. , intestinal endotoxins ) , and tnf- may be a major mediator of this damage . due to the damage that gal causes to liver cells , it is used in experimental models of acute liver failure ( alf ) . kepler et al . showed that gal administered to rats caused diffuse inflammation and liver damage . successive studies confirmed the hepatotoxic potential of gal , with a significant difference noted in the susceptibility of different animal species to the toxic effects of the compound . rats and rabbits proved to be sensitive to gal , but mice were resistant , and the sensitivity of dogs depended on the type of substance used for general anaesthesia [ 69 ] . an ideal model of although the gal - based model is not perfect and does not fully reflect the picture of human alf , the general consensus is that it is an appropriate model for studying the pathogenesis and treatment of alf , particularly in small experimental animals . few studies have examined renal complications in the course of experimental gal - induced alf . . showed that acute renal failure occurred in the absence of changes in renal histopathology , in addition to acute liver damage , in rats following administration of gal . . observed a significant reduction in renal blood flow , in addition to hemodynamic changes in the splanchnic vascular bed following the administration of gal to rats . we also found that animals rapidly developed functional acute renal failure , the development and the degree of which depended on the strain of animal tested , in addition to acute damage and liver failure , following intoxication with gal . however , the previous studies investigated the impact of other factors on the development of alf and associated complications rather than the alf model itself . thus , the evaluation of renal disorders in those studies was only a background , not a primary objective . the mechanisms underlying the transition from liver damage to renal failure have not been fully explained , and the lack of knowledge on these mechanisms makes it difficult to study the pathogenesis and treatment of alf and its complications in humans . therefore , the aim of this study was to describe all detectable kidney disorders that developed in the course of gal - induced experimental liver failure . the study included 24 randomly selected male sprague - dawley rats . all weighed 200250 g and were obtained from the department of laboratory animals of the polish mother s memorial hospital research institute , ld , poland . all animals were kept in standard cages , fed a standard diet , and had free access to water and food . they were all maintained under a natural 12-h diurnal day / night cycle , at a constant temperature of 222c and constant humidity of 4550% . the experiments were performed between 10.00 and 18.00 h on naturally moving animals performing normal activities . during the experiments , the animals were placed individually in metabolic cages with free access to food and water . all experiments were performed in accordance with the principles of the animals scientific procedures act 1986 . d--galactosamine hydrochloride ( sigma - aldrich , germany ) was administered intraperitoneally to the rats in the test group at a concentration of 200 mg / ml in physiological saline . the animals were randomly divided into 2 groups ( n=12 in each ) : a control group ( group 1 ) and a test group ( group 2 ) in which the acute liver damage was induced . group 1 received 1 ml of 0.9% saline intraperitoneally , and group 2 received 1.1 g of gal intraperitoneally per kg body weight in a solution of 0.9% saline at a concentration of 200 mg / ml . urine was collected daily for 24 h starting from the 24th to 48th hour after the administration of saline or gal and analyzed immediately after collection . blood samples of 6-ml volume were collected 48 h after the administration of gal or saline from the beating heart of the animals under deep general anaesthesia . biochemistry was determined in serum or urine using an integra 700 autoanalyzer ( roche , usa ) , and reagents for the determination of bilirubin , aspartate aminotransferase , alanine aminotransferase , albumin , sodium , potassium , creatinine and urea were obtained from roche ( germany ) . the ammonia level in the plasma was determined with edta - k3 anticoagulant using a vitros 5600 autoanalyzer , ( johnson & johnson , usa ) , and reagents for ammonia determination were obtained from johnson & johnson . an automatic osmometer ( knauer , germany ) was used to measure the osmolality of the urine and serum . urine specific gravity was measured with a junior ii miditron analyser ( roche diagnostics , france ) , and reagents were purchased from roche diagnostics ( france ) . proteinuria was measured daily using an au 680 analyser ( beckman coulter , usa ) , and reagents for the determination of proteinuria were purchased from beckman coulter ( usa ) . creatinine clearance was calculated according to the following formula : where creatu is the creatinine concentration in urine , creats is the creatinine concentration in blood serum , and vu 24 h is the daily urine volume . osmolality clearance was calculated according to the following formula : where u osm is the osmolality of the urine , v u 24 h is the daily urine volume , and p osm is the plasma osmolality . the plasma osmolality was calculated according to the following formula : where nas is the sodium concentration in the blood serum , ks is the potassium concentration in blood serum , and ureas is the urea concentration in blood serum . free water clearance was calculated according to the following formula : where v u 24 h is the daily urine volume , and osmol cl is the osmolality clearance . the excreted fraction of sodium was calculated according to the following formula : where nau is the sodium concentration in urine , nas is the sodium concentration in blood serum , creatu is the creatinine concentration in urine , and creats is the creatinine concentration in blood serum . free water reabsorption was calculated according to the following formula : where osmol cl is the osmolality clearance , and v u 24 h is the daily urine volume . after blood collection , the liver and kidney of each of the test animals were prepared for histopathological examination . the organs were placed in formalin , embedded in paraffin , cut into ultra - thin sections , and finally stained with hematoxylin and eosin . statistical analyses were performed with the student s t - test and an analysis of variance when multiple comparisons were needed . where necessary , the mann - whitney u test was applied for the analysis of nonparametric data . the study included 24 randomly selected male sprague - dawley rats . all weighed 200250 g and were obtained from the department of laboratory animals of the polish mother s memorial hospital research institute , ld , poland . all animals were kept in standard cages , fed a standard diet , and had free access to water and food . they were all maintained under a natural 12-h diurnal day / night cycle , at a constant temperature of 222c and constant humidity of 4550% . the experiments were performed between 10.00 and 18.00 h on naturally moving animals performing normal activities . during the experiments , the animals were placed individually in metabolic cages with free access to food and water . all experiments were performed in accordance with the principles of the animals scientific procedures act 1986 . d--galactosamine hydrochloride ( sigma - aldrich , germany ) was administered intraperitoneally to the rats in the test group at a concentration of 200 mg / ml in physiological saline . the animals were randomly divided into 2 groups ( n=12 in each ) : a control group ( group 1 ) and a test group ( group 2 ) in which the acute liver damage was induced . group 1 received 1 ml of 0.9% saline intraperitoneally , and group 2 received 1.1 g of gal intraperitoneally per kg body weight in a solution of 0.9% saline at a concentration of 200 mg / ml . urine was collected daily for 24 h starting from the 24th to 48th hour after the administration of saline or gal and analyzed immediately after collection . blood samples of 6-ml volume were collected 48 h after the administration of gal or saline from the beating heart of the animals under deep general anaesthesia . biochemistry was determined in serum or urine using an integra 700 autoanalyzer ( roche , usa ) , and reagents for the determination of bilirubin , aspartate aminotransferase , alanine aminotransferase , albumin , sodium , potassium , creatinine and urea were obtained from roche ( germany ) . the ammonia level in the plasma was determined with edta - k3 anticoagulant using a vitros 5600 autoanalyzer , ( johnson & johnson , usa ) , and reagents for ammonia determination were obtained from johnson & johnson . an automatic osmometer ( knauer , germany ) was used to measure the osmolality of the urine and serum . urine specific gravity was measured with a junior ii miditron analyser ( roche diagnostics , france ) , and reagents were purchased from roche diagnostics ( france ) . proteinuria was measured daily using an au 680 analyser ( beckman coulter , usa ) , and reagents for the determination of proteinuria were purchased from beckman coulter ( usa ) . creatinine clearance was calculated according to the following formula : where creatu is the creatinine concentration in urine , creats is the creatinine concentration in blood serum , and vu 24 h is the daily urine volume . osmolality clearance was calculated according to the following formula : where u osm is the osmolality of the urine , v u 24 h is the daily urine volume , and p osm is the plasma osmolality . the plasma osmolality was calculated according to the following formula : where nas is the sodium concentration in the blood serum , ks is the potassium concentration in blood serum , and ureas is the urea concentration in blood serum . free water clearance was calculated according to the following formula : where v u 24 h is the daily urine volume , and osmol cl is the osmolality clearance . the excreted fraction of sodium was calculated according to the following formula : where nau is the sodium concentration in urine , nas is the sodium concentration in blood serum , creatu is the creatinine concentration in urine , and creats is the creatinine concentration in blood serum . free water reabsorption was calculated according to the following formula : where osmol cl is the osmolality clearance , and v u 24 h is the daily urine volume . after blood collection , the liver and kidney of each of the test animals were prepared for histopathological examination . the organs were placed in formalin , embedded in paraffin , cut into ultra - thin sections , and finally stained with hematoxylin and eosin . statistical analyses were performed with the student s t - test and an analysis of variance when multiple comparisons were needed . where necessary , the mann - whitney u test was applied for the analysis of nonparametric data . a p value < 0.05 was assumed as the level of significance . table 1 shows the liver parameters of the rats , indicating the development of acute liver damage and failure . compared to the control group , the administration of gal at a dose of 1.1 g / kg body weight caused severe liver damage and the development of alf after 48 h , with a statistically significant increase in plasma levels of alanine aminotransferase ( p<0.001 ) , aspartate aminotransferase ( p<0.0001 ) , bilirubin ( p<0.004 ) , and ammonia ( p<0.005 ) and a statistically significant decrease in albumin ( p<0.001 ) ( table 1 , figure 1 ) . gal administration led to the development of hepatic encephalopathy in all animals tested , while 10 animals developed hepatic coma ( 83% ) in clinical assessment . histopathological evaluation of the liver of the test animals showed generalized and massive hepatocyte necrosis ( figure 2a ) . in addition , diffused degenerative changes were visible , including small and giant cells , infiltrations of eosinophilic granulocytes , portal tract fibrosis and hyperemia . no changes were observed in the control group , which received only saline ( figures 2b ) . table 2 shows the renal parameters of the rats , indicating the development of acute renal failure . compared to the control group , the administration of gal at a dose of 1.1 g / kg bodyweight resulted in elevated indices of liver damage and failure , as well as the development of acute renal failure . the test group also showed a significant increase in serum concentrations of creatinine ( p<0.001 ) and urea ( p<0.001 ) , a reduction in creatinine clearance ( p<0.0012 ) , and a reduction in daily urine volume ( p<0.003 ) ( table 2 , figure 3 ) . the water and electrolyte balance of the test group were typical of functional renal failure , with a significant decrease in osmotic clearance ( p<0.001 ) , daily natriuresis ( p<0.003 ) , fractional sodium excretion ( p<0.016 ) , and daily diuresis ( p<0.003 ) , and an increase in the urine specific gravity and osmolality , although the last 2 parameters were statistically insignificant . in addition , the ratio of urine osmolality to plasma osmolality did not change significantly ; the ratio of the concentration of creatinine in urine to serum creatinine significantly decreased ( p<0.0052 ) ; free water clearance increased ( p < 0.001 ) , and free water reabsorption decreased ( p<0.001 ) . moreover , the daily loss of proteins significantly decreased ( p<0.007 ) ( table 2 , figure 3 ) . other parameters of water and electrolyte balance dysfunction and acute renal failure are presented in table 2 . renal histopathology showed no changes in the test group ( figure 4a ) or the control group ( figure 4b ) . table 1 shows the liver parameters of the rats , indicating the development of acute liver damage and failure . compared to the control group , the administration of gal at a dose of 1.1 g / kg body weight caused severe liver damage and the development of alf after 48 h , with a statistically significant increase in plasma levels of alanine aminotransferase ( p<0.001 ) , aspartate aminotransferase ( p<0.0001 ) , bilirubin ( p<0.004 ) , and ammonia ( p<0.005 ) and a statistically significant decrease in albumin ( p<0.001 ) ( table 1 , figure 1 ) . gal administration led to the development of hepatic encephalopathy in all animals tested , while 10 animals developed hepatic coma ( 83% ) in clinical assessment . histopathological evaluation of the liver of the test animals showed generalized and massive hepatocyte necrosis ( figure 2a ) . in addition , diffused degenerative changes were visible , including small and giant cells , infiltrations of eosinophilic granulocytes , portal tract fibrosis and hyperemia . no changes were observed in the control group , which received only saline ( figures 2b ) . table 2 shows the renal parameters of the rats , indicating the development of acute renal failure . compared to the control group , the administration of gal at a dose of 1.1 g / kg bodyweight resulted in elevated indices of liver damage and failure , as well as the development of acute renal failure . the test group also showed a significant increase in serum concentrations of creatinine ( p<0.001 ) and urea ( p<0.001 ) , a reduction in creatinine clearance ( p<0.0012 ) , and a reduction in daily urine volume ( p<0.003 ) ( table 2 , figure 3 ) . the water and electrolyte balance of the test group were typical of functional renal failure , with a significant decrease in osmotic clearance ( p<0.001 ) , daily natriuresis ( p<0.003 ) , fractional sodium excretion ( p<0.016 ) , and daily diuresis ( p<0.003 ) , and an increase in the urine specific gravity and osmolality , although the last 2 parameters were statistically insignificant . in addition , the ratio of urine osmolality to plasma osmolality did not change significantly ; the ratio of the concentration of creatinine in urine to serum creatinine significantly decreased ( p<0.0052 ) ; free water clearance increased ( p < 0.001 ) , and free water reabsorption decreased ( p<0.001 ) . moreover , the daily loss of proteins significantly decreased ( p<0.007 ) ( table 2 , figure 3 ) . other parameters of water and electrolyte balance dysfunction and acute renal failure are presented in table 2 . renal histopathology showed no changes in the test group ( figure 4a ) or the control group ( figure 4b ) . this work is the first report in the current literature to present a detailed description of water and electrolyte balance disorders and the development of acute renal failure as a consequence of acute damage and liver failure . in the present study , renal failure developed rapidly in a typical manner , with a parallel increase in nitrogen retention parameters and a decrease in creatinine clearance and daily diuresis . the osmotic clearance , daily natriuresis , and fractional sodium excretion values decreased , whereas those of urine osmolality and its specific gravity increased . hence , the kidneys retained the ability to concentrate urine and retained osmotically effective osmolytes . the aforementioned , together with the lack of changes in the histopathological analyses of the kidneys , suggests that the kidneys remained functioning . the unchanged urine to plasma osmolality and urine to serum creatinine ratio after gal intoxication also indicate that gal was not directly toxic to the kidneys . the preserved renal tubular function and intact structure of the whole nephron also point to the functional nature of the kidney disorders . other studies found similar findings , reporting no adverse effect of gal in cell lines of renal tubules , even at much higher concentrations than those applied in our study . these observations are also consistent with previous studies of the effect of gal in experimental animals that reported only hepatotoxic effects . various methods can be used to induce acute renal failure in surgical , pharmacological , and infectious alf models . surgical models include partial or total hepatectomy , temporary or permanent hepatic artery ligation , or ligation of the common bile duct [ 1517 ] . pharmacological models are based on the hepatotoxic properties of compounds , such as gal , carbon tetrachloride , acetaminophen , thioacetamide , concanavaline a , or lipopolysaccharides [ 3,9,1820 ] . none of the aforementioned models provide an ideal alf model because they do not accurately reflect clinical , biochemical , and histopathological acf observed in humans . however , their diversity and the use of different animal species provide sufficient tools to study the pathogenesis and therapeutic possibilities of alf . terblanche and hickman described 6 features of an ideal model of alf : 1 ) it should be reversible ( i.e. , react to the potential treatment ; 2 ) it should be reproducible , with recurrent , characteristic endpoints ; 3 ) the damage and liver failure should be a direct cause of death in experimental animals ; 4 ) it should have a therapeutic window ( ie , sufficient time to implement the proposed treatment ) ; 5 ) it should be possible to use the model in large experimental animals , where the treatment could include artificial liver support systems ; and 6 ) the techniques and toxins or viruses applied should be as safe as possible for researchers and other individuals undertaking the experiments . the presented model of gal - induced intoxication of animals meets most of the above criteria . it is reproducibly established that liver damage and failure are the direct cause of death of the animals , it has an appropriate therapeutic window , it can be used in large experimental animals , and it is safe for scientists carrying out the experiments . although the partial reversibility may be a limitation , the results of some studies suggested that certain substances can slow down the development of gal - induced alf . as noted previously , the model of alf used in the present study is employed less frequently than other models , and its value is probably underappreciated . however , it has recently become more widely accepted due to standardization of methodology and standardization of clinical , morphological , and biochemical parameters of alf . in the model employed in the present study , the animals were intoxicated with a gal dose of approximately 1 g / kg bodyweight at a concentration of 200 mg / ml in saline solution , which was administered intraperitoneally . experiments can be performed after 48 h when acute damage of hepatic parenchyma develops , leading to liver failure . the damage results in a very significant increase in the level of cellular enzymes alanine and aspartate aminotransferases , as well as bilirubin , in serum . simultaneously , the synthetic activity ( decreased albumin ) and metabolic and detoxifying functions of the liver decline ( increased concentration of ammonia ) , coagulopathy develops , and the histopathological picture is dominated by massive necrosis of hepatocytes . severe liver damage leads to hemodynamic changes in the splanchnic vascular bed , with portal pressure , venous inflow , and intrahepatic portosystemic shunt all increased . mean arterial pressure is reduced , mainly due to decreased peripheral vascular resistance , and the myocardial ejection fraction increases , which leads to the development of hyperdynamic circulation . the vascular bed in the kidneys becomes contracted , particularly within the cortical vessels . renal function tests demonstrate a marked decline by more than 50% in renal blood flow and the glomerular filtration rate , with a simultaneous increase in urea and creatinine in serum . daily excretion of sodium is reduced , but the excretion of fractional sodium varies only slightly . the following parameters do not change : osmolality of the urine , ratio of urine osmolality to plasma osmolality , and free water clearance . it seems that the gal - induced experimental alf model in the present study meets the rigorous criteria of an appropriate alf model proposed by terblanche and hickman . however , taking into account previous descriptions of the model and , above all , our results , the question arises as to whether it is solely a model of acute liver failure . acute renal failure develops in this model in a relatively short time ( about 48 h ) after intoxication of animals with gal . the deterioration of the glomerular filtration rate is accompanied by a reduction in osmotic clearance and daily natriuresis and an increase in urine osmolality . therefore , the kidneys retain the ability to concentrate urine , and their histopathological examination shows no changes . the clinical situation in which acute functional renal failure develops in the course of acute damage and liver failure is known as hepatorenal syndrome ( hrs ) . in this syndrome , liver disease is the only etiological factor of kidney failure . in contrast , renal failure with oliguria , hyperazotemia , and hyponatremia develops in the absence of clinical , laboratory , and histological characteristics of any known renal disease , leading to renal failure . this is the case in the alf model described above and presented in our study . firstly , damage to the liver in the present study caused by gal is the only cause of acute renal failure . gal had no direct toxic effect on the structure of kidneys in common with that reported in previous studies . secondly , the pathogenesis of the development of renal failure in this model corresponds with the mechanisms observed in typical hrs . it progresses from damage to the liver parenchyma to the development of portal hypertension , enlargement of the splanchnic vascular bed , reduction of the effective volume of fluid in the systemic circulation , and subsequent vascular baroreceptor stimulation , followed by activation of numerous vasoconstriction factors , including the renin - angiotensin system , sympathetic nervous system , or arginine vasopressin system . . observed that the administration of gal to rats increased acute liver damage , portal pressure , venous inflow , and intrahepatic portosystemic shunting and that renal blood flow was subsequently reduced significantly , particularly in the renal cortical vessels . thirdly , the acute renal failure in the present study is of a purely functional nature . for example , anand et al . showed that following intraperitoneal administration of gal to rats , in addition to acute liver damage , acute renal failure occurred in the absence of changes in renal histopathology . furthermore , the kidneys in the current study retained the ability to concentrate urine , indicating the functional nature of the developing renal disorder . therefore , it seems that the presented model is not only a model of alf but also of hrs . however , opinions on this subject are divided , with some authors not sharing this view . the model discussed is not the hrs model that develops in patients with cirrhosis and ascites . the etiology , pathogenesis , time of the emergence of symptoms and prognosis differ in these cases . the model that can mimic the mechanism of cirrhotic hrs is ( e.g. , chronic intoxication with carbon tetrachloride [ ccl4 ] ) administered by inhalation or orally . ccl4 is administered for several weeks . after this time , ascites , elevated liver enzymes , hyponatremia , and hypoalbuminemia develop . peripheral vascular resistance is decreased , mean arterial pressure is reduced , and cardiac output is increased . the histopathological examination of the liver shows a typical picture of cirrhosis . renal disorders are dominated by an increase in sodium and water retention , with no apparent decrease in the glomerular filtration rate and renal blood flow . however , some researchers emphasize the possibility of a direct toxic effect of ccl4 on the kidneys . it is not known whether renal failure is derived solely from liver damage or additional toxic damage to the kidneys . in contrast , intoxication of animals with gal models the situation of patients with alf and the development of acute renal failure during the onset of alf . according to some authors , the model in the present study should be considered simply as a model of alf and its complications and not as a model of typical hrs . we believe that the difference between these opinions stems solely from the various classifications and definitions of hrs ( i.e. , whether acute renal failure per se falls within the definition of hrs ) . according to the stance and modified definition in 2007 of the international ascites club , hrs induced by acute liver damage is included in this classification as a type iv separate syndrome . in our opinion , this view is completely legitimate . as indicated above , despite the different etiology of the mechanisms leading to renal failure , they coincide , at least partially , in both chronic and acute liver damage . these include disorders of the splanchnic vascular bed and hemodynamic changes in the systemic circulation and renal cortical vessels . therefore , in our opinion , the experimental model of alf caused by gal intoxication can be simultaneously treated as a model of hrs in the course of acute damage and liver failure . in table 3 we present short specification of the experimental model of alf / hrs induced by galactosamine intoxication . in the present study , acute gal - induced intoxication of experimental animals caused acute damage and hepatic failure and secondary development of acute renal failure . this acute renal failure was of a functional nature , with the kidneys retaining the ability to concentrate urine and retain sodium , and no changes were observed in the histopathological examination . therefore , it seems that the gal - induced experimental model of alf can be simultaneously treated as a model of hrs that occurs in the course of acute damage and liver failure .

backgroundshort - term administration of galactosamine to experimental animals causes liver damage and acute liver failure ( alf ) , as well as acute renal failure in some cases . the aim of our study was to describe kidney disorders that developed in the course of galactosamine - induced liver failure.material/methodssprague-dawley rats were randomly divided into 2 groups : a study group administered galactosamine intraperitoneally and a control group administered saline.resultsall the animals in the study group developed liver damage and failure within 48 h , with significant increase of alanine ( p<0.001 ) , aspartate aminotransferases ( p<0.0001 ) , bilirubin ( p<0.004 ) , and ammonia ( p<0.005 ) and decrease of albumin ( p<0.001 ) concentrations . acute renal failure was observed in all test animals , with a significant increase in creatinine ( p<0.001 ) and urea ( p<0.001 ) concentrations and a decrease in creatinine clearance ( p<0.0012 ) . moreover , osmotic clearance ( p<0.001 ) , daily natriuresis ( p<0.003 ) , and fractional sodium excretion ( p<0.016 ) decreased significantly in this group of animals . the ratio of urine osmolality to serum osmolality did not change . histopathology of the liver revealed massive necrosis of hepatocytes , whereas renal histopathology showed no changes.conclusionsacute renal failure that developed in the course of galactosamine - induced alf was of a functional nature , with the kidneys retaining the ability to concentrate urine and retain sodium , and there were no renal changes in the histopathological examination . it seems that the experimental model of alf induced by galactosamine can be viewed as a model of hepatorenal syndrome that occurs in the course of acute damage and liver failure .

cardiovascular disease is the leading cause of mortality throughout the world , accounting for 17.3 million deaths / year . a large number of studies have proven that the epidemiology , clinical manifestations , and clinical prognosis of coronary artery disease ( cad ) have sex - related differences . in the past 30 years , the mortality of male patients with cad has declined gradually , but an increase had been observed in women . unfortunately , accurate diagnosis of cad might be more challenging in women than in men because women more frequently present with atypical symptoms . thus , women are at increased risk of delayed or incorrect diagnosis . according to a report from the american heart association ( aha ) in 2015 , the mortality rate of female patients with cardiovascular disease ( 50.6% ) has exceeded that of men ( 49.4% ) . conventional coronary angiography ( cag ) is the gold standard diagnostic modality for cad , but it is invasive and associated with several potential complications . moreover , nearly half of the women who underwent invasive cag were found to have nonobstructive cad . multislice computed tomography ( msct ) cag is highly accurate for the exclusion of significant coronary artery stenoses ( > 50% luminal narrowing ) , with high negative predictive values ( npvs ) , as compared with conventional cag . at present , the accuracy of msct diagnosis in women is still not clear . data are mainly based on the male population , with only about 20% of the included patients being female . many of the studies that have been conducted have small sample sizes and conflicting results . in addition , whether atypical presentation influences the diagnostic accuracy of msct in women remains unknown . this study aimed to investigate the effect of sex and atypical symptoms on the diagnostic accuracy of the current 64-slice msct cag technique using conventional cag as the reference standard . totally , 1246 in - patients who had performed cag from january 2007 to february 2011 in the peking university people 's hospital were included in the study . demographic information , medical history , and clinical data were obtained from the patients medical records . the exclusion criteria were nonsinus rhythm ( 145 patients ) , intolerance to iodine contrast ( 11 patients ) , renal dysfunction ( serum creatinine levels 15 mg / l , 87 patients ) , pregnancy ( 0 patients ) , history of percutaneous coronary intervention or coronary artery bypass surgery ( 46 patients ) , uninterpretable images due to poor quality ( 72 patients ) , and other complications not suitable for msct ( 489 patients ) . finally , a database of 396 patients with suspected or proven cad on admission , who successively underwent both msct and invasive cag , was reviewed . cad was diagnosed based on invasive cag results , indicating stenoses of 50% in any coronary artery . the patients were divided into typical and atypical groups based on their symptoms of angina pectoris . typical angina symptoms were defined as the sensation of chest pain or discomfort that may feel like tightness , heaviness , or burning in the central chest . the discomfort might move or radiate to the shoulder , arms , jaw , neck , and back . angina is typically precipitated by exertion or emotional stress , lasting only a few minutes and relieved by rest and nitroglycerin . this retrospective study was approved by the ethics committee of peking university people 's hospital . all the patients underwent coronary msct imaging using the general electric lightspeed volume computed tomography ( ge healthcare , milwaukee , wi , usa ) . patients with a heart rate of > 65 beats / min received additional blockers ( 25 or 50 mg of metoprolol ) 1 h before the ct examination . then , the patients were injected with 6080 ml of iodinated contrast medium in the antecubital vein at a high flow rate ( 5 ml / s ) , followed by saline flushing and coronary ct angiographic examinations . the collimation was 64 mm 0.625 mm ; gantry rotation time was 350 ms , tube current was 600750 ma , and voltage was 120 kv . automated bolus - tracking in the aortic root was used for timing of the scan . all the images were acquired during a single inspiratory breath hold of about 10 s , with simultaneous electrocardiographic recording . all images were interpreted in consensus by two experienced radiologists , who were unaware of the clinical presentations of the patients . the coronary arteries were divided into 13 separate segments according to the modified aha classification . coronary artery plaque was defined as an area of 1 mm , and a clearly recognizable structure associated with the coronary artery wall in at least two independent image planes . plaques with a ct density greater than that of the contrast - enhanced coronary lumen were defined as calcified plaques . for patients with suspected or proven cad who needed to undergo a second evaluation of coronary arteries , conventional invasive cag was performed according to the standard protocols . cags were visually assessed in consensus by two experienced observers who were unaware of the results of the msct cag . continuous variables were expressed as mean standard deviation ( sd ) and compared between the two groups using the t - test for independent samples . when not normally distributed , continuous data were expressed as median ( interquartile range ) and compared between the two groups using the nonparametric mann - whitney u - test . categorical variables were expressed as absolute numbers ( percentages ) and compared between the two groups using the chi - square test . the diagnostic performance of msct was assessed in comparison with that of cag , which was used as the reference standard . sensitivity , specificity , positive predictive value ( ppv ) , and npv were calculated . the youden index was calculated as ( sensitivity + specificity 1 ) to summarize both sensitivity and specificity in a single number between 0 and 1 . multivariate logistic regression analyses were performed to determine the influencing factors on the diagnostic accuracy of msct . the diagnostic performance of msct was assessed using the area under the receiver operating characteristic curve ( auc ) of the receiver operating characteristic ( roc ) curves , taking 50% or greater diameter stenoses at conventional invasive cag as the reference standard . all p values were two - tailed and a significance level of < 0.05 was used . all statistical analyses were performed using spss version 17.0 ( spss inc . , chicago , il , usa ) . totally , 1246 in - patients who had performed cag from january 2007 to february 2011 in the peking university people 's hospital were included in the study . demographic information , medical history , and clinical data were obtained from the patients medical records . the exclusion criteria were nonsinus rhythm ( 145 patients ) , intolerance to iodine contrast ( 11 patients ) , renal dysfunction ( serum creatinine levels 15 mg / l , 87 patients ) , pregnancy ( 0 patients ) , history of percutaneous coronary intervention or coronary artery bypass surgery ( 46 patients ) , uninterpretable images due to poor quality ( 72 patients ) , and other complications not suitable for msct ( 489 patients ) . finally , a database of 396 patients with suspected or proven cad on admission , who successively underwent both msct and invasive cag , was reviewed . cad was diagnosed based on invasive cag results , indicating stenoses of 50% in any coronary artery . the patients were divided into typical and atypical groups based on their symptoms of angina pectoris . typical angina symptoms were defined as the sensation of chest pain or discomfort that may feel like tightness , heaviness , or burning in the central chest . the discomfort might move or radiate to the shoulder , arms , jaw , neck , and back . angina is typically precipitated by exertion or emotional stress , lasting only a few minutes and relieved by rest and nitroglycerin . this retrospective study was approved by the ethics committee of peking university people 's hospital . all the patients underwent coronary msct imaging using the general electric lightspeed volume computed tomography ( ge healthcare , milwaukee , wi , usa ) . patients with a heart rate of > 65 beats / min received additional blockers ( 25 or 50 mg of metoprolol ) 1 h before the ct examination . then , the patients were injected with 6080 ml of iodinated contrast medium in the antecubital vein at a high flow rate ( 5 ml / s ) , followed by saline flushing and coronary ct angiographic examinations . the collimation was 64 mm 0.625 mm ; gantry rotation time was 350 ms , tube current was 600750 ma , and voltage was 120 kv . automated bolus - tracking in the aortic root was used for timing of the scan . all the images were acquired during a single inspiratory breath hold of about 10 s , with simultaneous electrocardiographic recording . all images were interpreted in consensus by two experienced radiologists , who were unaware of the clinical presentations of the patients . the coronary arteries were divided into 13 separate segments according to the modified aha classification . coronary artery plaque was defined as an area of 1 mm , and a clearly recognizable structure associated with the coronary artery wall in at least two independent image planes . plaques with a ct density greater than that of the contrast - enhanced coronary lumen were defined as calcified plaques . for patients with suspected or proven cad who needed to undergo a second evaluation of coronary arteries , conventional invasive cag was performed according to the standard protocols . cags were visually assessed in consensus by two experienced observers who were unaware of the results of the msct cag . continuous variables were expressed as mean standard deviation ( sd ) and compared between the two groups using the t - test for independent samples . when not normally distributed , continuous data were expressed as median ( interquartile range ) and compared between the two groups using the nonparametric mann - whitney u - test . categorical variables were expressed as absolute numbers ( percentages ) and compared between the two groups using the chi - square test . the diagnostic performance of msct was assessed in comparison with that of cag , which was used as the reference standard . sensitivity , specificity , positive predictive value ( ppv ) , and npv were calculated . the youden index was calculated as ( sensitivity + specificity 1 ) to summarize both sensitivity and specificity in a single number between 0 and 1 . multivariate logistic regression analyses were performed to determine the influencing factors on the diagnostic accuracy of msct . the diagnostic performance of msct was assessed using the area under the receiver operating characteristic curve ( auc ) of the receiver operating characteristic ( roc ) curves , taking 50% or greater diameter stenoses at conventional invasive cag as the reference standard . all p values were two - tailed and a significance level of < 0.05 was used . all statistical analyses were performed using spss version 17.0 ( spss inc . , chicago , il , usa ) . table 1 lists the baseline characteristics of the entire study population . in this study , 396 patients were enrolled , including 141 women and 255 men . according to the results of cag , cad diagnosis was established in 346 patients and ruled out in 50 patients . on average , the women were older than the men ( 67 10 vs. 62 11 years , t = 4.416 , p < 0.01 ) . the number of male smokers was significantly greater than that of female smokers , and more women had no cad . women showed a lower prevalence of lesions in mid - segments or side branches . with regard to calcified plaque on msct , and the number of diseased and culprit vessels on invasive cag clinical characteristics of the study group sd : standard deviation ; cad : coronary artery disease ; msct : multislice computed tomography ; nstemi : non - st elevation myocardial infarction ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . table 2 lists the diagnostic accuracy of msct in the total population . of the 396 patients , 328 had obstructive cad on conventional invasive cag that were correctly identified by msct , resulting in a sensitivity of 95% ( 95% confidence interval [ ci ] : 9397% ) . eighteen patients ( 36% , 18/50 ) were identified as false positive . on a per - patient level , the sensitivity , specificity , ppv , npv , total accuracy , and youden index were 95% , 64% , 95% , 64% , 91% , and 0.59 , respectively . diagnostic accuracy of multislice computed tomography in the total population ( n = 396 ) * p<0.001 , compared with all vessels ; p<0.05 , compared with all vessels ; p<0.001 , compared with all segments ; p<0.05 , compared with all segments . tn : true negative ; tp : true positive ; fn : false negative ; fp : false positive ; ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . the diagnostic performance of msct for detecting significant stenoses in a vessel - based analysis was good . most of the significantly diseased vessels ( 75% , 570/756 ) were correctly identified using ct . ninety - six vessels ( 12% , 96/828 ) with nonsignificant stenoses were incorrectly classified as significant stenoses using ct . the diagnostic accuracy of msct in detecting left main ( lm ) cad is the highest ( 91% vs. 82% , compared with all vessels , = 10.120 , p < 0.05 ) . msct had a lower diagnostic accuracy ( 74% vs. 82% , compared with all vessels , = 12.432 , p < 0.05 ) in detecting lesions of left circumflex coronary . on a per - segment basis , the diagnostic values of msct in detecting lesions in distal segments and side branches were low , and the youden indexes for each segment in comparison with all the segments were 0.22 versus 0.4 ( u = 7.493 , p < 0.05 ) and 0.15 versus 0.4 ( u = 6.767 , p < 0.05 ) , respectively . table 3 shows the diagnostic accuracy of msct in the women compared with that in the men . on a patient - based analysis , the ppv ( 91% vs. 97% , = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , = 5.093 , p < 0.05 ) of msct in detecting cad were lower in the women . the women had a lower youden index ( 0.45 vs. 0.70 , u = 2.584 , p < 0.05 ) in msct diagnosis . in the cad population diagnosed using conventional invasive cag , 93% ( 110/118 ) of the female patients and 96% ( 218/228 ) of the male patients were correctly identified using msct . eleven women ( 48% , 11/23 ) and 7 men ( 26% , 7/27 ) with nonsignificant cad were incorrectly classified as having significant coronary stenoses using ct . diagnostic accuracy of multislice computed tomography in women versus men * p<0.05 , compared with men . ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . on a per - vessel basis ( lm , left anterior descending , left circumflex , and right coronary arteries ) , 564 coronary arteries in women and 1020 vessels in men were included in the study . the youden index showed no significant difference . using msct , 76% ( 186/244 ) of vessels in the women and 75% ( 384/512 ) of vessels in the men thirty - eight vessels ( 13% , 38/290 ) in the women and 58 vessels ( 11% , 58/508 ) in the men with nonsignificant stenoses on invasive cag were misdiagnosed using ct . after exclusion of 104 segments ( 23 for women and 81 for men ) owing to small vessel size , motion artifacts , and undetectable distal segments due to total occlusion of the proximal vessel , 1534 coronary segments in the female population and 2718 segments in the male population were included in this study . on a per - segment basis , the npv ( 87% vs. 83% , = 8.563 , p < 0.05 ) and diagnostic accuracy ( 82% vs. 79% , = 5.916 , p < 0.05 ) of msct in detecting cad were higher in the women . however , the ppv was relatively low in both groups for all the segments ( 60% and 63% , respectively ) and extremely low in distal segments and side branches ( 3050% ) . thus , the diagnostic values were low for both groups ( youden index : 0.42 and 0.39 , respectively ) . according to the msct results with conventional invasive cag as the reference standard , patients with true positive and true negative results were defined as the accurate diagnosis group . table 4 shows that the percentage of men was significantly higher in the accurate diagnosis group . multivessel diseases were significantly more frequent in the accurate group . in the multivariate logistic regression analysis , atypical presentation was an independent influencing factor of the diagnostic accuracy of msct in the women ( odds ratio [ or ] = 4.94 , 95% ci : 1.1620.92 , walds = 4.69 , p < 0.05 ) . multivessel disease ( or = 31.34 , 95% ci : 3.6272.6 , walds = 9.7 , p < 0.01 ) and noncalcified plaque ( or = 4.96 , 95% ci : 1.0124.51 , walds = 3.87 , p < 0.05 ) were also independent influencing factors . clinical characteristics based on the accuracy of multislice computed tomography cad : coronary artery disease ; msct : multislice computed tomography ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery ; ldl - c ; low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol . the whole population was divided into two groups , namely , the typical and atypical groups , according to angina pectoris symptoms . table 5 shows that in a patient - based analysis , the ppv ( 98% vs. 74% , = 17.283 , p < 0.001 ) and diagnostic accuracy ( 93% vs. 72% , = 9.571 , p < 0.001 ) of msct in detecting female cad were significantly higher in the typical group than in the atypical group . the women in the typical group had a higher youden index ( 0.69 vs. 0.29 , u = 2.359 , p < 0.05 ) in msct diagnosis . not much difference was observed for msct in the diagnosis of male cad between the two groups . diagnostic accuracy of multislice computed tomography in patients with typical or atypical angina symptoms * p<0.001 ; p<0.05 , compared with atypical group . ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value the diagnostic performance of msct to detect coronary stenoses of 50% was further investigated with respect to the roc curve . for all the patients , the auc was 0.84 ( 95% ci : 0.770.90 ) , suggesting a good accuracy . the auc were 0.90 ( 95% ci : 0.830.97 ) for men and 0.74 ( 95% ci : 0.620.87 ) for women ( z = 2.194 , p < 0.05 ) . in women , patients with typical symptoms had a higher auc ( 0.91 , 95% ci : 0.830.99 ) than those with atypical symptoms ( 0.64 , 95% ci : 0.450.82 , z = 2.690 , p < 0.01 ) . however , in the men , no significant difference in auc was found between the typical and atypical groups . table 1 lists the baseline characteristics of the entire study population . in this study , 396 patients were enrolled , including 141 women and 255 men . according to the results of cag , cad diagnosis was established in 346 patients and ruled out in 50 patients . on average , the women were older than the men ( 67 10 vs. 62 11 years , t = 4.416 , p < 0.01 ) . the number of male smokers was significantly greater than that of female smokers , and more women had no cad . women showed a lower prevalence of lesions in mid - segments or side branches . with regard to calcified plaque on msct , and the number of diseased and culprit vessels on invasive cag clinical characteristics of the study group sd : standard deviation ; cad : coronary artery disease ; msct : multislice computed tomography ; nstemi : non - st elevation myocardial infarction ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . table 2 lists the diagnostic accuracy of msct in the total population . of the 396 patients , 328 had obstructive cad on conventional invasive cag that were correctly identified by msct , resulting in a sensitivity of 95% ( 95% confidence interval [ ci ] : 9397% ) . eighteen patients ( 36% , 18/50 ) were identified as false positive . on a per - patient level , the sensitivity , specificity , ppv , npv , total accuracy , and youden index were 95% , 64% , 95% , 64% , 91% , and 0.59 , respectively . diagnostic accuracy of multislice computed tomography in the total population ( n = 396 ) * p<0.001 , compared with all vessels ; p<0.05 , compared with all vessels ; p<0.001 , compared with all segments ; p<0.05 , compared with all segments . tn : true negative ; tp : true positive ; fn : false negative ; fp : false positive ; ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . the diagnostic performance of msct for detecting significant stenoses in a vessel - based analysis was good . most of the significantly diseased vessels ( 75% , 570/756 ) were correctly identified using ct . ninety - six vessels ( 12% , 96/828 ) with nonsignificant stenoses were incorrectly classified as significant stenoses using ct . the diagnostic accuracy of msct in detecting left main ( lm ) cad is the highest ( 91% vs. 82% , compared with all vessels , = 10.120 , p < 0.05 ) . msct had a lower diagnostic accuracy ( 74% vs. 82% , compared with all vessels , = 12.432 , p < 0.05 ) in detecting lesions of left circumflex coronary . on a per - segment basis , the diagnostic values of msct in detecting lesions in distal segments and side branches were low , and the youden indexes for each segment in comparison with all the segments were 0.22 versus 0.4 ( u = 7.493 , p < 0.05 ) and 0.15 versus 0.4 ( u = 6.767 , p < 0.05 ) , respectively . table 3 shows the diagnostic accuracy of msct in the women compared with that in the men . on a patient - based analysis , the ppv ( 91% vs. 97% , = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , = 5.093 , p < 0.05 ) of msct in detecting cad were lower in the women . the women had a lower youden index ( 0.45 vs. 0.70 , u = 2.584 , p < 0.05 ) in msct diagnosis . in the cad population diagnosed using conventional invasive cag , 93% ( 110/118 ) of the female patients and 96% ( 218/228 ) of the male patients were correctly identified using msct . eleven women ( 48% , 11/23 ) and 7 men ( 26% , 7/27 ) with nonsignificant cad were incorrectly classified as having significant coronary stenoses using ct . diagnostic accuracy of multislice computed tomography in women versus men * p<0.05 , compared with men . ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . on a per - vessel basis ( lm , left anterior descending , left circumflex , and right coronary arteries ) , 564 coronary arteries in women and 1020 vessels in men were included in the study . the youden index showed no significant difference . using msct , 76% ( 186/244 ) of vessels in the women and 75% ( 384/512 ) of vessels in the men thirty - eight vessels ( 13% , 38/290 ) in the women and 58 vessels ( 11% , 58/508 ) in the men with nonsignificant stenoses on invasive cag were misdiagnosed using ct . after exclusion of 104 segments ( 23 for women and 81 for men ) owing to small vessel size , motion artifacts , and undetectable distal segments due to total occlusion of the proximal vessel , 1534 coronary segments in the female population and 2718 segments in the male population were included in this study . on a per - segment basis , the npv ( 87% vs. 83% , = 8.563 , p < 0.05 ) and diagnostic accuracy ( 82% vs. 79% , = 5.916 , p < 0.05 ) of msct in detecting cad were higher in the women . however , the ppv was relatively low in both groups for all the segments ( 60% and 63% , respectively ) and extremely low in distal segments and side branches ( 3050% ) . thus , the diagnostic values were low for both groups ( youden index : 0.42 and 0.39 , respectively ) . according to the msct results with conventional invasive cag as the reference standard , patients with true positive and true negative results were defined as the accurate diagnosis group . table 4 shows that the percentage of men was significantly higher in the accurate diagnosis group . multivessel diseases were significantly more frequent in the accurate group . in the multivariate logistic regression analysis , atypical presentation was an independent influencing factor of the diagnostic accuracy of msct in the women ( odds ratio [ or ] = 4.94 , 95% ci : 1.1620.92 , walds = 4.69 , p < 0.05 ) . multivessel disease ( or = 31.34 , 95% ci : 3.6272.6 , walds = 9.7 , p < 0.01 ) and noncalcified plaque ( or = 4.96 , 95% ci : 1.0124.51 , walds = 3.87 , p < 0.05 ) were also independent influencing factors . clinical characteristics based on the accuracy of multislice computed tomography cad : coronary artery disease ; msct : multislice computed tomography ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery ; ldl - c ; low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol . the whole population was divided into two groups , namely , the typical and atypical groups , according to angina pectoris symptoms . table 5 shows that in a patient - based analysis , the ppv ( 98% vs. 74% , = 17.283 , p < 0.001 ) and diagnostic accuracy ( 93% vs. 72% , = 9.571 , p < 0.001 ) of msct in detecting female cad were significantly higher in the typical group than in the atypical group . the women in the typical group had a higher youden index ( 0.69 vs. 0.29 , u = 2.359 , p < 0.05 ) in msct diagnosis . not much difference was observed for msct in the diagnosis of male cad between the two groups . diagnostic accuracy of multislice computed tomography in patients with typical or atypical angina symptoms * p<0.001 ; p<0.05 , compared with atypical group . ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value the diagnostic performance of msct to detect coronary stenoses of 50% was further investigated with respect to the roc curve . for all the patients , the auc was 0.84 ( 95% ci : 0.770.90 ) , suggesting a good accuracy . the auc were 0.90 ( 95% ci : 0.830.97 ) for men and 0.74 ( 95% ci : 0.620.87 ) for women ( z = 2.194 , p < 0.05 ) . in women , patients with typical symptoms had a higher auc ( 0.91 , 95% ci : 0.830.99 ) than those with atypical symptoms ( 0.64 , 95% ci : 0.450.82 , z = 2.690 , p < 0.01 ) . however , in the men , no significant difference in auc was found between the typical and atypical groups . our findings suggest that the ppv ( 91% vs. 97% , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , p < 0.05 ) of msct in detecting cad were lower in the women than in the men . a more important finding is that our results suggest that both gender and atypical symptoms may influence the diagnostic accuracy of msct . numerous studies have proven that significant sex - related differences exist not only in the incidence and prognosis of atherosclerosis disease but also in the accuracy of different clinical examination methods . anatomic and physiological differences , including body composition , heart rate , coronary calcium level , and coronary diameter between women and men , may affect the diagnostic performance of msct . our study proved sex - related differences in the performance of msct in detecting obstructive cad . in our study , the sensitivity , specificity , ppv , npv , and accuracy of msct in detecting cad were 95% , 64% , 95% , 64% , and 91% , respectively , on a per - patient level . the per - patient - based specificity and the sensitivity , specificity , ppv , npv , and diagnostic accuracy of msct on a per - segment level were 50% , 90% , 62% , 85% , and 80% , respectively . its sensitivity and npv were also relatively low . besides the technical problems , smaller body size and vascular lumen diameter may be possible reasons . in addition , our study population was almost 5 years older than that of other studies . the prevalence of calcified plaques was also relatively high in our study . in our study , the women with typical angina pectoris had higher ppv and diagnostic accuracy in msct diagnosis . thus , the diagnostic accuracy of msct may be different in women with atypical symptoms . several studies previously investigated the diagnostic accuracy of msct in female coronary heart disease , yielding inconclusive results . some studies found no significant difference in the performance of msct between women and men . while the study of meijboom et al . suggested a similar sensitivity in diagnosing coronary stenoses between the sexes , the specificity for detection of obstructive cad ( especially in distal and side coronary branches ) was significantly lower in the women than in the men . another study reported that msct has similar specificity between the sexes but lower sensitivity and accuracy in women . the inconsistent results between diagnostic studies may be related to the remarkable difference in the number of patients in each study . however , we should consider that the prevalence of cad is remarkably lower in women than in men . nonobstruction artery disease and positive remodeling of blood vessels are more common in women . after adjustment for body surface , the diameter of the female coronary artery was still smaller than that of the male coronary artery . studies have proven sex - related differences in plaque morphology on coronary computed tomography angiography . males tended to have more calcified plaques than females , with a greater tendency to have multiple vessel involvement . all of these factors might contribute to the significant differences in the diagnostic accuracy of msct between women and men . in our study , the diagnostic value of msct in the detection of cad was lower in the women than in the men . however , it is still a reliable modality for the exclusion of significant coronary artery stenoses in both sexes . our study suggests that both gender and atypical symptoms might influence the diagnostic accuracy of msct . our study is limited by its single - center retrospective design and the relatively small number of patients included . quantitative analysis of calcification and morphologies of plaques were not studied due to technical limitations . meanwhile , age and the prevalence rates of hypertension and diabetes were significantly higher in the women than that in the men , which might influence the results in our study . this work was supported by the grant from the beijing science and technology project in china ( no . this work was supported by the grant from the beijing science and technology project in china ( no .

background : multislice computed tomography ( msct ) coronary angiography ( cag ) is a noninvasive technique with a reported high diagnostic accuracy for coronary artery disease ( cad ) . women , more frequently than men , are known to develop atypical angina symptoms . the purpose of this study was to investigate whether the diagnostic accuracy of msct in women with atypical presentation differs from that in men.methods:we enrolled 396 in - hospital patients ( 141 women and 255 men ) with suspected or proven cad who successively underwent both msct and invasive cag . cad was defined as any coronary stenosis of 50% on conventional invasive cag , which was used as the reference standard . the patients were divided into typical and atypical groups based on their symptoms of angina pectoris . the diagnostic accuracy of msct , including its sensitivity , specificity , negative predictive value , and positive predictive value ( ppv ) , was calculated to determine the usefulness of msct in assessing stenoses . the diagnostic performance of msct was also assessed by constructing receiver operating characteristic ( roc ) curves.results:the ppv ( 91% vs. 97% , 2 = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , 2 = 5.093 , p < 0.05 ) of msct in detecting cad were lower in women than in men . atypical presentation was an independent influencing factor on the diagnostic accuracy of msct in women ( odds ratio = 4.94 , 95% confidence intervals : 1.1620.92 , walds = 4.69 , p < 0.05 ) . compared with those in the atypical group , women with typical angina pectoris had higher ppv ( 98% vs. 74% , 2 = 17.283 . p < 0.001 ) , diagnostic accuracy ( 93% vs. 72% , 2 = 9.571 , p < 0.001 ) , and area under the roc curve ( 0.91 vs. 0.64 , z = 2.690 , p < 0.01 ) in msct diagnosis.conclusions:although msct is a reliable diagnostic modality for the exclusion of significant coronary artery stenoses in all patients , gender and atypical symptoms might have some influence on its diagnostic accuracy .

according to the world health organization ( who ) , a maternal death is defined as death of a woman while pregnant or within 42 days of termination of pregnancy , irrespective of the duration and site of pregnancy , from any cause related to or aggravated by pregnancy or its management ( icd-10 ) . about 99% of these women are from developing world with over 90% concentrated in africa and asia . the risk of a woman dying as a result of pregnancy and childbirth during her lifetime is about 1 in 6 in afghanistan compared with 1 in 30,000 in northern europe . united nation ( un ) report card on millennium development goal-5 concluded that little progress had been made in sub - saharan africa where half of all maternal deaths take place . the progress shown by the south asian countries including india which accounts for 25% of all maternal deaths is also not impressive . most of the evidence for maternal mortality is obtained through hospital data and community based reports , which are situated mostly in urban areas , whereas most of the maternal deaths are from rural areas . this study was done to assess maternal mortality in a tertiary medical college hospital situated in semi urban part of western maharashtra where large numbers of patients are referred from rural parts of western maharashtra and north karnataka . this study was done to assess the local causes of maternal mortality and suggest remedial measures to reduce the same . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital.to assess the epidemiological aspects of maternal mortality.to assess the causes of maternal mortality.to suggest ways to reduce the mmr . to calculate the maternal mortality rate in our hospital . to assess the epidemiological aspects of maternal mortality . to assess the causes of maternal mortality . to suggest ways to reduce the mmr our hospital is a semi - urban , tertiary care center ; situated in the southernmost part of western maharashtra . it gets a large number of referrals from maternity homes , primary health centers from rural parts of western maharashtra , and also from north karnataka . the present study was a retrospective study , conducted in the department of obstetrics and gynecology of this hospital . data regarding maternal mortality was collected from maternal mortality register after obtaining permission from the medical superintendent of the hospital . the details of maternal deaths from january 2001 to december 2010 were collected and analyzed with respect to following epidemiological parameters . locality wise distribution of maternal deaths.literacy wise distribution of maternal deaths.gravidity wise distribution of maternal deaths.maternal deaths according to receipt of antenatal care.distribution of maternal deaths according to socioeconomic status.causes of maternal deaths . the details of number of live births from january 2001 to december 2010 were collected from labor ward register . maternal mortality rate for the study period was calculated by using the formula- mean maternal mortality ratio for the study period was calculated by calculating the mean of yearly mmr of the entire study period . during the study period , january 2001 to december 2010 , there were a total of 39704 live births and 120 maternal deaths . maximum maternal deaths ( 49.16% ) were reported in the age group of 20 to 24 years . ( 43.33% ) , more maternal deaths were reported in women from rural areas ( 69.16% ) as compared to women from urban areas ( 30.83%).maximum maternal deaths were reported in unbooked patients ( 83.33% ) as compared to booked patients ( 16.66% ) . epidemiological characteristics of maternal deaths ( n = 120 ) in the study period , 72.5% of maternal deaths were due to direct causes . year - wise distribution of direct causes of maternal deaths is shown in table 2 . the classical triad of hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) was the major direct causes of maternal deaths , whereas only one maternal death ( 0.83% ) was due to obstructed labor . in the study period , anemia , jaundice , and heart disease accounted for 10% , 9.16% , and 3.33% of maternal deaths respectively and miscellaneous cause like acute gastroenteritis accounted for 5% of maternal deaths as shown in table 3 . high incidence of maternal deaths reflects poor quality of maternal services , late referral and low socioeconomic status of the community . various studies done in india in the last 15 years have shown wide variation in mmr ranging from 47/100000 to 625/100000 births.[49 ] madhu jain has reported a very high mmr of 2270/100000 . this study has comparatively high mmr , which could be due to the fact , that our hospital is a tertiary care hospital and receives a lot of complicated referrals from rural areas of southern maharashtra and also from north karnataka at a very late stage . in our study , 70% of maternal deaths were in the age group of 20 to 29 years , as highest numbers of births are reported in this age group . more maternal deaths were reported in women from rural areas ( 69.16% ) , unbooked patients ( 83.33% ) , illiterate women ( 65% ) , and women belonging to low socioeconomic status . ( 83.33% ) all our findings were similar to studies by jain , jadhav , pal , onakewhor . in our study , hemorrhage ( 26.66% ) , eclampsia ( 26.66% ) , and sepsis ( 18.33% ) were the major direct causes of maternal deaths . our findings were consistent with studies by jain , jadhav , pal , onakewhor , and shah . even today large number of maternal deaths is due to the classical triad of hemorrhage , sepsis , and eclampsia . all these are preventable causes of maternal mortality provided the treatment is instituted in time . unfortunately , in many cases , patients were referred very late , in critical condition , unaccompanied by health care worker . many patients had to travel a distance of 70 to 80 kilometers in a private vehicle to reach our tertiary center . most of these deaths are preventable if patients are given appropriate treatment at periphery and timely referred to higher centers . training of medical officers and staff nurses working in rural areas by programs like basic emergency obstetrics care ( bemoc ) and skilled attendant at birth ( sab ) training gives a ray of hope of reducing maternal mortality . anemia , jaundice , and heart disease were responsible for 10% , 9.16% , and 3.33% of maternal deaths , respectively . these findings were consistent with studies by jain , jadhav , pal , and onakewhor . maternal deaths can be prevented by improving the health care facilities in rural areas by ensuring round the clock availability of certain basic drugs like injection magnesium sulfate , tablet misoprostol as most maternal deaths in rural areas are still due to eclampsia and post partum hemorrhage . early detection of high risk pregnancies and referring them to a tertiary center at the earliest can reduce the complications of high risk pregnancies . national rural health mission ( nrhm ) can play a major role in reducing maternal mortality by advocating institutional deliveries and timely referral of high risk cases . although we have not actually evaluated the impact of aforementioned educational programs on maternal mortality , it would be interesting to direct future studies in this regard . even today most maternal deaths are seen in patients from rural areas , unbooked , illiterate patients and patients from low socioeconomic status . hemorrhage , eclampsia and sepsis are the major causes of maternal deaths . improvement in primary health care in rural areas and proper implementation of nrhm programs and up gradation of hospitals in rural areas can definitely bring down the number of maternal deaths .

background : epidemiological data pertaining to maternal mortality is valuable in each set up to design interventional programs to favourably reduce the ratio . this study was done to evaluate the maternal mortality rate in our hospital , to assess the epidemiological aspects and causes of maternal mortality , and to suggest recommendations for improvement.methods:this was a 10 year retrospective study . epidemiological data was collected from the hospital register and maternal mortality ratio , epidemiological factors and causes affecting maternal mortality were assessed.results:a total of 120 maternal deaths occurred . most maternal deaths occurred in the age group of 2024 years , multiparous women ( 56.66% ) , women from rural areas ( 69.16% ) , illiterate women ( 65% ) , unbooked patients ( 83.33% ) , and patients of low socioeconomic status ( 83.33% ) . direct causes accounted for 72.5% of maternal deaths where as 27.5% of maternal deaths were due to indirect causes.conclusion:there is a wide scope for improvement as a large proportion of the observed deaths are preventable .

original description of pacemaker syndrome was done for the first time by mitsui et al . in 1969 as a collection of symptoms associated with right ventricular ( rv ) pacing . since its first description , several definitions have been proposed for the pacemaker syndrome . the mode selection trial ( most ) investigators defined the pacemaker syndrome as occurring if either one of two different criteria was met : the first criterion was new or worsened dyspnea , orthopnea , rales , elevated jugular venous pressure , and edema with ventriculoatrial conduction during ventricular pacing . the second criteria was symptoms of dizziness , weakness , presyncope , or syncope , and a > 20 mmhg reduction of systolic blood pressure when the patient had vvir pacing compared with atrial pacing or normal sinus rhythm . these symptoms lead to significant decrease in quality of life , and sometimes surgical intervention was required to change the pacing mode from vvir to dddr . despite the significant progress , understanding of the cause of pacemaker syndrome is still under investigation . role of atrioventricular ( av ) synchrony in the emergence of this syndrome has been carefully addressed , but the role of rv - left ventricle ( lv ) dyssynchrony has not been studied yet . there are speculations that the pacemaker syndrome may be etiologically related to interventricular ( vv ) dyssynchrony imposed by the high percentage of ventricular pacing commonly seen in the dddr pacing . this data shortage is mainly related to fact that standard antibradycardia pacing only allowed for univentricular rv pacing . consequently , we enrolled a cohort of the patients with biventricular pacing to permit univentricular pacing from rv and lv , av sequential and ventricular - only biv pacing , and no pacing ( sinus rhythm ) . utility of beat - to - beat finger plethysmography for hemodynamic assessment of different pacing modes has been demonstrated previously . therefore , we designed a study to noninvasively assess the hemodynamic effects of different ventricular pacing sites with and without av and vv dyssynchrony using beat - to - beat finger plethysmography and to observe the patients for clinical symptoms of the pacemaker syndrome during the different av sequential and ventricular - only pacing modes . between march 2009 and february 2010 , a total of 40 patients ( 28 men , mean age , 61 15 years ) with cardiac resynchronization therapy ( crt ) device were enrolled in this cross - sectional study . the resynchronization devices consist of 31 biventricular defibrillators ( crt - d ) and 9 biventricular pacemakers ( crt - p ) . the patients with pacemaker dependency , atrial fibrillation , and recent heart failure decompensation were excluded . the study protocol was approved by the local ethics committee , and all patients gave written consent prior to the study . after attaching the plethysmography probe to patient 's finger , systolic and diastolic blood pressures ( bp ) were recorded in different pacing modes , namely , right ventricle pacing ( vvi - rv ) , left ventricular pacing ( vvi - lv ) , biventricular pacing ( vvi - biv ) , biventricular sequential pacing ( ddd - biv ) , and sinus rhythm ( odo ) . average of five beats blood pressure ( bp ) in each mode was accepted for comparison . five minutes was allowed between each mode change to ensure that the effect of previous pacing mode on measured bp had vanished . patients symptoms were recorded simultaneously ( palpitation , dyspnea , chest pain , dizziness , presyncope , and syncope ) . the student t - test was used to compare the data between the two groups with a normal distribution . between march 2009 and february 2010 , a total of 40 patients ( 28 men , mean age , 61 15 years ) with cardiac resynchronization therapy ( crt ) device were enrolled in this cross - sectional study . the resynchronization devices consist of 31 biventricular defibrillators ( crt - d ) and 9 biventricular pacemakers ( crt - p ) . the patients with pacemaker dependency , atrial fibrillation , and recent heart failure decompensation were excluded . the study protocol was approved by the local ethics committee , and all patients gave written consent prior to the study . after attaching the plethysmography probe to patient 's finger , systolic and diastolic blood pressures ( bp ) were recorded in different pacing modes , namely , right ventricle pacing ( vvi - rv ) , left ventricular pacing ( vvi - lv ) , biventricular pacing ( vvi - biv ) , biventricular sequential pacing ( ddd - biv ) , and sinus rhythm ( odo ) . average of five beats blood pressure ( bp ) in each mode was accepted for comparison . five minutes was allowed between each mode change to ensure that the effect of previous pacing mode on measured bp had vanished . patients symptoms were recorded simultaneously ( palpitation , dyspnea , chest pain , dizziness , presyncope , and syncope ) . the data were recorded in spss ( chicago , il , usa ) ver . 16 software package . the student t - test was used to compare the data between the two groups with a normal distribution . hemodynamic data in different pacing modes are depicted in figures 1 and 2 . paired comparisons of systolic bp in vvi - rv , vvi - lv , and vvi - biv systolic bp in ddd - biv mode is similar to sinus rhythm ( odo mode ) ; however , the former had a significantly higher systolic bp than ventricular - only ( vvi - rv , vvi - lv , and vvi - biv ) modes [ figure 1 ] . the diastolic bp was comparable in different ventricular - only modes [ table 1 ] and there were no significant differences between the av sequential biventricular and any of the ventricular - only modes [ figure 2 ] . paired comparisons of the systolic blood pressure between the atrioventricular sequential biventricular pacing ( ddd - biv ) and ventricular - only pacing modes ( vvi - rv , vvi - lv , and vvi - biv ) . note that systolic blood pressure is higher in ddd - biv pacing mode compared with any of ventricular - only pacing modes . in addition , systolic blood pressure is comparable between the ddd - biv and sinus rhythm paired comparisons of the diastolic blood pressure between the atrioventricular sequential biventricular pacing ( ddd - biv ) and ventricular - only pacing modes ( vvi - rv , vvi - lv , and vvi - biv ) . there is no difference in the diastolic blood pressure between the ddd - biv and ventricular - only pacing modes . in addition , diastolic blood pressure is similar between the ddd - biv and sinus rhythm comparison of hemodynamic data between different ventricular - only pacing modes none of the patients developed syncope , presyncope , or dizziness after mode change . mode change from ddd - biv to vvi - rv was associated with palpitations and dyspnea in 22.5% of the patients . similarly , 22.5% of the patients experienced palpitations and dyspnea after ddd - biv to vvi - lv mode change . ddd - biv to vvi - biv resulted in palpitations and dyspnea in 12.5% of the patients . however , incidence of palpitations and dyspnea was similar between the mode changes from ddd - biv to all vvi modes [ table 2 ] . hemodynamic data in different pacing modes are depicted in figures 1 and 2 . paired comparisons of systolic bp in vvi - rv , vvi - lv , and vvi - biv systolic bp in ddd - biv mode is similar to sinus rhythm ( odo mode ) ; however , the former had a significantly higher systolic bp than ventricular - only ( vvi - rv , vvi - lv , and vvi - biv ) modes [ figure 1 ] . the diastolic bp was comparable in different ventricular - only modes [ table 1 ] and there were no significant differences between the av sequential biventricular and any of the ventricular - only modes [ figure 2 ] . paired comparisons of the systolic blood pressure between the atrioventricular sequential biventricular pacing ( ddd - biv ) and ventricular - only pacing modes ( vvi - rv , vvi - lv , and vvi - biv ) . note that systolic blood pressure is higher in ddd - biv pacing mode compared with any of ventricular - only pacing modes . in addition , systolic blood pressure is comparable between the ddd - biv and sinus rhythm paired comparisons of the diastolic blood pressure between the atrioventricular sequential biventricular pacing ( ddd - biv ) and ventricular - only pacing modes ( vvi - rv , vvi - lv , and vvi - biv ) . there is no difference in the diastolic blood pressure between the ddd - biv and ventricular - only pacing modes . in addition , diastolic blood pressure is similar between the ddd - biv and sinus rhythm comparison of hemodynamic data between different ventricular - only pacing modes mode change from ddd - biv to vvi - rv was associated with palpitations and dyspnea in 22.5% of the patients . similarly , 22.5% of the patients experienced palpitations and dyspnea after ddd - biv to vvi - lv mode change . ddd - biv to vvi - biv resulted in palpitations and dyspnea in 12.5% of the patients . however , incidence of palpitations and dyspnea was similar between the mode changes from ddd - biv to all vvi modes [ table 2 ] . major findings of the current study are as follows : ( 1 ) non - av sequential biventricular pacing , univentricular rv pacing , and univentricular lv pacing produced similar systolic and diastolic bps ; ( 2 ) av sequential biventricular pacing and sinus rhythm had similar hemodynamic profile and produced higher systolic bp than ventricular - only pacing modes ; ( 3 ) mode downgrading from av sequential biventricular ( ddd - biv ) to all ventricular - only modes ( vvi - rv , vvi - lv , and vvi - biv ) were associated with similar rate of palpitations and dyspnea , but mode change from ddd - biv to odo was not associated with development of new symptoms . to the best of our knowledge , this is the first quantitative study performed in different pacing modes to evaluate for av and vv dyssynchrony in patients with biventricular device . similar to our study , varma et al . showed no hemodynamic benefit for biv and lv pacing compared with rv pacing . previously , it was shown that in patients having dual - chamber pacemaker , mode change from ddd to vvi leads to systolic bp decline with no significant change in diastolic bp , which is related to symptomatic intolerance of vvi pacing and may have potential utility as an aid to diagnosis or as a predictor of pacemaker syndrome . in our study , diastolic bp was similar in different pacing modes ; this is in accordance with prior quantitative studies . diastolic blood pressure is not directly linked to cardiac output and ventricular systolic function , but on the contrary it is derived from systemic vascular resistance , peripheral run - off , and heart rate . besides the decisive role for av dyssynchrony in the emergence of pacemaker syndrome since early reports of vvi - rv pacing , another role for interventricular dyssynchrony caused by rv pacing was raised . since the first nomination of cardiac synchronization by cazeau et al . in 1994 for placement of four epicardial leads on all four cardiac chambers , role of av synchrony and optimization was not less than v - v synchrony . it is hypothesized that rv pacing with or without av synchrony induces a nonphysiologic contraction similar to that caused by left bundle branch block . this dyssynchrony leads to disturbed lv diastolic filling , reduction of cardiac output , and increase in mitral regurgitation.[1012 ] our analysis showed that decrease in systolic bp accompanied by dyspnea and palpitation ( as an index of pacemaker syndrome ) is related to loss of av synchrony rather than site of ventricular pacing ( rv , lv or biv ) . farmer et al . believed that the majority of the symptoms of pacemaker syndrome are likely attributable to the reduction in ejection fraction and cardiac output . increase in mitral regurgitation associated with right ventricular pacing is guilty for pacemaker syndrome , and if biv pacing is performed , pacemaker syndrome is less likely . we observed that with mode change from ddd - biv to odo ( when device was turned off ) , no new symptom or change in blood pressure was observed . considering the design of our study ( quantitative assay accompanied by reproduction of symptoms , high fidelity measurement with fingertip plethysmography ) , we believe that pacemaker syndrome is derived mainly from av dyssynchrony rather than vv dyssynchrony . this study was an acute hemodynamic study ; real value of these findings should be tested in a long - term study . this study is also limited by the fact that we did not evaluate the role of different rv pacing sites ( mid septal , low septal , or apical ) on the hemodynamic data and the clinical symptoms . this study was an acute hemodynamic study ; real value of these findings should be tested in a long - term study . this study is also limited by the fact that we did not evaluate the role of different rv pacing sites ( mid septal , low septal , or apical ) on the hemodynamic data and the clinical symptoms . the present acute hemodynamic study showed that location of ventricular pacing and vv dyssynchrony may have no important role in the pathogenesis of the pacemaker syndrome . this study also confirmed the fundamental role of av synchrony in preventing the pacemaker syndrome .

background / objectives : pacemaker syndrome was mainly described as the sequel of atrioventricular ( av ) dyssynchrony . the role of interventricular ( vv ) dyssynchrony has not been studied yet . the aims of this study were to noninvasively assess the hemodynamic effects of different ventricular pacing sites with and without av and vv dyssynchrony and to observe the patients for clinical symptoms of the pacemaker syndrome during the av sequential and ventricular - only pacing modes.materials and methods : between march 2009 and february 2010 , 40 patients ( 28 men ; mean age , 61 15 years ) with biventricular ( biv ) device were enrolled . mean systolic and diastolic blood pressures ( bp ) of 5 beats were measured 5 minutes after each mode change using fingertip plethysmography . the patients were also observed for the occurrence of symptoms suggestive of the pacemaker syndrome , including dyspnea , palpitations , dizziness , presyncope , and syncope.results:there was no difference in mean systolic bp among different ventricular - only pacing modes ( all p = ns ) . however , mean systolic bp was significantly higher in av sequential biventricular pacing ( ddd - biv ) compared with ventricular - only pacing modes ( all p<0.05 ) . in addition , there was no difference in terms of pacemaker syndrome - related symptoms following mode change from ddd - biv to ddd - rv or ddd - lv ( all p>0.05).conclusions : the present study showed that the non - av sequential biv and lv pacing may have no significant benefit compared with rv pacing in terms of systolic blood pressure . however , there was marked hemodynamic improvement following mode change to av sequential biv pacing . this study may have important implications for pathogenesis of pacemaker syndrome .

cardiac adenosine stress perfusion magnetic resonance ( mr ) imaging is a highly accurate diagnostic tool for coronary artery diseases ( 1 - 3 ) . it measures the contrast enhancement of the myocardium during the first pass of a contrast agent bolus as it is sensitive to changes in the myocardial blood flow . during the imaging of a patient after a coronary artery bypass graft ( cabg ) surgery , the first pass kinetics of a contrast agent bolus are complex due to the altered distance of bypass graft vessels to the myocardial territory . herein , we report about a case in which a delay in contrast agent arrival was observed at the myocardial territory whilst the patent bypass graft , mimicking a perfusion defect on adenosine stress perfusion mr images . the report also compares the semiquantitative perfusion parameters of this myocardial territory by the patent bypass graft with those of normal myocardium and of another myocardial territory with a stenotic bypass graft in the same patient . a 50-year - old man visited our hospital for a 3 month - follow - up examination after coronary bypass graft surgery . in this surgery a left internal thoracic artery ( lita ) graft to the distal left anterior descending artery ( lad ) , and a right saphenous venous y graft from the lita to the diagonal branch and posterior descending artery ( pda ) was performed through sequential anastomosis . the cardiac adenosine stress perfusion mr imaging revealed two perfusion defects at the mid - anterior and mid - inferior walls ( fig . one perfusion defect at the mid - anterior wall was reversible on rest perfusion images . however , the other perfusion defect at the mid - inferior wallwas persistent on rest perfusion images . but later it disappeared on both stress and rest perfusion images . on the myocardial single - photon emission computed tomography , the inferior wall showed a mild degree of perfusion decrease which was considered as due to the diaphragmatic attenuation effect rather than an ischemic perfusion defect while the mid - anterior wall showed a reversible perfusion defect indicating a reversible ischemia ( fig . no regional wall motion abnormalities and on late gadolinium enhancement images no delayed myocardial enhancement were observed . signal time curve analyses at stress and rest perfusion mr were performed at three different regions : normal myocardium at the mid - lateral wall and two perfusion defects at the mid - anterior and mid - inferior walls ( fig . the perfusion index and ratio index of each region were calculated using a previously described method ( 4 ) . in brief , a smooth - fit corrected signal - time curve within the time window of the first pass was analyzed using the gamma - variate function after subtraction of the baseline signal intensity ( si ) value from the mean si of each region . the time window of the first pass was determined from the si curve of the left ventricular cavity . the time window of the first pass in the myocardium at the same level as the left ventricular cavity was determined by shifting the time window determined from the left ventricular cavity to when the si in the myocardium started to rise . the maximal upslope indicating the perfusion index was computed from the peak value of the time derivatives of the fit function in the myocardial region , normalized by the maximal upslope in the left ventricular cavity . the ratio index , widely accepted to represent myocardial perfusion reserve , was defined as the ratio of maximal upslope at stress to that at rest ( 4 ) . to calculate perfusion parameters an in - house software was developed using matlab 2012a ( mathworks , natick , ma , usa ) . the corrected signal - time curves demonstrated that the maximal upslope of the anterior wall decreased from 0.089 during rest to 0.022 during stress perfusion with a ratio index of 0.25 , indicating typical reversible ischemia ( fig . 1d , e ) . in contrast , the maximal upslope of the mid - inferior wall was similar to that of normal myocardium during stress and rest perfusion . but the time to delivery and time to peak enhancement were delayed 7 seconds on both stress and rest images approximately ( fig . the maximal upslope of the mid - lateral wall increased with a ratio index by 1.10 from 0.094 during rest perfusion to 0.104 during stress perfusion . also for the mid - inferior wall the maximal upslope increased with a ratio index by 1.10 from 0.079 during rest to 0.087 during stress perfusion ( fig . coronary angiography revealed a focal tight stenosis at the anastomosis site between the right saphenous venous graft and diagonal branch , limiting the flow to the diagonal branch . with this can be explained the reversible ischemia at the apical to the mid - anterior wall on perfusion mri . however , there was no significant stenosis at the right saphenous venous graft to the pda . the contrast delivery in the right saphenous venous graft was slightly delayed compared to the lita to distal lad graft . it may indicate that the early perfusion defect at the mid - inferior wall was a result of delayed contrast media delivery due to the wide and long pathway of the bypass graft ( fig . six - month follow - up images showed still a persistent perfusion defect without significant stenosis of the saphenous venous y graft in the mid - inferior myocardial wall ( fig . this case report illustrates focal delayed myocardial perfusion at the mid - inferior wall after cabg surgery mimicking a perfusion defect on adenosine stress perfusion mr images . there was a significant delay of contrast agent arrival ( 7 seconds ) observed on the semiquantitative analysis of the perfusion at the mid - inferior wall myocardium . however , the maximal upslope of the corrected signal - time curves and rest - to - stress ratio index of maximal upslope were similar to those of normal myocardium . on coronary angiography , there was no significant stenosis at the bypass graft to the mid - inferior myocardial territory . therefore , the early perfusion defect at the mid - inferior wall on stress and rest perfusion mris can be interpreted as a result of delayed delivery of contrast media due to the wide and long pathway of the venous graft . since an exercise electrocardiogram is limited in many post - bypass patients , non - invasive stress imaging tests are often preferred to assess the patency of the graft ( 5 ) . although adenosine stress perfusion mr has been accepted as an accurate diagnostic tool , there have been a limited number of studies in post - bypass patients only with mixed results ( 6 , 7 ) . one study reported about reduced diagnostic accuracy in patients with cabg and attributed this to the different flow and perfusion kinetics involved ( 6 ) . another study reported about good diagnostic accuracy for the detection and localization of significant stenosis , but also about a reduced sensitivity compared with published data for patients without cabg ( 7 ) . these results were obtained by visual assessment of myocardial perfusion . in the present study , a semiquantitative analysis method was employed to better evaluate regional perfusion abnormalities . among the semiquantitative parameters , upslope at stress is known to best detect ischemic myocardial segments and has been shown to correlate well with angiography and positron emission tomography ( 2 , 8) . in our case , maximal upslopes at stress in both the normal myocardium and myocardium perfused by the patent graft with a long pathway were higher than that of ischemic myocardium . similar to a previous study the rest - to - stress ratio index of the maximal upslope of the inferior wall perfused by the patent graft with a long pathway was also similar to that of normal myocardium in the presented case ( 4 , 9 ) . ( 4 ) reported that upslope significantly increased from rest to stress in normal myocardium , whereas in ischemic myocardium the change was insignificant . in another study , 9 ) calculated the semiquantitative parameters of 38 patients after cabg surgery and reported similar wash - in kinetics ( upslope ) with a short delay in contrast arrival in areas perfused by bypasses compared to native coronaries . they observed that the mean contrast delay was below one beat in heartbeats and therefore concluded that differing contrast kinetics through graft vessels might not be a limiting factor in the accuracy of adenosine stress perfusion mr in post - cabg patients . our case exhibited a contrast delay of 7 seconds which is much more delayed than in kelle et al . the authors therefore postulate that the longer pathway of the graft and the size discrepancy between the graft and anastomotic native vessels may cause a greater delay in contrast delivery . first , the delayed perfusion may be influenced by the collateral flow from the native coronary artery . second , the pda graft is a sequential y graft of the saphenous vein and there is a tight stenosis at the proximal anastomotic site of diagonal branch . thus , flow disturbances in the distal sequential graft due to proximal stenosis may have caused this perfusion defect more than the long graft course and size discrepancy . however , in this case , the tight stenosis was located in the proximal diagonal branch affecting the diagonal branch flow only . interestingly , on follow - up coronary angiography taken after 6 months , the diagonal branch regressed due to tight stenosis and no stenosis was detected in the y graft of saphenous vein . follow - up perfusion mr images at both stress and rest after spontaneous regression of the diagonal branch still showed the same feature of a persistent perfusion defect in the inferior myocardial wall , indicating a delayed perfusion rather than a true perfusion defect . this case illustrates focal delayed perfusion after bypass surgery mimicking a perfusion defect on adenosine stress perfusion mr images . the delayed perfusion observed in our study also mimicked a perfusion defect in the myocardial spect study . however , the semiquantitative analysis of corrected signal - time curves revealed that the myocardial territory in question was not ischemic but presented a delayed perfusion due to the long pathway of the bypass graft .

herein we report about the adenosine stress perfusion mr imaging findings of a 50-year - old man who exhibited two different perfusion defects resulting from two different mechanisms after a coronary artery bypass surgery . an invasive coronary angiography confirmed that one perfusion defect at the mid - anterior wall resulted from an ischemia due to graft stenosis . however , no stenosis was detected on the graft responsible for the mid - inferior wall showing the other perfusion defect . it was assumed that the perfusion defect at the mid - inferior wall resulted from delayed perfusion owing to the long pathway of the bypass graft . the semiquantitative analysis of corrected signal - time curves supported our speculation , demonstrating that the rest - to - stress ratio index of the maximal slope of the myocardial territory in question was similar to those of normal myocardium , whereas that of myocardium with the stenotic graft showed a typical ischemic pattern . a delayed perfusion during long graft pathway in a post - bypass graft patient can mimick a true perfusion defect on myocardial stress mr imaging . radiologists should be aware of this knowledge to avoid misinterpretation of graft and myocardial status in post bypass surgery patients .

as producers and decomposers , bacterioplankton are a key component of microbial food webs and play significant roles in biogeochemical cycles . in algae bacteria symbiosis , bacteria extensively participate in substance cycles , oxidation reduction activities , and regulate the balance of phytoplankton ecosystems , which is the most basic and active link in the whole phycosphere habitat . in recent years , chemical ecology has emerged as a new approach to evaluate the structural and functional diversity , as well as the dynamic equilibrium of the algae many key life processes , including food acquisition , movement behavior , defense , and signal communication are mediated by signal interactions at the individual , population , and community levels , . understanding signal regulated ecological processes will help to define the crucial molecular interactions between algae and bacteria . infochemicals are frequent in the phycosphere , and various cross - talking behaviors of heterotrophic bacteria have been identified in algae these processes are often controlled by cell density - dependent regulation of gene expression , which is mediated by diffusible signal molecules whose concentration correlates with the population density . this process is termed quorum sensing ( qs ) , and one of the most well - known qs signals is n - acyl - homoserine lactone ( ahl ) . ahls can induce gene expression either directly by interacting with a transcriptional regulator , or indirectly by activating a signal cascade . bacteria use ahl to regulate a variety of phenotypes such as biofilm formation , exopolysaccharide production , virulence factor production , and motility , which are essential for the successful establishment of a symbiotic relationship with their eukaryotic hosts . previous studies have shown that alga - associated bacterial isolates ( of epi- or endophytic origin ) produce ahls and exhibit various functions , including facilitating the settlement of zoospores , regulating the morphogenesis pattern , affecting individual reproduction , and promoting the liberation of carpospores . recently , our group isolated an enterobacter sp . st3 strain from the dinoflagellate scrippsiella trochoidea ( the sample environmental features list in table 1 ) . the electron micrograph images showed that st3 strain is a motile and long rod - shaped bacterium ( fig . it has broad environmental suitability and the optimal growth temperature and ph values were 30 c and 8.0 , respectively ( fig . 2 ) . as a member of -proteobacteria , enterobacter sp . play multi - roles in symbiosis environment , such as exopolysaccharide produce , iron metabolism , heavy metals biosorption , and pollutant biodegradation , , , . in algae bacteria symbionts , enterobacter sp . is often found as a main species and participant in the matter cycles of the phycosphere . of particular note , this isolate st3 possesses cross - talking language activities , and can secrete short - chain ( c6 ) ahl molecules ( fig . although the phenotype was previously observed , this type of functionality has not been elucidated at the gene level with genomic approaches . in addition , we speculated that its ecological function was regulated by quorum sensing , but there is a lack of direct evidence for confirmation on the genomic level . in order to better understand genetic underpinning of the bacterium roles , here , we performed whole - genome sequencing of this bacterium and searched for its ahl encoding gene(s ) . the genomic dna of enterobacter sp . st3 was extracted using the dna extraction kit ( mo bio , ca , usa ) according to the manufacturer 's instructions . the whole - genome shotgun project of enterobacter sp . st3 was performed using pair - end sequencing in an illumina miseq sequencing platform ( illumina , ca , usa ) , which was performed by shenzhen hengchuan gene - tech . the reads were assembled with soapdenovo ( v.2.04 ) , and the sequence was annotated using the rast annotation server . trna and rrna genes were predicted by trnascan - se and rnammer , respectively . genes were predicted using glimmer 3.02 and annotated by searching against the nvbi - nr and kegg databases . the whole genome of enterobacter cloacae st3 comprised 4,815,521 nucleotides and the g + c content was 55.59% ( table 2 ) . it contains 75 contigs with an n50 contig length of 127,978 bp . the whole genome encodes nine 5 s rrna , two 16 s rna , five 23 s rna , and seventy - nine trna genes . based on functional categories of cog ( http://www.ncbi.nlm.nih.gov/cog/ ) , a total of 426 genes were annotated to be involved in carbohydrate transport and metabolism , which enables the strain to adapt to and compete in algae we found an ahl - related gene ( luxr family transcriptional regulator ) located in the upstream position ( contig 1 ) , which indicates that enterobacter sp . st3 may be able to mediate its cell density with ahl signals and exhibit its functions in a fluctuating phycosphere environment . this gene showed relatively high sequence identity to the luxr gene of another enterobacter species ( genbank : wp_017692678.1 ) . some scientists found some bacterial ecological function ( for example algicidal activity ) was regulated by quorum sensing from the genomic level . the genome sequence of enterobacter sp . st3 should enable further research to gain deeper insights into the molecular mechanisms of algae bacteria interactions , and may facilitate the development of new ideas for improving the understanding of algae bloom formation and carbon cycle processes . the authors declare that there is no conflict of interests on work published in this paper .

phycosphere environment is a typical marine niche , harbor diverse populations of microorganisms , which are thought to play a critical role in algae host and influence mutualistic and competitive interactions . understanding quorum sensing - based acyl - homoserine lactone ( ahl ) language may shed light on the interaction between algal - associated microbial communities in the native environment . in this work , we isolated an epidermal bacterium ( was tentatively named enterobacter sp . st3 , and deposited in soa china , the number is mccc1k02277-st3 ) from the marine dinoflagellate scrippsiella trochoidea , and found it has the ability to produce short - chain ahl signal . in order to better understand its communication information at molecular level , the genomic map was investigated . the genome size was determined to be 4.81 mb with a g + c content of 55.59% , comprising 6 scaffolds of 75 contigs containing 4647 protein - coding genes . the functional proteins were predicted , and 3534 proteins were assigned to cog functional categories . an ahl - relating gene , luxr , was found in upstream position at contig 1 . this genome data may provide clues to increase understanding of the chemical characterization and ecological behavior of strain st3 in the phycosphere microenvironment .

desmoid tumors ( dts ) , also known as aggressive fibromatoses , are characterized as rare , slow - growing tumors . although strictly benign , they are locally invasive with a high rate of recurrence even after seemingly total resection.1 dts typically develop sporadically , often in the abdomen , hip / buttocks area , and shoulder girdle . some populations are at higher risk , including individuals with episodes of antecedent trauma at the site of the tumor.2 a particularly rare subset of these cases includes dts that develop adjacent to the spinal column . a total of five reported cases have described this phenomenon in the absence of gardner syndrome , a variant of familial adenomatous polyposis ( fap ) , or any other predisposing genetic diseases.3 4 5 6 7 this report describes an instance of postoperative aggressive fibromatosis that developed 10 months after corpectomy and spinal fusion for treatment of an atypical vertebral hemangioma in the thoracic spine . a 57-year - old woman presented with lower extremity clumsiness , balance , and ambulation difficulty resulting from spinal cord compression due to an upper thoracic atypical vertebral hemangioma . 1 ) . the resection involved a transpedicular approach for a t4 corpectomy , placement of an expandable cage extending from t3 to t5 and instrumented posterior fixation from t2 to t7 . ( a ) sagittal computed tomography ( ct ) demonstrating the hemangioma in the t4 vertebral body ( arrow ) . ten months postoperatively , the patient reported moderate pain at the rostral end of the surgical incision . a mass reported by the patient to have enlarged was found on palpation , just superior to the surgical incision . as the mass continued to grow , the patient suffered from increasing local pain and pressure , but there were no neurological deficits . magnetic resonance imaging ( mri ) revealed a 5.7 1.6 3.1 cm t2 hyperintense mass within the right subcutaneous tissues and paraspinal muscles at the t1t3 levels ( fig . the mass was located superficial to a pedicle screw at the rostral end of the fusion construct . negative margins were achieved through added removal of the spinous processes of t1t2 and part of c7 . magnetic resonance images ( mri ) showing the postlaminectomy desmoid tumor ( dt ) and results after surgical excision . ( a ) preoperative sagittal stir mri showing the dt ( arrow ) . ( b ) the firm rubbery mass was in some areas histologically compact and appeared to exclude preexisting elements . the degrees of cellularity and cytological atypia varied from region to region , but both were low in most areas . in a few regions , however , there was a modest degree of cytological atypia and low - level mitotic activity . 3 ) . immunostaining for -catenin was confined to the cytoplasm in most cells , but there were scattered cells in which nuclei were also positive . ( a ) the paucicellular , somewhat fascicular and lobular mass incorporates regional tissues such as skeletal myocytes ( 40 ) . ( b ) cell density here is low and there is only slight - to - moderate cytological atypia . serial mri was performed to follow for tumor recurrence . at 2-year follow - up , aggressive fibromatosis comprises 0.03% of all neoplasms and less than 3% of all soft tissue tumors.2 several conditions such as fap and other hereditary cancer syndromes predispose individuals to developing these fibroblastic growths.8 a summary of the five previously reported cases of dt formation soon after laminectomy in patients without a unique predisposing condition are presented in table 1.3 4 5 6 7 some mild postoperative ( procedure before development of desmoid tumor ) paresthesias or weakness . all cases occurred in adult women ranging in age from 39 to 50 years , with our patient , a 57-year - old woman , having similar demographics . this is concordant with previous studies that have asserted that 67 to 80% of all dts arise in women , with 50% between the ages of 30 to 50 years . it has been previously suggested that aggressive fibromatosis may be hormonally related.2 in fact , middle - aged mothers may be at heightened risk because of characteristic hormone profiles . in addition , none of the five previous cases developed neurological deficits as a result of the dt . two patients did have mild residual neurological side effects as a result of the initial laminectomy procedure , but these symptoms were unrelated to the dt . the number of reported cases of paraspinous aggressive fibromatosis cases occurring postlaminectomy indicates that this may not be as rare of a phenomenon as originally suspected . the scar tissue surrounding a laminectomy may be predisposed to neoplastic transformation due to underlying inflammation from both the treated malignancy and tissue manipulation from surgery . recent studies have demonstrated that sporadic dts often have mutations in adenomatosis polyposis coli and -catenin genes . a study by lazar et al demonstrated that 85% of sporadic dts have mutations in the ctnnb1 gene , which encodes -catenin . furthermore , different ctnnb1 mutations were associated with differing rates of progression and recurrence.9 -catenin has been observed to play a significant role in postfracture bone healing . a study by chen et al demonstrated that -catenin signaling is activated during bone healing . furthermore , resultant t cell factor - dependent transcription is only activated in later stages of repair , after mesenchymal cells have differentiated into fibroblastic cells.10 this would suggest a unique molecular and cellular environment within the surgical bed during both the acute and chronic stages of healing . theoretically , any bone fracture has the potential to trigger dt growth that could explain the development of aggressive fibromatosis after clavicle and radius fractures . dts are often difficult to treat because of their proliferative nature and capacity for local invasion . treatment becomes even more challenging when these masses are found proximal to sensitive anatomical structures , such as in the spine . in the presented case and this was seen to be the best option given the high local recurrence rate associated with positive margin resections ( 54% over 10 years ) as compared with those with negative margins ( 27% over 10 years ) . if the mass were located proximal to functionally important anatomic entities such that negative margins were impossible to achieve , adjuvant radiation therapy ( 25% recurrence over 10 years ) would have been another option.1 middle - aged women undergoing laminectomies have been observed to develop postoperative dts of the spine . although these tumors do not cause neurological deficits , they tend to be locally invasive and are best treated with wide excision with negative margins . although it has been speculated that hormonal triggers could be involved in postsurgical dt formation , it is also likely that inflammatory cytokines released in the surgical bed during the acute and chronic phases of healing also play a role in growth of aberrant tissue . this may be particularly true with the continued presence of surgical instrumentation for fixation , which represents foreign material within the surgical bed . national center for spinal disorders , budapest , hungary postsurgical desmoid formation at the site of surgery is not a common pathology . puvanesarajah et al describe a case of a 57-year - old woman who underwent resection and stabilization because of spinal cord compression due to a high thoracic ( t4 ) hemangioma . ten months after the surgery , they observed a rapidly growing mass in the surgical scar , which was histologically determined to be a desmoid fibromatosis . they illustrate nicely the preoperative pathology and the desmoid tumor ( dt ) in the rostral part of the pathology . performing en bloc resection of the dt with strict local control of the disease , succeeded in preventing local recurrence after 2 years . dts are benign , deep - seated monoclonal myofibroblastic neoplasms that grow slowly , infiltrative and arise from musculoaponeurotic stromal elements . in the past few years , the term aggressive fibromatosis also has come into use . genetic , endocrine , and physical factors play a role because abdominal wall tumors often arise in young parous women following childbirth or in a postsurgical scar . dts also occur commonly as part of hereditary syndromes , such as familial adenomatous polyposis ( fap ) , which are often associated with a germ - line mutation in the adenomatosis polyposis coli ( apc ) gene.1 as the authors pointed out , there are just five reported cases in the literature that describe de novo dt formation in the postsurgical scar in patients with an absence of any predisposing genetic syndromes . there are some generally accepted principles regarding the treatment of dts that are as follows.2 when medically and technically feasible , growing and symptomatic dts should be treated surgically.3 en bloc resection of these tumors give the lowest rate of local recurrence , while positive surgical margins or intralesional resections show a much higher rate of recurrence.4 in the case of oncologically improper surgical resection , adjuvant radiotherapy would be an option.5 the same principles are dominant in postsurgical desmoid - formation . this special appearance in the postoperative scar was described after breast,6 7 abdominal,8 and other orthopedic3 procedures . according to the literature , the first detection of the tumor formation showed a wide range , between 10 months and 7 years after the local surgery . the first clinical sign is a growing , well palpable ( most frequently painless ) tissue in the postoperative scar or inside the muscles dissected during the surgical approach . the myofibroblast is the cell considered to be responsible for the development of tumorous malformation , but the etiological factor to initiate this process is unknown ( as the authors indicated , probably the activation of -catenin signaling by different local tissue - healing factors could result in aggressive fibroblast proliferation , supported or promoted by inflammatory cytokines in the surgical bed ) . all of them were treated surgically by en bloc resection without local recurrence . in certain cases , after the extensive resection , a myocutan flap was necessary for the local soft tissue reconstruction . in the cases described in the literature , the local pain caused by the postsurgical desmoid formation is more significant in the breast and the abdominal wall even in the earlier stage with smaller tumor size , whereas after spine surgery , the level of local pain caused by the tumors with paraspinal locations shows a very wide range . ( in one case in our institutional experience , a 47-year - old female patient returned 11 months after a lower lumbar instrumented posterolateral fusion with a large , well palpable mass later histologically proved desmoid which was painless at the time of the recognition . ) the functional disturbances due to the tumor growth highly depend on the region the spinal surgery was performed , the muscle , and where the tumor appears first . restricted spinal range of motion ( especially with increasing local pain provoked by the movement ) appearing after a certain stage of normal functional rehabilitation could also indicate abnormal tissue changes in the mass of the postoperative scar or the surrounding paraspinal muscles . in the cervical spine ( despite the benign histological appearance and negligible metastatic potential ) , the growing dt infiltrating the surrounding tissues could lead to deformity , morbidity , and mortality resulting from pressure effects and also by the potential obstruction of vital structures and organs.9 the authors recognized and diagnosed the pathology in proper time and selected the state - of - the - art treatment method . the successful postoperative course and the lack of the local recurrence after 2 years show effective decision making . this case report of aggressive fibromatosis in the posterior cervico - thoracic junction following an otherwise uneventful hemangioma resection underscores a number of important considerations : it is helpful to have advanced postoperative imaging early after tumor resections of the spine as a baseline comparison study for any recurrences , reconstructive failures , or as happened in this case , a secondary neoplasia . a ct scan is most commonly chosen for this type of reference study ( which is also helpful in checking adequacy of resection margins among other factors ) , but an mri scan may be helpful by about 3 months after surgery when soft tissue lesions are of concern and the initial postsurgical changes have largely settled down . this case also underscores the value of scheduled longer term follow - up following neoplasia care of the spinal column , as pointed out in the commentary by dr . there is hope that members of the aospine international community who increasingly treat spinal neoplasia join the aospine neoplasia knowledge forum to place not only routine cases into their well - governed registry but especially rare cases like this one , preferably with tissue samples to allow for genetic studies later on . this may offer improved insights into the oncogenic circumstances of certain forms of neoplasia , such as the one found described here .

study design case report . objective the objective of the article is to illustrate a case of desmoid tumor ( dt ) formation after posterior instrumentation of the thoracic spine . methods a 57-year - old woman presented with lower extremity clumsiness , balance , and ambulation difficulty resulting from spinal cord compression due to an upper thoracic atypical vertebral hemangioma . ten months after undergoing embolization , resection , and placement of instrumentation for this lesion , the patient developed a growing mass at the rostral end of the incision . biopsy revealed desmoid fibromatosis . the mass was removed via an en bloc resection . histology revealed an infiltrative dt above the laminectomy site abutting the instrumentation . results at 2-year follow - up , there was no evidence of recurrence of the tumor . conclusion paraspinal dts have been reported in the literature to develop after surgical procedures of the spine . often times , patients attribute swelling or fullness at the site of their surgery to scar tissue formation or instrumentation . one must consider the possibility of a dt in the setting of reported surgical site fullness or mass after spine surgery . it is thought that postoperative inflammation present in the surgical bed may promote formation of dts . instrumentation may also contribute to inflammation and increase the likelihood of developing a dt . generous margins must be taken to prevent recurrence .

oral health has a great impact on our overall health including both physical and psychological . the who defines quality of life as individual 's perception of their position in life in the context of culture value system , in which they live and in relation to their goal , standards , and concerns . a comprehensive national health survey conducted in 2004 in india clearly symbolizes that dental diseases are a significant public health burden in india . the psychosocial impact of oral diseases often significantly diminishes quality of life and children are the worst affected group . children are more prone to numerous oral conditions that may have a negative impact on quality of life which may be quantified in terms of dropping out from school ; disruption to physical activity , eating , sleeping , and studying , which may affect their emotions adversely . the gravity of the situation further deepens when we talk in terms of oral health conditions of differently abled children . visual impairment is one such condition which relates to a person 's eyesight , which can not be corrected to normal vision . visual impairment can be a major hindrance in maintenance of proper oral hygiene and absence of proper training further worsens this . clinical indicators of oral diseases were not entirely suitable to capture the new concept of health declared by the who . hence , researchers started to develop alternative measures that came in the form of standardized questionnaires . oral health - related quality of life ( ohrqol ) is a relatively new but rapidly growing phenomenon which has emerged over the past two decades . pediatric oral disorders are numerous ( surgeon general 's report , 2000 ) and are likely to have a negative effect on the quality of life , five of these tools were designed to assess the ohrqol in children . these include child perception questionnaire , the michigan ohrqol scale , the child oral health impact profile , the early childhood oral health impact scale , and the child - oral impact on daily performances ( c - oidps ) . although c - oidp assessment has recently penetrated into the pediatric dental literature , assessment of c - oidp in special populations such as visually impaired children has been hindered by several factors mostly lack of awareness about the same . thus , an effort was made to appraise the impact of oral diseases on daily activities of these visually impaired children in our study by evaluating the existing oral health status of 915 year children with visual impairment in districts of uttarakhand , india , with regard to dental caries experience , dental trauma , and dentofacial anomalies and assessing the impact of the oral health status if any , on quality of life of these children using c - oidp index . the present descriptive cross - sectional study had 423 male and female visually impaired children . only 915-year - old children who could read and write in braille and were ready to answer the questionnaire were considered for the study . the study was conducted after due clearance from the ethical committee and the institutional research board . prior permissions were also taken from the head of the department and principals of the special schools who participated in the study . there are 22 schools working in the field of education , training , employment , and rehabilitation of disabled children in different states of uttarakhand . only five schools of them ( from both garhwal and kumaon divisions of uttarakhand state , i.e. from haridwar , dehradun , and nainital districts ) had children who were only visually impaired without any other form of disability , and so they were shortlisted for the study . the study was carried out in a span of 6 months , i.e. from february 2015 to july 2015 . all the children studying in the schools were examined were legally visually impaired ( both partially and completely visually impaired ) , i.e. , one who , with the best optical correction , can see less at 20 feet than a person with normal vision can at 200 feet ( visual acuity is 20/200 ) ; or whose field of vision is limited to a narrow - angle and had severe visual impairment since birth was considered in this study . uncooperative children , those with severe systemic diseases , mentally challenged , or having any disability other than visual impairment were excluded from the study . baseline information of these visually impaired children was collected by means of questionnaire - record form 1 and clinical examination - record form 2 . the principal investigator and teachers filled out form 1 individually for all the children as scribes to avoid discrepancy and while doing so they took care not to influence the participant 's answer in any way . for recording of form 2 , two house surgeons were identified and trained . the principal investigator and the two house surgeons were then calibrated for the recording the indices , based on which the kappa coefficient was calculated and was found to be ranging from 0.85 to 0.95 , which was good . for the braille version of c - oidp questionnaire , internal reliability was tested using the standardized cronbach 's alpha coefficient which was found to be 0.86 . dental caries was assessed using dmft index given by henry t klein , carrole e palmer , and knutson jw in 1938 for permanent dentition and deft index given by gruebbel in 1944 for primary dentition . traumatic injuries were recorded with the help of traumatic dental injury ( tdi ) index ( based on who classification of dental trauma ) . presence of dentofacial anomalies , i.e. , open bite , cross bite , deep bite , and crowding of teeth were also recorded . overjet more than 3 mm and overbite more than 2 mm were considered increased . crowding similarly , crossbite was considered present , if one or more maxillary teeth were placed palatal or lingual to mandibular teeth . the braille version of c - oidp index was used to assess the final impact of oral health - related conditions which can affect one 's daily life . the data were retrieved from precoded survey pro forma to a computer . the excel and spss version 21.0 ( spss inc . , chicago , il , usa ) software packages were used for data entry and analysis . correlations among sociodemographic , clinical variables , and c - oidp impact were assessed using pearson 's correlation coefficient , and bivariate analysis was applied between the independent variables , i.e. , sociodemographic and oral health indicators , and the outcome of c - oidp to find out the significance and odds ratio . significance for all statistical tests was predetermined at a probability value of 0.05 or less . there was a high dental caries prevalence of 57.7% among the visually impaired children with a mean dmft of 1.64 and mean deft of 1.53 [ graph 1 ] . the prevalence of traumatic dental injuries was 50.6% among these visually impaired children [ graph 1 ] . the study sample showed the highest prevalence of angle 's class i molar relation ( 62.6% ) . crowding ( 61.5% ) of all the dentofacial anomalies , there was a significant male predilection in deep bite among visually impaired children . the most commonly perceived oral health problem among visually impaired group was toothache , while on the other hand , children rated missing teeth as the least perceived oral health problem . toothache was the most common oral condition affecting almost all the daily activities and had maximum impact on sleeping , studying , and eating . the overall severity of impact on daily activities was 30.45% and the overall frequency of impact on daily activities was 29.7% . the c - oidp impact showed highly significant positive correlation with age , study group , and significant negative correlation with dmft [ table 2 ] . the male participants tended to have higher c - oidp scores as compared to females thus suggesting less favorable ohrqol in males as compared to females . children who presented with dentofacial deformities , dental caries , and traumatic dental injuries reported higher c - oidp scores suggestive of unfavorable ohrqol . prevalence of dental caries , tooth trauma , and dentofacial anomalies in the study population . prevalence of different types of dentofacial anomalies and self - perceived oral health problems among the visually impaired group correlations among sociodemographic , clinical variables , and child - oral impact on daily performances impact and bivariate analysis between the independent variables : sociodemographic and oral health indicators , and the outcome child - oral impact on daily performances with odds ratio and 95% confidence interval the first objective of our study was to measure components of oral health status among visually impaired children through various indices . the second objective was to find out the impact of the oral health status if any , on quality of life of these children using c - oidp index . the perceptions of the shape , color , and alignment of teeth can vary from person to person and can affect people accordingly . keeping this in mind , the self - reported or patient - reported health outcomes such as ohrqol were used in our study . of all the available ohrqol measures , c - oidp inventory has the ability to provide information on condition - specific impacts whereby the respondent attributes the impacts to specific oral conditions or diseases ; thus contributing to the needs assessment and the planning of oral health - care services . hence , we used c - oidp questionnaire in our study . in the classical questionnaire , the participating children were first presented with a list of 16 impairments : toothache , sensitive teeth , tooth decay ( cavity in teeth ) , exfoliating primary teeth , tooth space ( due to a nonerupted permanent tooth ) , fractured permanent tooth , color of tooth , shape or size of tooth , position of tooth , bleeding gum , swollen gum , calculus , oral ulcers , bad breath , deformity of mouth or face , erupting permanent tooth , and missing permanent tooth . however , in this study , visually impaired participants were not prompted on all the 16 impairments . those that were dropped were color , shape and size , position , and deformity of mouth or face assuming that the participants could not make a fair judgment based on their visual challenge . the questionnaire was first translated into hindi as it 's the most commonly spoken language in this region and for ease of understanding by the children . the braille version was distributed to them , and scribes were used to record their answers . there were concerns that children 's cognitive capacities and communication skills may compromise the validity and reliability of their qol reports . however , wilson - genderson et al . in their review suggested that self - report of qol and health status is feasible in 915-year - old children as age effects should no longer be significant . at this age , children have a good capacity to remember , retrieve , and apply information related to specific events and experiences . their matured language skills and ease in independent reading allow for the comprehension of items and meet the demands of self - reported questionnaires . further , children at this stage , reflective of piaget 's stage of formal operations , have matured intellectual functioning and are capable of making the comparative judgments required for representations of oral health status and qol . 915-year - old children view health as a multidimensional concept organized around constructs such as being functional , adhering to good lifestyle behaviors , and having a general sense of well - being and relationships with others . in addition , they have a greater appreciation of illness , disease , and disability in that each can be understood based on causality and multisystem understanding . the prevalence of dental caries in the present study was 57.7% in visually impaired group . it was more than that seen in a similar study conducted among institutionalized visually impaired children in south india by reddy and sharma in 2011 , where the caries prevalence was 40% and mean dmft / deft was 1.1 and 0.17 . another study conducted by chand et al . in 2014 in indore , madhya pradesh , had found mean dmft / deft of 0.97/0.46 in visually impaired children . furthermore , similar studies conducted in central india ( udaipur ) by jain et al . in 2013 where mean dmft / deft was 1.9/1.7 in visually impaired had similar results for dental caries prevalence as that present in our study . sanjay et al . in 2014 conducted a study in maharashtra which also showed similar results with mean dmft / deft of 2.1/2.0 in visually impaired population . probable reason for some variations seen in different studies conducted through the latitudes and longitudes in india could be variable access to dental care , inadequate oral hygiene , and many other disability - related factors , their diet , medications , physical limitations , lack of oral hygiene , and attitude of caretaker / parent and health - care providers . the sample group showed the highest prevalence of class i molar relation ( 62.6% ) followed by angle 's class ii at 32.9% in visually impaired and finally class iii which was seen in 4.5% visually impaired . this was similar to the results reported by avasthi et al . in 2011 in sensory impaired children in delhi - gurgaon region where they had used similar parameters to check for dentofacial anomalies . crowding was most commonly seen dentofacial anomaly in the study group present in 61.5% of the study population . of all the dentofacial anomalies , there was a significant male predilection in deep bite among visually impaired children . these results corroborate with survey carried out by avasthi et al . in 2011 in delhi in 2013 , which reported that there was anterior crowding in 27.37% of the total sample size , deep bite in 20.5% , class i in 14.34% , anterior spacing in 12.9% , class ii in 9.95% , class iii in 5.33% , anterior crossbite in 4.98% , and open bite in 4.62% . the discrepancy in the percentages could be because of difference in the genetic pattern of both the study populations . in our study , another study conducted by bhat et al . in 2011 in udaipur showed a prevalence of 33% of traumatic dental injuries among 1215 year age group . a probable cause for this variation in prevalence could be the difference in the sample size and facilities and supervision present at the institution . in our study , eating was the most common performance affected by poor oral health as per c - oidp inventory this was similar to the results seen for normal school - going children aged 1215 year in a survey done by usha et al . in 2013 ( davangere ) using c - oidp questionnaire and also in another survey done among the national cadet corps aged 1215 years of udupi district , india , in 2013 using c - oidp inventory . the c - oidp impact showed highly significant positive correlation with age , study group , and significant negative correlation with dmft . the male participants tend to have higher coidp scores as compared to females thus suggesting less favorable ohrqol in males . participants > 12 years of age had severe impact of poor oral health on their day - to - day activities as compared to those < 12 years of age . children who presented with dentofacial deformities , dental caries , and traumatic injuries reported higher coidp scores suggestive of unfavorable ohrqol . there was only one study conducted in khartoum state , sudan by tagelsir et al . in 83 visually impaired children where they tested the c - oidp questionnaire . furthermore , there was a difference in the mode of delivery of c - oidp questionnaire which was verbal in their case , and we had given a braille version of c - oidp questionnaire to children . thus , it makes our study first of its kind to use the braille version of c - oidp questionnaire which has not been done anywhere in the world . the children in our study were able to read and write in braille which could also have an impact on their understanding of the questions and thereby generating an improved response to the questionnaire . limitation of this study is that factors such as parent education and socioeconomic status could not be considered as it was conducted among institutionalized children of whom most were orphans and/or abandoned by family very early in life . in hilly terrain of uttarakhand , dental diseases still exist as a smoldering disease that has ingressed its tentacles deep due to lack of public awareness and motivation . within the limitations of the study , visually impaired individuals here showed a higher prevalence of dental caries , traumatic dental injuries , and dentofacial anomalies . hence , special oral health care measures tailor - made for this special population must be implemented at the earliest . furthermore , provision of oral health education in special schools including proper instructions on oral hygiene practices in braille language must be implemented at the earliest . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or nonfinancial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or nonfinancial in this article .

background : to assess the prevalence of dental diseases among 915-year - old visually impaired children and find out its impact on their daily activities using the child - oral impact on daily performance ( c - oidp ) questionnaire in districts of uttarakhand , india.materials and methods : a total of 423 visually impaired institutionalized children between the age group of 915 years were included in the study . stratified random sampling technique was used to obtain the study population . dental caries was recorded using dmft for primary dentition and dmft for permanent dentition , traumatic dental injuries were assessed using traumatic dental injury index , and dentofacial anomalies were recorded using angle 's classification of malocclusion . the hindi braille version of c - oidp questionnaire was used to gather information regarding oral health - related quality of life ( ohrqol).results : there was a high dental caries prevalence of 57.7% in visually impaired children . the prevalence of traumatic dental injuries was 50.6% . crowding ( 61.5% ) was the most commonly seen dentofacial anomaly and the most commonly perceived oral health problem was toothache . there was less favorable ohrqol in males as compared to females.conclusion:there was a high prevalence of dental diseases in this group and higher c - oidp scores suggestive of unfavorable ohrqol .

this study assessed whether a current medical school curriculum is adequately preparing medical students to diagnose and treat common dermatologic conditions . a 15-item anonymous multiple choice quiz covering fifteen diseases was developed to test students ability to diagnose and treat common dermatologic conditions . the quiz also contained five items that assessed students confidence in their ability to diagnose common dermatologic conditions , their perception of whether they were receiving adequate training in dermatology , and their preferences for additional training in dermatology . the survey was performed in 2014 , and was completed by 85 students ( 79.4% ) . many students ( 87.6% ) felt that they received inadequate training in dermatology during medical school . on average , students scored 46.6% on the 15-item quiz . proficiency at the medical school where the study was performed is considered an overall score of greater than or equal to 70.0% . students received an average score of 49.9% on the diagnostic items and an average score of 43.2% on the treatment items . the findings of this study suggest that united states medical schools should consider testing their students and assessing whether they are being adequately trained in dermatology . then schools can decide if they need to re - evaluate the timing and delivery of their current dermatology curriculum , or whether additional curriculum hours or clinical rotations should be assigned for dermatologic training .

the online version of this article ( doi:10.1007/s13300 - 014 - 0071 - 1 ) contains supplementary material , which is available to authorized users . type 2 diabetes mellitus ( t2 dm ) is a heterogeneous disease characterized by insulin resistance and defective insulin secretion . several dipeptidyl peptidase-4 ( dpp-4 ) inhibitors are available for the treatment of t2 dm . dpp-4 inhibitors work by inhibiting dpp-4 , which degrades incretin hormones such as glucagon - like peptide ( glp)-1 and glucose - dependent insulinotropic polypeptide , stabilizing the intact ( active ) forms of the hormones . dpp-4 inhibitors were first reported to improve glycemic control , insulin secretion , and -cell function in rodents . in patients with t2 dm , dpp-4 inhibitors were reported to decrease postprandial glucose excursions , fasting plasma glucose ( fpg ) , and hemoglobin a1c ( hba1c ) , without affecting insulin levels . in another study , sitagliptin significantly increased insulin levels ( 2122% ) and decreased glucagon levels ( 714% ) at 2 h in an oral glucose tolerance test ( ogtt ) relative to placebo , but the acute insulin response ( 30 min ) was not significantly increased . other studies have also demonstrated that postprandial insulin and c - peptide levels were not significantly affected by sitagliptin . in that study , the sensitivity of pancreatic cells to glucose , evaluated as c - peptide at 0 min ( cp0)/plasma glucose at 0 min ( pg0 ) and cp30/pg30 , did not improve in the sitagliptin or control groups , but cp240/pg240 increased in the sitagliptin group with borderline statistical significance . another study showed that vildagliptin lowered postprandial glucagon levels by 16% , and that insulin sensitivity and glucose clearance , as determined using glucose clamps , were improved by vildagliptin , but the postprandial insulin levels did not change . the mean body mass index ( bmi ) of patients in these studies was 27.5 kg / m , 29.5 kg / m , 33.2 kg / m , and 31.4 kg / m . therefore , these results suggest that dpp-4 inhibitors mainly improved insulin resistance in obese populations , with no or only small improvements in the acute insulin response . in japan , approximately half of all patients with diabetes have a genetic predisposition to the disease , and insulin secretion is often impaired in lean patients with diabetes mellitus [ 9 , 10 ] . additionally , japanese and asian patients often show reduced -cell function and it is thought that the innate insulin secretory capacity of east asians is limited [ 11 , 12 ] . the possible instability and vulnerability of canalization due to reduced -cell function may contribute to the increased prevalence of diabetes in east asia in recent decades . a prior study of japanese patients revealed that once - daily administration of 100-mg sitagliptin for 12 weeks improved the insulinogenic index compared with placebo during a mtt in which the subjects consumed two nutritional bars and a nutritional drink . however , that study was a phase 2 trial , and sitagliptin was administered before a meal at a dose of 100 mg / day . in japan , the package insert for sitagliptin recommends a dose of 50 mg / day . therefore , the effects of a standard dose of sitagliptin ( 50 mg / day ) on the postprandial metabolic control of patients in real - world settings are currently unknown . furthermore , japanese patients usually take sitagliptin after a meal in accordance with the package insert , which is based on the results of peak concentration time ( tmax ) and apparent terminal half - life ( t1/2 ) that were not altered significantly by food . based on these results , we conducted a study to determine whether a standard dose of sitagliptin ( 50 mg / day ) after a meal could improve the impaired acute insulin response in japanese patients with t2 dm in a real - world setting . we used a meal tolerance test ( mtt ) that was developed by the japan diabetes society specifically for japanese patients with t2 dm . the results of the mtt show a strong correlation with those of glucose tolerance tests . the purpose of this study is to evaluate whether a standard dose of sitagliptin ( 50 mg / day ) after a meal could improve the impaired acute insulin response in japanese patients with t2 dm in a real - world setting . twenty - one outpatients with t2 dm participated in this study at tottori university hospital . this study was a prospective , open - label , non - randomized , clinical study . the patients were consecutively assigned to the control group and the sitagliptin group where the first 10 patients were assigned to the control group and the next 11 patients were assigned to the sitagliptin group . patients in the sitagliptin group administered sitagliptin at a dose of 50 mg / day once daily after breakfast throughout the treatment period . patients with pancreatic disease , liver disease , renal failure ( estimated glomerular filtration rate < 30 ml / min/1.73 m ) , or those taking diabetogenic medications such as corticosteroids were excluded from this study . the duration of diabetes and presence of diabetic microangiopathy were retrieved from the patients medical records . the adverse events were monitored by evaluating liver function , amylase , renal function , common blood count , and hypoglycemic events and abdominal symptoms , and so on . this study was approved by the ethics committee of the faculty of medicine , tottori university . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 . this study was registered with the university hospital medical information network ( identifier : umin000011189 ) . after fasting for at least 12 h , the participants visited the hospital in the morning and consumed a test meal prepared by the japan diabetes society ( janef e460f18 : total calories 460 kcal , carbohydrates 56.5 g , fat 18.0 g , protein 18.0 g ; kewpie corporation , tokyo , japan ) . patients consumed the test meal within 15 min , and took 50-mg sitagliptin after the meal . plasma glucose and insulin were measured at 0 min ( fasting ) , and 30 , 60 , 120 , and 180 min after the test meal . plasma insulin levels were measured using a human chemiluminescent immunoassay kit ( kyowa medix , tokyo , japan ) . this mtt is a well - established method that has been used in our hospital in prior studies [ 1921 ] . hba1c ( jds , japan diabetes society ) was measured by high - performance liquid chromatography . hba1c ( jds ) values are about 0.4% lower than those of ngsp values , which are the global standard , although measurements of hbalc in japan are well calibrated with certified serial reference materials from lot 1 to lot 4 using a high resolution ion - exchange hplc method ( ko500 ) . hba1c ( jds ) was converted to national glycohemoglobin standardization program ( ngsp ) values using the following officially certified equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{ngsp } } \left(\% \right ) = 1.0 2 \times { \text{jds } } \left(\% \right ) + 0 . 2 5 \ , \% $ $ \end{document } . the reverse equation is : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{jds } } \left ( \% \right ) = 0 . 2 4 5\ , \% $ $ \end{document}. the insulinogenic index was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{insulinogenic index } } = { { \left\ { { \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } \mathord{\left/ { \vphantom { { \left\ { { \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } { \left\ { { \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } } } \right . \kern-\nulldelimiterspace } { \left\ { { \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } } $ $ \end{document}. an impaired acute insulin response was defined as an insulinogenic index of < 43.2 ( < 0.4 based on glucose in mg / dl and insulin in u / ml ) . homeostasis model assessment of insulin resistance ( homa - ir ) was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{homa}}{\text{-}}{\text{ir } } = { \text{fpg } } \left ( { { \text{mmol}}/{\text{l } } } \right)\left]\ ; { \times } \ ; \right[{\text{fasting iri } } \left ( { { \text{f}}{\text{-}}{\text{iri}};{\text { pmol}}/{\text{l } } } \right)]/135 $ $ \end{document}. the insulin sensitivity index ( isi ) was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{isi } } = 10,000/\surd \left\ { { \left [ { { \text{fpg } } \left ( { { \text{mmol}}/{\text{l } } } \right ) \times \ , { \text{fasting plasma insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right ) } \right ] \times \left [ { { \text{mean glucose}}\ , \times\ , { \text{mean insulin during the mtt } } } \right ] } \right\}. $ $ \end{document } data are expressed as mean standard deviation . the area under the curve ( auc ) was calculated according to the trapezoidal rule . smirnov test . because isi and f - iri showed non - normal distributions , the variables f - iri , homa - ir , isi , and insulinogenic index were analyzed using non - parametric tests . the baseline characteristics were compared between the two groups using unpaired student s t test . f - iri , homa - ir , isi , and insulinogenic index were compared between the two groups using the mann differences in continuous variables were compared between before and 3 months of treatment using paired student s t test , except for the changes in f - iri , homa - ir , isi , and insulinogenic index , which were analyzed using the wilcoxon rank - sum test . the changes in continuous variables from before to 3 months of treatment were compared between the two groups using unpaired student s t test , except for f - iri , homa - ir , isi , and insulinogenic index , which were compared using the mann values of p < 0.05 were considered statistically significant . spss software version 15.0 ( spss , chicago , il , usa ) was used for all analyses . twenty - one outpatients with t2 dm participated in this study at tottori university hospital . this study was a prospective , open - label , non - randomized , clinical study . the patients were consecutively assigned to the control group and the sitagliptin group where the first 10 patients were assigned to the control group and the next 11 patients were assigned to the sitagliptin group . patients in the sitagliptin group administered sitagliptin at a dose of 50 mg / day once daily after breakfast throughout the treatment period . patients with pancreatic disease , liver disease , renal failure ( estimated glomerular filtration rate < 30 ml / min/1.73 m ) , or those taking diabetogenic medications such as corticosteroids were excluded from this study . the duration of diabetes and presence of diabetic microangiopathy were retrieved from the patients medical records . the adverse events were monitored by evaluating liver function , amylase , renal function , common blood count , and hypoglycemic events and abdominal symptoms , and so on . this study was approved by the ethics committee of the faculty of medicine , tottori university . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 . this study was registered with the university hospital medical information network ( identifier : umin000011189 ) . after fasting for at least 12 h , the participants visited the hospital in the morning and consumed a test meal prepared by the japan diabetes society ( janef e460f18 : total calories 460 kcal , carbohydrates 56.5 g , fat 18.0 g , protein 18.0 g ; kewpie corporation , tokyo , japan ) . patients consumed the test meal within 15 min , and took 50-mg sitagliptin after the meal . plasma glucose and insulin were measured at 0 min ( fasting ) , and 30 , 60 , 120 , and 180 min after the test meal . plasma insulin levels were measured using a human chemiluminescent immunoassay kit ( kyowa medix , tokyo , japan ) . this mtt is a well - established method that has been used in our hospital in prior studies [ 1921 ] . hba1c ( jds , japan diabetes society ) was measured by high - performance liquid chromatography . however , hba1c ( jds ) values are about 0.4% lower than those of ngsp values , which are the global standard , although measurements of hbalc in japan are well calibrated with certified serial reference materials from lot 1 to lot 4 using a high resolution ion - exchange hplc method ( ko500 ) . hba1c ( jds ) was converted to national glycohemoglobin standardization program ( ngsp ) values using the following officially certified equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{ngsp } } \left(\% \right ) = 1.0 2 \times { \text{jds } } \left(\% \right ) + 0 . the reverse equation is : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{jds } } \left ( \% \right ) = 0 . 9 80 \times { \text{ngsp } } \left ( \% \right ) - 0 . 2 4 5\ , \% $ $ \end{document}. the insulinogenic index was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{insulinogenic index } } = { { \left\ { { \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } \mathord{\left/ { \vphantom { { \left\ { { \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } { \left\ { { \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } } } \right . \kern-\nulldelimiterspace } { \left\ { { \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at 3}}0{\mkern 1mu } { \text{min } } } \right ] - \left [ { { \text{glucose } } \left ( { { \text{mmol}}/{\text{l } } } \right){\text { at } } 0{\mkern 1mu } { \text{min } } } \right ] } \right\ } } } $ $ \end{document}. an impaired acute insulin response was defined as an insulinogenic index of < 43.2 ( < 0.4 based on glucose in mg / dl and insulin in u / ml ) . homeostasis model assessment of insulin resistance ( homa - ir ) was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{homa}}{\text{-}}{\text{ir } } = { \text{fpg } } \left ( { { \text{mmol}}/{\text{l } } } \right)\left]\ ; { \times } \ ; \right[{\text{fasting iri } } \left ( { { \text{f}}{\text{-}}{\text{iri}};{\text { pmol}}/{\text{l } } } \right)]/135 $ $ \end{document}. the insulin sensitivity index ( isi ) was calculated as previously described : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{isi } } = 10,000/\surd \left\ { { \left [ { { \text{fpg } } \left ( { { \text{mmol}}/{\text{l } } } \right ) \times \ , { \text{fasting plasma insulin } } \left ( { { \text{pmol}}/{\text{l } } } \right ) } \right ] \times \left [ { { \text{mean glucose}}\ , \times\ , { \text{mean insulin during the mtt } } } \right ] } \right\}. $ $ \end{document } the area under the curve ( auc ) was calculated according to the trapezoidal rule . smirnov test . because isi and f - iri showed non - normal distributions , the variables f - iri , homa - ir , isi , and insulinogenic index were analyzed using non - parametric tests . the baseline characteristics were compared between the two groups using unpaired student s t test . f - iri , homa - ir , isi , and insulinogenic index were compared between the two groups using the mann differences in continuous variables were compared between before and 3 months of treatment using paired student s t test , except for the changes in f - iri , homa - ir , isi , and insulinogenic index , which were analyzed using the wilcoxon rank - sum test . the changes in continuous variables from before to 3 months of treatment were compared between the two groups using unpaired student s t test , except for f - iri , homa - ir , isi , and insulinogenic index , which were compared using the mann spss software version 15.0 ( spss , chicago , il , usa ) was used for all analyses . the mean age of the patients in sitagliptin group was 67.4 years , the mean bmi was 23.0 kg / m , the mean fpg was 8.05 mmol / l ( 144.8 mg / dl ) , the mean hba1c was 7.73% , and the mean insulinogenic index was 20.1 ( table 1 ) . there were eight males and three females in sitagliptin group , and seven males and three females in the control group . the mean age of the patients in the control group was 64.2 years , the mean bmi was 24.4 kg / m , the mean fpg was 8.12 mmol / l ( 146.3 mg / dl ) , the mean hba1c was 7.20% , and the mean insulinogenic index was 21.8 ( table 1 ) . in the sitagliptin group , four patients were on diet therapy alone and seven were using oral hypoglycemic agents ( ohas ) , including sulfonylurea ( 7 patients ) , -glucosidase inhibitors ( 3 ) , biguanides ( 3 ) , and thiazolidinediones ( 1 ) . in the control group , two patients were on diet therapy alone and eight were using oral hypoglycemic agents ( ohas ) , including sulfonylurea ( 6 patients ) , -glucosidase inhibitors ( 3 ) , biguanides ( 4 ) , thiazolidinediones ( 1 ) . the duration of diabetes was 9.8 years in the sitagliptin group versus 7.7 years in the control group . in each group , seven patients had diabetic neuropathy , and two patients had simple diabetic retinopathy . two patients also had diabetic nephropathy , the two patients in the sitagliptin group had macroalbuminurea , and the two patients in the control group had microalbuminurea . there were no significant differences in any of the baseline characteristics between the sitagliptin and control groups . the adverse events were not detected in the research period.table 1comparison of clinical variables before and after treatment in the sitagliptin and control groupsclinical variablesitagliptincontrolchange from baseline to 3 monthsbaseline3 months p * baseline3 months p * sitagliptincontrol p * * body weight , kg61.5 12.461.2 12.30.5266.1 11.066.1 11.70.750.25 1.280.0 1.160.49bmi , kg / m 23.0 3.022.9 3.00.5224.4 1.524.4 1.700.840.10 0.470.01 0.400.53wc , cm83.4 9.084.1 8.20.4986.4 5.388.0 5.40.12 + 0.77 3.61 + 1.48 3.120.52fpg , mmol / l(mg / dl)8.05 1.56(144.8 28.2)7.26 1.04(130.6 18.6)0.098.12 1.34(146.3 24.5)7.88 1.36(141.8 24.5)0.290.79 1.38(14.2 25.0)0.25 0.71(4.5 12.8)0.282-h ppg , mmol / l(mg / dl)13.37 2.74(240.7 49.3)10.32 1.36(185.8 24.4)<0.00511.50 4.2(207.3 76.4)12.0 3.66(216.0 65.8)0.583.05 2.25(54.9 40.5)+0.48 2.7(+8.7 48.7)<0.05hba1c ( % , ngsp)7.73 0.546.86 0.47<0.0017.20 1.427.37 1.650.200.88 0.60 + 0.17 0.39<0.0005f - iri , pmol / l(ng / ml)20.8 9.0(3.5 1.5)28.4 16.8(4.7 2.8)<0.0528.7 19.3(4.8 3.2)32.9 14.1(5.5 2.4)0.36 + 7.56 8.52(+1.26 1.42)+4.14 13.6(+0.69 2.27)0.49homa - ir1.27 0.711.53 0.40<0.051.68 1.131.82 0.590.65 + 0.27 0.33 + 0.14 0.930.48isi ( matsuda s index)10.4 5.710.4 7.30.958.15 4.16.63 2.540.340.08 4.351.54 4.950.72insulinogenic index20.1 15.736.9 24.1<0.0521.8 11.522.0 13.90.96 + 16.7 23.7 + 0.1 7.9<0.005auc glucose , mmol / lh23.2 3.919.1 2.8<0.00521.7 5.221.7 4.90.954.01 3.50 + 0.05 2.43<0.01auc insulin , pmol / lh166.9 76.2204.7 114.40.14221.5 103.4228.6 99.80.76 + 37.7 78.7 + 7.10 72.50.36auc insulin / auc glucose7.2 19.310.7 41.2<0.0510.2 19.910.5 20.20.63 + 3.5 0.3 + 0.5 3.10.12ldl - c , mmol / l3.28 0.563.20 0.630.613.29 0.982.92 0.760.100.08 0.570.36 0.620.31hdl - c , mmol / l1.67 0.371.57 0.490.121.62 0.261.83 0.590.290.09 0.18 + 0.21 0.610.13tg , mmol / l1.19 0.531.36 0.770.271.63 1.311.36 0.780.24 + 0.17 0.490.28 0.700.10data are mean standard deviation auc area under the concentration time curve , bmi body mass index , f - iri fasting immunoreactive insulin , fpg fasting plasma glucose , hba1c hemoglobin a1c , hdl - c high - density lipoprotein cholesterol , homa - ir homeostasis model assessment of insulin resistance , isi insulin sensitivity index , ldl - c low - density lipoprotein cholesterol , ngsp national glycohemoglobin standardization program , ppg postprandial glucose , tg triglyceride , wc waist circumference * paired t test , except homa - ir , isi , and insulinogenic index ( wilcoxon rank - sum test ) * * unpaired t test , except homa - ir , isi , and insulinogenic index ( mann whitney u test ) comparison of clinical variables before and after treatment in the sitagliptin and control groups data are mean standard deviation auc area under the concentration time curve , bmi body mass index , f - iri fasting immunoreactive insulin , fpg fasting plasma glucose , hba1c hemoglobin a1c , hdl - c high - density lipoprotein cholesterol , homa - ir homeostasis model assessment of insulin resistance , isi insulin sensitivity index , ldl - c low - density lipoprotein cholesterol , ngsp national glycohemoglobin standardization program , ppg postprandial glucose , tg triglyceride , wc waist circumference * paired t test , except homa - ir , isi , and insulinogenic index ( wilcoxon rank - sum test ) * * unpaired t test , except homa - ir , isi , and insulinogenic index ( mann whitney u test ) the mean fpg did not change significantly from baseline to 3 months of treatment in the sitagliptin group ( from 8.05 to 7.26 mmol / l , p = 0.09 ) and the control group ( from 8.12 to 7.88 mmol / l , p = 0.29 ) ( table 1 ) . the mean hba1c decreased significantly in the sitagliptin group ( from 7.73 to 6.86% , p < 0.001 ) , but not in the control group ( from 7.20 to 7.37% , p = 0.20 ) . there were no significant changes in body weight or bmi , and lipid profile in either group . the mean change in fpg from baseline to 3 months was not significantly different between the sitagliptin and control group ( 0.79 vs. 0.25 mmol / l , p = 0.28 ) , but hba1c decreased significantly in the sitagliptin group compared with the control group ( 0.88% vs. + 0.17% , p < 0.0005 ; table 1 ) . the changes in body weight , bmi , and lipid profile were not significantly different between the two groups . there were no significant differences in glucose levels and insulin responses between the two groups at baseline . after 3 months of treatment , the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin ( fig . 1a ) , whereas no significant changes were observed in the control group ( fig . the plasma insulin levels at 0 and 30 min were significantly increased by sitagliptin ( fig . 2a ) , whereas no changes in plasma insulin were found in the control group ( fig . 2b).fig . . the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline ( paired t test)fig . 2plasma insulin responses during the meal tolerance test . plasma insulin levels at 0 and 30 min were significantly increased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline [ paired t test , except for fasting plasma insulin ( wilcoxon rank - sum test ) ] plasma glucose responses during the meal tolerance test . the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline ( paired t test ) plasma insulin responses during the meal tolerance test . plasma insulin levels at 0 and 30 min were significantly increased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline [ paired t test , except for fasting plasma insulin ( wilcoxon rank - sum test ) ] the insulinogenic index increased significantly from baseline to 3 months of treatment in the sitagliptin group ( from 20.1 to 36.9 , p < 0.05 ) ( table 1 ) , but not in the control group ( from 21.8 to 22.0 , p = 0.96 ) . although homa - ir increased significantly in the sitagliptin group ( from 1.27 to 1.53 , p < 0.05 ) , the isi did not ( from 10.4 to 10.4 , p = 0.95 ) . homa - ir and isi did not significantly change in the control group ( homa - ir from 1.68 to 1.82 , p = 0.65 ; isi from 8.15 to 6.63 , p = 0.34 ) . time curve ( auc ) for glucose decreased significantly in the sitagliptin , but did not change in the control group . the auc for insulin / auc for glucose ratio increased significantly in the sitagliptin group , but not in the control group ( table 1 ) . the change in the insulinogenic index from baseline to 3 months was significantly greater in the sitagliptin group than in the control group ( + 16.7 vs. + 0.1 , p < 0.005 ; table 1 ) . however , the changes in homa - ir and isi were not significantly different between the two groups . although the auc for glucose decreased significantly in the sitagliptin group compared with the control group , the auc for insulin and the auc for insulin / auc for glucose were not significantly different between the two groups . the mean age of the patients in sitagliptin group was 67.4 years , the mean bmi was 23.0 kg / m , the mean fpg was 8.05 mmol / l ( 144.8 mg / dl ) , the mean hba1c was 7.73% , and the mean insulinogenic index was 20.1 ( table 1 ) . there were eight males and three females in sitagliptin group , and seven males and three females in the control group . the mean age of the patients in the control group was 64.2 years , the mean bmi was 24.4 kg / m , the mean fpg was 8.12 mmol / l ( 146.3 mg / dl ) , the mean hba1c was 7.20% , and the mean insulinogenic index was 21.8 ( table 1 ) . in the sitagliptin group , four patients were on diet therapy alone and seven were using oral hypoglycemic agents ( ohas ) , including sulfonylurea ( 7 patients ) , -glucosidase inhibitors ( 3 ) , biguanides ( 3 ) , and thiazolidinediones ( 1 ) . in the control group , two patients were on diet therapy alone and eight were using oral hypoglycemic agents ( ohas ) , including sulfonylurea ( 6 patients ) , -glucosidase inhibitors ( 3 ) , biguanides ( 4 ) , thiazolidinediones ( 1 ) . the duration of diabetes was 9.8 years in the sitagliptin group versus 7.7 years in the control group . in each group , seven patients had diabetic neuropathy , and two patients had simple diabetic retinopathy . two patients also had diabetic nephropathy , the two patients in the sitagliptin group had macroalbuminurea , and the two patients in the control group had microalbuminurea . there were no significant differences in any of the baseline characteristics between the sitagliptin and control groups . the adverse events were not detected in the research period.table 1comparison of clinical variables before and after treatment in the sitagliptin and control groupsclinical variablesitagliptincontrolchange from baseline to 3 monthsbaseline3 months p * baseline3 months p * sitagliptincontrol p * * body weight , kg61.5 12.461.2 12.30.5266.1 11.066.1 11.70.750.25 1.280.0 1.160.49bmi , kg / m 23.0 3.022.9 3.00.5224.4 1.524.4 1.700.840.10 0.470.01 0.400.53wc , cm83.4 9.084.1 8.20.4986.4 5.388.0 5.40.12 + 0.77 3.61 + 1.48 3.120.52fpg , mmol / l(mg / dl)8.05 1.56(144.8 28.2)7.26 1.04(130.6 18.6)0.098.12 1.34(146.3 24.5)7.88 1.36(141.8 24.5)0.290.79 1.38(14.2 25.0)0.25 0.71(4.5 12.8)0.282-h ppg , mmol / l(mg / dl)13.37 2.74(240.7 49.3)10.32 1.36(185.8 24.4)<0.00511.50 4.2(207.3 76.4)12.0 3.66(216.0 65.8)0.583.05 2.25(54.9 40.5)+0.48 2.7(+8.7 48.7)<0.05hba1c ( % , ngsp)7.73 0.546.86 0.47<0.0017.20 1.427.37 1.650.200.88 0.60 + 0.17 0.39<0.0005f - iri , pmol / l(ng / ml)20.8 9.0(3.5 1.5)28.4 16.8(4.7 2.8)<0.0528.7 19.3(4.8 3.2)32.9 14.1(5.5 2.4)0.36 + 7.56 8.52(+1.26 1.42)+4.14 13.6(+0.69 2.27)0.49homa - ir1.27 0.711.53 0.40<0.051.68 1.131.82 0.590.65 + 0.27 0.33 + 0.14 0.930.48isi ( matsuda s index)10.4 5.710.4 7.30.958.15 4.16.63 2.540.340.08 4.351.54 4.950.72insulinogenic index20.1 15.736.9 24.1<0.0521.8 11.522.0 13.90.96 + 16.7 23.7 + 0.1 7.9<0.005auc glucose , mmol / lh23.2 3.919.1 2.8<0.00521.7 5.221.7 4.90.954.01 3.50 + 0.05 2.43<0.01auc insulin , pmol / lh166.9 76.2204.7 114.40.14221.5 103.4228.6 99.80.76 + 37.7 78.7 + 7.10 72.50.36auc insulin / auc glucose7.2 19.310.7 41.2<0.0510.2 19.910.5 20.20.63 + 3.5 0.3 + 0.5 3.10.12ldl - c , mmol / l3.28 0.563.20 0.630.613.29 0.982.92 0.760.100.08 0.570.36 0.620.31hdl - c , mmol / l1.67 0.371.57 0.490.121.62 0.261.83 0.590.290.09 0.18 + 0.21 0.610.13tg , mmol / l1.19 0.531.36 0.770.271.63 1.311.36 0.780.24 + 0.17 0.490.28 0.700.10data are mean standard deviation auc area under the concentration time curve , bmi body mass index , f - iri fasting immunoreactive insulin , fpg fasting plasma glucose , hba1c hemoglobin a1c , hdl - c high - density lipoprotein cholesterol , homa - ir homeostasis model assessment of insulin resistance , isi insulin sensitivity index , ldl - c low - density lipoprotein cholesterol , ngsp national glycohemoglobin standardization program , ppg postprandial glucose , tg triglyceride , wc waist circumference * paired t test , except homa - ir , isi , and insulinogenic index ( wilcoxon rank - sum test ) * * unpaired t test , except homa - ir , isi , and insulinogenic index ( mann whitney u test ) comparison of clinical variables before and after treatment in the sitagliptin and control groups data are mean standard deviation auc area under the concentration time curve , bmi body mass index , f - iri fasting immunoreactive insulin , fpg fasting plasma glucose , hba1c hemoglobin a1c , hdl - c high - density lipoprotein cholesterol , homa - ir homeostasis model assessment of insulin resistance , isi insulin sensitivity index , ldl - c low - density lipoprotein cholesterol , ngsp national glycohemoglobin standardization program , ppg postprandial glucose , tg triglyceride , wc waist circumference * paired t test , except homa - ir , isi , and insulinogenic index ( wilcoxon rank - sum test ) * * unpaired t test , except homa - ir , isi , and insulinogenic index ( mann whitney u test ) the mean fpg did not change significantly from baseline to 3 months of treatment in the sitagliptin group ( from 8.05 to 7.26 mmol / l , p = 0.09 ) and the control group ( from 8.12 to 7.88 mmol / l , p = 0.29 ) ( table 1 ) . the mean hba1c decreased significantly in the sitagliptin group ( from 7.73 to 6.86% , p < 0.001 ) , but not in the control group ( from 7.20 to 7.37% , p = 0.20 ) . there were no significant changes in body weight or bmi , and lipid profile in either group . the mean change in fpg from baseline to 3 months was not significantly different between the sitagliptin and control group ( 0.79 vs. 0.25 mmol / l , p = 0.28 ) , but hba1c decreased significantly in the sitagliptin group compared with the control group ( 0.88% vs. + 0.17% , p < 0.0005 ; table 1 ) . the changes in body weight , bmi , and lipid profile were not significantly different between the two groups . there were no significant differences in glucose levels and insulin responses between the two groups at baseline . after 3 months of treatment , the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin ( fig . 1a ) , whereas no significant changes were observed in the control group ( fig . the plasma insulin levels at 0 and 30 min were significantly increased by sitagliptin ( fig . 2a ) , whereas no changes in plasma insulin were found in the control group ( fig . . the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline ( paired t test)fig . plasma insulin levels at 0 and 30 min were significantly increased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline [ paired t test , except for fasting plasma insulin ( wilcoxon rank - sum test ) ] plasma glucose responses during the meal tolerance test . the plasma glucose levels at 60 , 120 , and 180 min were significantly decreased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline ( paired t test ) plasma insulin responses during the meal tolerance test . plasma insulin levels at 0 and 30 min were significantly increased in the sitagliptin group ( a ) , but not in the control group ( b ) . * p < 0.05 vs. baseline [ paired t test , except for fasting plasma insulin ( wilcoxon rank - sum test ) ] the insulinogenic index increased significantly from baseline to 3 months of treatment in the sitagliptin group ( from 20.1 to 36.9 , p < 0.05 ) ( table 1 ) , but not in the control group ( from 21.8 to 22.0 , p = 0.96 ) . although homa - ir increased significantly in the sitagliptin group ( from 1.27 to 1.53 , p < 0.05 ) , the isi did not ( from 10.4 to 10.4 , p = 0.95 ) . homa - ir and isi did not significantly change in the control group ( homa - ir from 1.68 to 1.82 , p = 0.65 ; isi from 8.15 to 6.63 , p = 0.34 ) . time curve ( auc ) for glucose decreased significantly in the sitagliptin , but did not change in the control group . the auc for insulin / auc for glucose ratio increased significantly in the sitagliptin group , but not in the control group ( table 1 ) . the change in the insulinogenic index from baseline to 3 months was significantly greater in the sitagliptin group than in the control group ( + 16.7 vs. + 0.1 , p < 0.005 ; table 1 ) . however , the changes in homa - ir and isi were not significantly different between the two groups . although the auc for glucose decreased significantly in the sitagliptin group compared with the control group , the auc for insulin and the auc for insulin / auc for glucose were not significantly different between the two groups . in this study , treatment with 50 mg / day sitagliptin after a meal for 3 months significantly reduced postprandial glucose levels and increased the insulinogenic index measured during mtts . these results suggest that sitagliptin improved the acute insulin response in a cohort of japanese patients with t2 dm . in prior studies , dpp-4 inhibitors mainly improved insulin resistance in obese populations , but did not improve the acute insulin response [ 58 ] . the mean bmis of patients in these earlier studies ranged from 27.5 to 33.2 kg / m . a recent study revealed that sitagliptin improved the insulinogenic index determined during an ogtt in korean patients . these results imply that dpp-4 inhibitors improve the acute insulin response in asian patients with t2 dm . the mean bmi was 23.0 kg / m for the sitagliptin group in our study . the diabetes epidemic in asia is characterized by the onset of diabetes at lower bmi levels and younger ages compared with caucasian populations . although the mean bmi is still relatively low in asian populations , abdominal or central obesity is also highly prevalent , resulting in a widespread metabolically obese phenotype . notable , a meta - analysis revealed that dpp-4 inhibitors lowered hba1c to a greater extent in studies with 50% asian participants ( weighted mean difference 0.92% ; 95 % ci 1.03 , 0.82 ) than in studies with < 50% asian participants ( weighted mean difference 0.65% ; 95 % ci 0.69 , 0.60 ) . the meta - analysis found no correlation between bmi and the reduction in hba1c in studies where the mean bmi was 30 kg / m , but bmi was significantly correlated with the reduction in hba1c in studies where the mean bmi was < 30 kg / m . there were no differences in the change in body weight from baseline between the asian- and the non - asian - dominant studies , which suggest that the baseline bmi might influence the glucose - lowering effects of dpp-4 inhibitors . these results suggest that a lower bmi at baseline predicts at greater response to a dpp-4 inhibitor . accordingly , the difference in bmi among ethnic groups may contribute to the differences in the glucose - lowering response to dpp-4 inhibitors . another meta - analysis revealed that dpp-4 inhibitors achieved a mean reduction in hba1c of 0.65% in non - japanese randomized controlled trials ( n = 55 ) , and a greater reduction of 1.67% in japanese randomized controlled trials ( n = 7 ) . the authors suggested that there may be pharmacogenetic or cultural / lifestyle differences that may contribute to the greater reduction in hba1c in japanese patients than in studies performed in other countries . a recent article reported that dpp4 , a novel adipokine , has a higher release from visceral adipose tissue that is particularly pronounced in obese and insulin - resistant patients . dpp4 from adipose tissue explants was higher in visceral adipose tissue than in subcutaneous adipose tissue in both lean and obese patients , with obese patients displaying higher dpp4 release than lean controls . these results might be able to explain that dpp4 inhibitor is effective for non - obese patients . our results also suggest that sitagliptin might be effective in non - obese japanese and other asian patients with t2 dm . it has been proposed that the restoration of the early phase of insulin release and postprandial hyperglycemia has important implications on the improvements of metabolic control and reducing the risk of macrovascular complications . our results suggest that sitagliptin contributes to improvements in metabolic control and a reduction in cardiovascular risk in patients with t2 dm . however , a recent study indicated that the top quartile of the matsuda isi was negatively associated with coronary artery calcification , but the insulinogenic index was not associated with coronary artery calcification . the effects of treating impaired early phase insulin secretion and postprandial hyperglycemia on macroangiopathy are still controversial , and more studies are needed in this field . moreover , in japan , approximately half of all patients with diabetes have a genetic predisposition to the disease , and insulin secretion is often impaired in lean patients with diabetes mellitus [ 9 , 10 ] . additionally , japanese and asian patients often show reduced -cell function [ 11 , 12 ] . however , a recent study indicated that disposition indices were similar in japanese and caucasian patients , and that the differences in insulin sensitivity and -cell responses between japanese and caucasian patients are mainly explained by differences in body composition . although further studies are needed to investigate these issues , it is notable that a large proportion of japanese patients exhibit impaired acute insulin responses . in our study , sitagliptin significantly reduced hba1c and the auc for glucose but not auc for insulin and lipid profile , suggesting that a reduction in glucotoxicity helped to improve insulin secretion . treatment with sitagliptin also had a significant effect on homa - ir , but not on isi . we suspect that the increase in homa - ir was due to an increase in insulin secretion rather than worsening of insulin resistance . the peak concentration time ( tmax ) for 50 mg / day sitagliptin in japanese patients is 2.0 h when taken after a meal and 2.5 h when taken before a meal . moreover , the half - life ( t1/2 ) of sitagliptin is 12.3 h when taken after a meal and 12.2 h when taken before a meal . therefore , we consider that the insulin responses at 0 and 30 min were due to the acute and chronic effects of sitagliptin . although we must be careful when interpreting these findings , our results demonstrate that sitagliptin is effective in japanese patients with t2 dm by reducing postprandial hyperglycemia in particular . because only 11 patients were treated with sitagliptin , our results require confirmation in a larger study . furthermore , because our study was small and was conducted in an open - label , non - randomized manner , it is difficult to exclude the risk of bias . design of assignment of patients may have influenced the tendency of difference of postprandial glucose , hba1c , and f - iri , although the difference was not significant . the sample size of our study was very small because of the limited numbers of potentially eligible patients at our clinic . accordingly , the results of this study should be confirmed in a larger study with an appropriate power calculation . therefore , we added a small scale real - world study in the title . however , it is difficult to measure these hormones because they are very unstable and are rapidly degraded . they recently proposed that samples should be extracted using a specific procedure to provide more accurate values , and they are currently developing a method to standardize and improve the accuracy of measuring of incretin levels . in lieu of these recommendations , it is very difficult to measure incretin levels . it is possible that the different medications used by the subjects modified the insulin response in the mtt . many of the patients were treated with a sulfonylurea , which might affect the insulin response . however , in a recent study , treatment with sitagliptin for 60 weeks decreased hba1c by 0.9% ( 10 mmol / mol ) when used in combination with metformin and by 1.0% ( 11 mmol / mol ) in combination with metformin and glimepiride . these results suggest that treatment with a sulfonylurea did not markedly affect the reduction in hba1c during treatment with sitagliptin . because the insulinogenic index was originally derived from the results of ogtts , it may not be appropriate for assessing acute insulin secretion during mtts . indeed , in one study , the insulinogenic index was higher during mtts than during ogtts . however , another study found no difference in the insulinogenic index between ogtts and mtts . considering the results of these earlier studies , we defined impaired acute insulin secretion during mtt as an insulinogenic index of < 43.2 . therefore , it is possible that the effects of sitagliptin were augmented by the fat content of the mixed meal used in this study . despite these limitations , we think that our study may aid routine clinical treatment of japanese and other asian patients with t2 dm . treatment with 50 mg / day sitagliptin after a meal for 3 months improved the acute insulin response and suppressed postprandial hyperglycemia measured during mtts . whereas the study is rather small and the design is suboptimal as it is not randomized and not blinded , these results suggest that sitagliptin may be effective in japanese patients with t2 dm , many of whom display impaired acute insulin responses . supplementary material 1 ( pdf 207 kb ) supplementary material 1 ( pdf 207 kb ) tsuyoshi ohkura , youhei fujioka , keisuke sumi , risa nakanishi , hideki shiochi , naoya yamamoto , kazuhiko matsuzawa , shoichiro izawa , hiroko ohkura , masahiko kato , shin - ichi taniguchi , and kazuhiro yamamoto declare no conflict of interest . this study was approved by the ethics committee of the faculty of medicine , tottori university . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 . this study was registered with the university hospital medical information network ( identifier : umin000011189 ) . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

introductionseveral studies have shown that dipeptidyl peptidase-4 ( dpp-4 ) inhibitors improve insulin secretion during oral glucose tolerance tests . however , the effects of dpp-4 inhibitors on impaired acute insulin responses in the postprandial state in real - world settings are unknown . therefore , we evaluated the effects of sitagliptin on the acute insulin responses in japanese patients with type 2 diabetes mellitus ( t2 dm ) using meal tolerance tests.methodstwenty-one patients with t2 dm were given a test meal ( 460 kcal ) , and plasma glucose and insulin were measured at 0 , 30 , 60 , 120 , and 180 min after the meal . the insulinogenic index of all of these patients was below 43.2 . the postprandial profiles were assessed at baseline and after 3 months of treatment with 50 mg / day sitagliptin after a meal ( n = 11 ) or were untreated ( control group ; n = 10 ) . this study was a prospective , open - label , non - blinded , non - randomized , clinical study.resultssitagliptin significantly decreased the plasma glucose levels at 60 , 120 , and 180 min , and significantly increased the plasma insulin levels at 0 and 30 min . there were no significant changes in glucose or insulin in the control group . the insulinogenic index increased significantly in the sitagliptin group compared with the control group ( + 16.7 vs. + 0.1 , p < 0.005 ) . however , homeostasis model assessment of insulin resistance and the insulin sensitivity index were not significant different between the two groups.conclusionadministration of sitagliptin at 50 mg / day after a meal improved the impaired acute insulin response and suppressed postprandial hyperglycemia . whereas the study is rather small and the design is suboptimal as it is not randomized and not blinded , these results suggest that sitagliptin is effective in japanese patients with t2 dm , many of whom display impaired acute insulin responses after a meal.electronic supplementary materialthe online version of this article ( doi:10.1007/s13300 - 014 - 0071 - 1 ) contains supplementary material , which is available to authorized users .

renewable energy policies have become embodied legislation in a number of countries ( e.g. the eu , directive 2009/28/eu eu , 2009 ) as part of the drive to reduce reliance upon fossil fuels and mitigate greenhouse gas emissions ( goldemberg , 2007 ) . while first generation biofuels ( typically derived from crops which can be processed into food or energy [ e.g. cereals , oilseed , sugar crops ] lovett et al . , 2014 ) initially gained wide political support ( boucher , 2012 ) , concerns over their legitimacy ( upham et al . , 2011 ) and increasingly negative media coverage ( sengers et al . , 2010 ) quickly surfaced . these concerns included food versus fuel land use change ( luc ) ( boucher , 2012 ; rathmann et al . , 2010 ) , indirect land use change ( iluc ) ( kim et al . , 2009 ) and the potential for biofuel induced land use change to lead to increased greenhouse gas emissions ( searchinger et al . , 2007 ) . consequently , interest emerged in advanced , or second generation , biofuels that can make use of waste streams and co - products ( e.g. corn stover , cereal [ wheat , barley , rice ] straw ) , or dedicated energy crops ( decs , e.g. miscanthus , short rotation coppice willow [ src ] ) . hence second generation biofuels utilise biomass that is derived from non - food crops with greater energy generation efficiency ( lovett et al . , a commercial second generation processing plant now exists in the eu , in italy ( anon , 2013 ) , with development plans for other second generation plants , for example in the usa and europe , already in place ( walker , 2013 ) . however , in light of luc and iluc concerns , recent literature distinguishes between co - product ( e.g. cereal straw ) second generation biofuels ( cpsgb ) and dedicated energy crop second generation biofuels ( desgb ; glithero et al . , 2012 ) , providing clarity between feedstock sources used within different second generation biofuel supply chains . however , cpsgbs still have resource use implications that must be considered : straw is utilised in livestock bedding and feeding , soil conditioning , and nutrient provision for arable crops ( copeland and turley , 2008 ; glithero et al . , 2013a , 2013b ; powlson et al . , 2011 ) cereal straw is currently used within the uk in electricity power generation ( e.g. the ely power station ) and recent research investment ( e.g. bbsrc sustainable bioenergy centre ) has explored the potential to use cereal straw as a feedstock for lignocellulosic biofuel . with respect to desgb , uk policies to encourage dec production have until recently ( august 2013 ) existed in the form of perennial bioenergy crop establishment grants ( natural england , 2013 ) . however , despite the financial assistance that establishment grants provide , areas of these crops currently grown in the england are small and declining : src 2600 ha ( declining from 6200 ha to 2600 ha over the 20082012 period ) , miscanthus 7000 ha ( increasing from 7400 ha in 2008 to a peak of 9200 ha in 2009 , followed by a decline to current levels ) ( defra , 2013 ) . it should be noted that these data are derived from non - national statistics approved approaches and are additionally associated with large confidence intervals around the point estimates provided . however , these data do indicate that financial assistance alone , in the form of establishment grants , is insufficient to incentivise large scale production change . moreover , the collapse of bioenergy companies that held contracts to purchase decs has generated increased business uncertainty for those farmers willing to produce these crops ( sherrington et al . this paper seeks to provide an understanding of english livestock farmer attitudes towards using their land for dec production and their use of cereal straw when faced by an increased straw input price . this understanding will complement previous research for the arable sector in england ( glithero et al . 2013c ) and be of direct relevance to policy makers seeking to achieve an increased supply of biomass production . the rationale for examining attitudes of livestock farmers in part flows from calls to produce decs on land not needed , or unsuitable , for food crops . however , issues of land use appropriate for energy production represent global concerns , and are not restricted to a european or western view alone ( fritsche et al . , 2010 ; zhuang et al . , 2011 ; tang et al . , 2010 marginal land is potentially problematic ; marginality can be defined in terms of economic output or reduced crop yield potential ( e.g. shortall , 2013 ; gopalakrishnan et al . , 2011 ) , unsuitability for food crop production ( e.g. royal society , 2008 ) or of low value for agricultural or biodiversity use ( royal society , 2008 ) . more structural definitions of land restrictions placed on energy crop production include excluding grade 1 and 2 land ( the most productive for arable cropping ) and land with slopes of > 15% ( lovett et al . , 2014 ; ( 2011 ) question whether marginal land can be made available at sufficiently low cost , while garnett ( 2009 ) argues that livestock farms on marginal agricultural land may provide an important role in maintaining grasslands and the carbon sinks associated with these areas . within the uk , grazing livestock production systems are predominantly located in western and upland areas ( fogerty et al . , 2013 ; harvey and scott , 2013 ) , where respectively higher annual rainfall and poorer quality agricultural land exists relative to the main arable cropping areas . hence , while a standard definition of land does not exist , within the context of bioenergy production , the approach of considering agricultural land grades 35 as appropriate for bioenergy crops ( lovett et al . , 2014 ) highlights the need to understand farm decision making within the livestock sector . several authors have examined the environmental consequences of livestock production on marginal land ( e.g. acs et al . 2010 ; oglethorpe , 2005 ) with respect to understanding livestock farmer behaviour and decision making in response to market and policy signals . to complement understanding of farmer behaviour in the arable sector ( glithero et al . , 2013c ) , we therefore need a greater understanding of marginality with respect to livestock farmer decision - making and dec production , particularly as livestock production is both an important component of the uk s agricultural economy and its land use . with respect to cereal straw , glithero et al . ( 2013a ) note the potential supply for larger scale use of straw in lignocellulosic processing facilities in england , estimating that 2.5 mt of cereal straw could be made available for bioenergy purposes . such volumes of feedstock supply to biofuel uses will affect current straw markets ( glithero et al . , 2013a ) , driving up product prices ; the response of livestock farmers to this price increase is currently unknown but is of fundamental importance to any competing cpsgb industry , because the input feedstock cost is likely to form a substantial proportion of the overall costs of biofuel production . previous research examining farmer attitudes towards dec production identified that availability of land ( adams et al . , 2011 ) , committing land to a single crop for a long time period ( glithero et al . , 2013c ) , 2008 ) , relative financial return and cash - flow considerations ( adams et al . , 2011 ; glithero et al . , 2013c ) , and knowledge of , or familiarity with , the crop ( glithero et al . , 2013c significant effects relating to managerial biographical factors ( e.g. farmer age ; paulrud and laitlia , 2010 ) , managerial attitudes ( e.g. objectives towards the environment ; augustenborg et al . , 2012 ) and farm business physical factors ( e.g. farm size and location ; paulrud and laitlia , 2010 ) on attitudes towards dec production have also been found . conversely , other researchers have not identified significant relationships between farm and farmer characteristics and attitudes towards dec production ( glithero et al . , 2013c ) . additional farmer attitude factors towards the production of decs include the remoteness or location of their farm in relation to a bioenergy plant , the topography of their farm land , and prevailing climatic conditions that impact on soil moisture content ( for both crop production and harvesting ) . the presence of farm advisors has been cited as a mechanism by which dec production can be encouraged ( velandia et al . , 2010 ; glithero et al . , 2013c ; alexander et al . with respect to quantifying the potential production of dec within england , taking into account farmer willingness to consider growing src and miscanthus , either separately or jointly , glithero et al . ( 2013c ) estimate that arable farms in england could potentially supply 50,700 ha of src and 89,900 ha of miscanthus , assuming farmers would convert less than 10% of their land area . other researchers have identified that farmers in the uk are only likely to convert a small proportion of the land area to dec , and on their least productive land , even where interest in these crops exists ( sherrington et al . , 2008 ) . understanding both the factors that influence the supply of biomass feedstock and the competing demands for feedstock are therefore crucial to the development of a commercial bioenergy sector , in particular for second generation feedstock in its current embryonic commercial stage ( walker , 2013 ) . by using survey techniques , this paper examines some of these potentially influential factors for livestock farmers in england . the specific objectives of this paper are to ( a ) describe the survey methodology adopted ; ( b ) indicate the numbers of farmers willing to grow src and miscanthus and analyse the responses in relation to a number of farmer characteristics ; ( c ) identify farmer attitudes and the main reasons given for growing and not growing these decs ; ( d ) estimate potential areas of these crops that could be grown on livestock farms in england based on the survey results and ( e ) draw national conclusions from these results in relation to the potential barriers / incentives identified to growing and not growing decs and potential bioenergy supply . the survey methodology and design and results are outlined in sections 2 and 3 , respectively ; this is put into the context of the uk bioenergy sector in section 4 . a summary , overall conclusions and policy recommendations building upon a body of attitudinal evidence that , for the arable sector , has captured attitudes towards straw production , straw incorporation / on - farm use or sale , and willingness to consider growing energy crops ( glithero et al . , 2013a ; 2013b , 2013c ) , a telephone survey was undertaken to capture data on livestock farmers attitudes towards dec production and their responses to an increased price for cereal straw . the survey was conducted in england on three livestock farm types : dairy , less favoured area [ lfa ] grazing livestock and lowland grazing livestock.1 these farm types dominate land use in the upland and western regions of england and on grazing land ( permanent pasture , temporary grassland and rough grazing ) ( fogerty et al . , 2013 ; harvey and scott , 2013 ) . taken together with evidence from previous research ( glithero et al . , 2013a ; 2013b ; 2013c ) conducting the survey on these three farms types will capture data for the majority of the agricultural land use area in england . in order to ensure direct comparability of data with previously published results for the arable sector in england specifically , information was gathered on : ( i ) attitudes towards straw use given increased purchase price for the product ( closed question responses to eight possible options ) ; ( ii ) whether farmers would consider growing the decs miscanthus and src at the present time ( closed question : yes , no , already growing ; for each crop ) ; ( iii ) factors that are important in their attitudes towards dec production ( closed question : [ selecting all options that apply ] ) ; ( iv ) the percentage farm area on which they would be willing to grow decs ( closed question : percentage of farm area , for each crop ) ; ( v ) importance rankings for key objectives relating to their farm and farm business ( ranking questions : 5 point scale ) ; and ( vi ) additional comments that the farmer wished to convey in relation to decs or use of straw for bioenergy purposes ( open question ) . further details on the individual questions are detailed below in the description of methods of analysis . the survey was carried out in conjunction with the farm business survey ( fbs ) by rural business research ros ( research officers ) between december 2012 and february 2013 . the english fbs consists of a sample of approximately 3% of farm businesses across all regions of england , stratified to match the population of farm businesses , with a minimum standard output from their business activities of 25,000 , across farm types defined by the cropping and livestock activity on the farm , and business size . farm businesses are invited to take part in the fbs based upon randomly selected farm business address details supplied by defra . the stratification for the fbs is based upon the annual june survey returns ( department for the environment , food and rural affairs ) , and permits data aggregation , via population weighting , to estimate national production and financial returns for the sector . within the fbs ros visit the farm businesses that take part , on an annual basis , to collect financial and physical data relating to their farm and business activities . the surveyed farms for this study were a sub - sample of the full fbs sample ( approximately 31% of the livestock farm types detailed above ) across eight government office regions ( gors ) of england and two business size groups per farm type category , stratified by sample numbers within the fbs . crucially , this sampling procedure ensured that the farms surveyed in this study were unbiased with respect to their opinions towards the production of energy crops , which overcomes any potential sampling bias when undertaken more general random sampling approaches , for example , via postal surveys . the sub - set of fbs farm businesses which took part in this telephone survey were randomly selected from the fbs sample for the farm types of interest . each respondent invited to take part in the telephone survey was previously known to the ro who conducted the survey . this respondent - researcher relationship was crucial to ensuring that all of the 263 respondents who were invited to take part completed the telephone survey ; hence respondents neither agreed nor declined to take part because they had either a particular interest , or no interest , in the survey topic ( e.g. decs ) . the number of farms surveyed in each farm type and gor is given in table 1 . reflecting the geographical nature of livestock production in england , the south west region of england represents the largest number of sample returns ( 31% ) , followed by the north west ( 19% ) ; note that less than 4% of the sample were from the east of england . following the approach of glithero et al . ( 2013c ) and drawing upon data held about the farm in the fbs , responses relating to willingness to grow src and miscanthus ( yes , no ; per crop ) have been analysed with respect to categories for : farmer age ( years : under 50 ; 5064 ; 65 and over ) ; eu england region location ( north , east or west ) ; land ownership ( greater than 50% area owned ; less than 50% owned ) ; farm type ( dairy , lfa grazing livestock , lowland grazing livestock ) ; farm size ( large , medium , small ; as defined by fbs ) ; and farmer educational attainment ( school level only [ gcse s , a - levels , apprenticeships and other ] ; college level ; university level [ degree or postgraduate ] ) . chi - squared tests were undertaken to test the hypothesis that there is no significant relationship between each of the above factors and farmer attitudes towards growing src and miscanthus . where farmers noted a willingness to grow src or miscanthus ( or both ) , data were obtained on the percentage land area ( per crop ) that they would consider growing across the following categories ( up to 10% ; > 1025% ; > 2550% ; > 5075% ; > 7599% ; 100%).2 combining data held about the farm s utilised agricultural area ( uaa ) from the fbs , with the responses to percentage farm area that the farmer would be willing to grow , minimum and maximum crops area per farm were calculated by taking the lower and upper bounds of the above percentage categories ( lower bound of up to 10% was assumed as 0% ) . these data were then aggregated to national ( england ) levels following a standard aggregation weighting procedure ( see glithero et al . , 2013b ) which calculates aggregation weights as the defra june survey total farmed area per farm type , per gor divided by the survey farm area per farm type , per gor ; the individual farm data are then weighted by this aggregation factor and estimates of potential crop production across the eight gors of england estimated . once farmers had indicated their willingness , or otherwise , towards growing src or miscanthus , they were asked to select which factors were important in their decision making across a range of practical , environmental , financial and knowledge factors . practical factors consisted of : lack of appropriate machinery ; use of known machinery ; ease of crop management ; committing land for a long period of time ; need planning permission from landlord. environmental factors examined were : positive environmental impact ( of crop ) ; negative environmental impact ; nitrate vulnerable zone ( nvz ) restrictions ; land quality aspects. financial and knowledge factors examined were : time to financial return for crop ; market for crop ; no market for crop ; profitability ; local working example ; no local working example . these responses were then analysed with respect to willingness to grow the energy crop or not , for each crop of src and miscanthus , to indicate the percentage of responses per importance factor . in addition to the pre - determined factors within the questionnaire , farmers could also add additional factors that were important in their decision making ; these factors were grouped together in broad categories with specific comments detailed as issues raised by farmers in the survey . in order to capture data on potential responses to an increased purchase price of straw , farmers were asked to indicate if , and how , they would change their practices towards straw purchase and use in light of a change in market conditions for straw . farmers were provided with a realistic practical business scenario of an increase straw price of 100/t for big square bales ( prevailing market price at the time of the survey was approximately 40/t ) . farmers were asked to select from a range of responses encompassing increasing own cereal crop production , start own cereal crop production , change livestock bedding material from straw to another product , change from loose housing to cubicles to reduce or remove the need for straw , continue to buy in straw , reduce livestock production , stop livestock production , and changing to grow taller cereal varieties ( for additional straw ) . in addition , farmers could indicate other options as responses to this scenario . the livestock farmers surveyed were asked if they would be willing to consider growing src willow and miscanthus , requiring separate responses for each crop ; farmers could respond with yes , no or already growing the crop. in total from the 263 responses received , 19 farmers ( 7.2% ) indicated they would consider growing src willow , while 17 ( 6.3% ) noted a willingness to consider growing miscanthus ; 12 farmers ( 4.6% ) indicated a willingness to grow both crops . none of the farmers surveyed were already growing either of these two crops . using chi - squared tests , responses were analysed to test the hypotheses that there was no significant relationship between farmers age , farmers education , farm ownership , farm type , farm size and farm eu region location to the farmer response to attitudes towards growing src or miscanthus . however , it is informative to note that at the 90% level of significance land ownership ( greater or less than 50% of farm area owned ) has a significant impact on response ; farmers with greater than 50% farm area owned are more willing to consider growing src ( 9.6% compared with 4.3% for farmers with less than 50% owned area ) and miscanthus ( 8.9% compared with 3.4% for farmers with less than 50% owned area ) . additionally , at the 90% level of significance , there is a significant relationship between eu region and willingness to consider growing miscanthus ( eu east region more willing to consider growing miscanthus 13.1% compared with 4% eu north , 4.8% eu west ) : p - values for these results are given in table 2 . 1 presents reasons given for being willing or not willing to consider growing src or miscanthus following the approach of glithero et al . ( 2013c ) . for both src and miscanthus common issues emerge as reasons cited for deciding to not consider growing these crops . within the practical reasons , committing land for a long period of time , followed by lack of appropriate machinery , the use of known machinery and ease of crop management were cited by very small numbers of respondents with respect to either being willing or not willing to consider growing these crops . with respect to environmental reasons the major issue cited relates to land quality aspects , with damage to drains and cost of changing land back to agricultural use identified to respondents as the definition for this category . approximately 15% of respondents cited negative environmental impacts as a reason for choosing not to consider growing either crop . nitrate vulnerable zone restrictions were not a key determining factor for farmers , and very limited numbers of respondents cited positive environmental impact of miscanthus or src as an important aspect in their decision making . profitability was cited as a key financial driver against considering growing these crops , followed by time to financial return and no market for the crop. it is informative to note that while no local working example was cited by over 10% of farmers as a reason for not being willing to consider growing either of these crops , the presence of a local working example has also been cited by a small number of respondents as a reason for not being willing to consider growing either crop . from the responses citing willingness to consider src or miscanthus , profitability of the crop market for the crop and time to financial return. note however , that the modest number of positive responses towards growing the crops leads to single responses having a relatively large influence on the overall percentage influence recorded . interest and morality ( 11 responses ) , current and future farming / business activities ( 19 ) , land resource availability ( 32 ) , land quality / topography ( 41 ) , knowledge ( 14 ) , other ( 14 ) . it is informative to note the large number of comments relating to land quality / topography which frequently highlighted aspects of unsuitability of land for crop production , steepness of slopes or wet land and weather conditions . those livestock farmers willing to consider growing src or miscanthus were asked to indicate the percentage of their farm area that they would be willing to commit to these crops . these responses were then combined with data from the fbs on farm area , and aggregated to provide gor and hence national ( england ) estimates of the minimum and maximum crop areas that would potentially be grown on livestock farms . 2 presents the results of this analysis ; the largest potential maximum crop growth area for both src and miscanthus is the south east of england . more modest areas of crop production possibilities were identified in the north east , north west , and yorkshire and the humber . for england , the maximum ( minimum ) potential area of src that would be grown on dairy , lfa grazing livestock and lowland grazing livestock farms is 54,603 ha ( 17,156 ha ) ; the respective results for miscanthus are 43,859 ha ( 12,321 ha ) . livestock farmers were asked to consider how they would respond to an increased price of straw as detailed in the methodology section ; on a number of farms a negative response was recorded as not applicable for specific possible responses , for example where farmers viewed starting own crop production , increasing crop production or growing taller varieties , as not applicable to their farm or business situation . fig . responses to strategies for responding to the increased straw price question , across the three farm types examined ; significant differences in the number of positive responses to the strategies by farm type groups were found ( chi - squared test , p - value < 0.001 ) . key results demonstrate that across all farm type groups the majority of farmers would not stop keeping livestock in response to this increased price of straw , as noted by less than 5% in each farm type group choosing this option . in response to increased straw prices however , lowland grazing livestock farmers are more likely to reduce livestock production given increased straw prices ; this response may in part be driven by the greater flexibility in agricultural and land - use production possibilities that lowland grazing livestock producers have in comparison to dairy or lfa grazing livestock farms . own cereal crops , lowland grazing livestock farmers are the group most likely to undertake this option . changing the bedding from straw to another product was a more popular response than changing infrastructure ( e.g. from loose housing to cubicles ) . our findings demonstrate that only modest numbers of livestock farmers are willing to consider growing decs ( 7.2% and 6.3% for src and miscanthus , respectively ) ; this is lower than observed for arable farmers in england ( 11.9% and 17.2% for src and miscanthus , respectively ; glithero et al . , 2013c ) and is argued to flow from a range of geographic , land tenure , land quality and financial considerations as explored further below . however , a central outcome from the results presented is that the lower level of interest shown from livestock farmers with respect to growing decs represents a substantial challenge to policy makers seeking to encourage dec production , in particular from geographic areas that are characterised as being more agriculturally marginal . in part the difference between livestock and arable farmer willingness to grow decs may be explained by the proximity of current concentrations of markets for decs , where greater concentrations of land use for dec production are found within the east midlands , yorkshire and humberside and the south west ( lovett et al . , 2014 ) ; while the latter region is predominantly livestock based , the former regions are predominantly arable and represent areas where farmers may have been more exposed to the possibility of growing these crops via information from their farmer neighbours , or from companies seeking to secure crop supplies . the combination of greater interest in growing decs from arable farmers , and the greater interest from livestock farmers in the eu east region of england , together indicate that developments in the bioenergy sector that rely upon decs or cereal straw should be located in the east of england ( glithero et al . , 2013a ) . while this may contrast with calls for dec production on marginal land , it will result in bioenergy facilities being located in areas where supply of both cereal straw and decs have greater potential . for livestock farmers , land ownership influences decisions about dec production , with those farmers owning more than 50% of their land area being more willing to consider growing decs ; in part these land ownership influences reflect restrictions placed upon tenants by landlords who wish to maintain particular types of livestock based land use activities on their land . livestock farmers in the eu east region of england are more likely to consider growing miscanthus than livestock farmers in the other eu regions of england ; this is possibly a reflection of the more marginal nature of livestock production in the east , where lower precipitation levels limit grass forage production and hence increase the profitability of crop production relative to grazing livestock enterprises . the land ownership and geographic influences observed for livestock farmers contrast with those for arable farmers in england , where land ownership and regional influences were not found to have a significant influence on attitudes towards growing decs ( glithero et al . , 2013c ) ; however , the results for livestock farmers presented here do reinforce findings for attitudes towards production of bioenergy feedstocks in other countries ( e.g. switchgrass in tennessee , jensen et al . , 2007 ; src on marginal land in germany , schweier and becker , 2013 ) . from the results presented above , issues relating to the impact of decs on land quality , committing land for a long period of time , lack of appropriate machinery , relative profitability of the crop and time to financial return were cited as common reasons for not considering growing these crops , confirming attitudes reported in previous studies ( e.g. glithero et al . , 2013c ; schweier and becker , 2013 ; sherrington et al . , 2008 ; 2012 ) , and providing clear signals to policy makers that there are constraints and barriers to the take up of dec crops by farmers . some of these are actual : relative profitability of existing farm enterprises will vary from farm to farm and those farmers with healthy enterprise gross margins ( financial value of output less direct , variable costs of production ) will be less willing to switch to decs , other things being equal . however , some barriers to take - up may be more perceived than real - for example , the effect of decs on land quality over time once established . for livestock farmers , land quality was also given as a reason for not starting production : topography , poor cropping ability of hill land , and within the context of dec production , these findings contrast directly with calls for dec production on marginal land . specifically , livestock farmers citing these concerns demonstrate that the marginality of the land , with respect to use for mechanised crop production , makes the prospect of dec production in these areas less viable , and hence less likely , than in non - marginal land areas . the dichotomy between policy objectives that aim to minimise food versus food conflicts and the potential uptake of dec production on english livestock farms , some of whom operate in challenging geographical and climatic conditions , is clearly evident here . where livestock farmers were willing to consider growing src and miscanthus , profitability , having a market for the crop , time to financial return and ease of crop management were cited as important factors influencing their decision making . while representing small numbers of respondents in this study , these factors are of direct interest to policy makers seeking to incentivise dec production . in particular , these farmers implicitly consider their land to be marginal with respect to the alternatives that they face within their own farm businesses . in some respects it would , from a policy perspective , be more productive to target these marginal farmers , rather than to attempt to identify , from a perspective external to the farm business , marginal land using a pre - conceived measure , such as soil quality or yield potential . placing the above points in wider context , it is informative to recognise that the results presented represent findings from a survey of stated attitudes at a single point in time towards a series of pre - determined questions . moreover , the use of a telephone survey during the winter months may have influenced farmer responses . previous authors have criticised such linear decision making models as not seeking to understand underlying motivations . within economic geography approaches , radwan and kinder ( 2013 ) and jones and murphy ( 2011 ) argue that innovation or adoption of different practices are more complex than can be elicited from the approaches adopted within this study , and that adoption involves aspects such as learning , social norms and networks , all of which are dynamic . within the specific case of energy crops , alexander et al . ( 2014a ) note that adoption is likely to follow a typical s - shaped curve over time , hence it is not directly possible to capture the dynamics of dec supply in the future from the approach adopted here . however , survey approaches provide a succinct method of data capture across a large number of observations and have been used in previous research that has direct relevance to this current study ( glithero et al . ( 2014b ) note that within an agent - based modelling framework the first stage in the decision making process for farmers is appropriately captured by willingness to consider growing the crops. accepting the restrictions outlined above as caveat to the survey findings , we provide estimates of the national potential for dec supply from these farm types which are argued to be of policy and industry relevance . specifically , on the assumption that livestock farmers in england grew decs in line with their stated willingness to consider growing these crops identified by the representative livestock farmers in this survey , the maximum ( minimum ) area of src that would be produced on livestock farms in england is 54,603 ha ( 17,156 ha ) ; the respective results for miscanthus are 43,859 ha ( 12,321 ha ) . ( 2013c ) estimated a potential supply 50,700 ha of src and 89,900 ha of miscanthus from arable farmers in england . taken together these data indicate potential for approximately 100,000 ha of src and 130,000 ha of miscanthus production in england . these estimates represent a small proportion of the estimated areas available for dec production cited in previous studies ( 3.1 mha for decs , haughton et al . ( 2014 ) ) reinforcing the finding that the constraint to dec supply will be economic considerations rather than planning or structural constraints . maximum potential supply in england of 81,000 ha of src and 141,000 ha of miscanthus , with supply levels being output price sensitive . while alexander et al . ( 2014a ) s area estimates are more directly in line with the results presented above , alexander et al . ( op cit ) estimate dec supply to largely occur in livestock dominated areas of england , with small crop areas likely to be found in the arable dominated regions . ( 2014a ) s estimate is based upon a comparison of conventional arable cropping with dec production in all regions ; in reality the agricultural activities in the main areas they identified for dec production are predominantly livestock based , and farmer decision making will therefore be based upon a comparison of livestock production against decs . however , it is informative to note that both the estimates from this current study , and from alexander et al . ( 2014a ) represent considerable increases from current dec areas and would require changes in policy support ( glithero et al . such policy support will need to be cognisant of the range of issues identified by farmers as highlighted above . , 2013c ) have noted the need for enhanced extension services to farmers in order to encourage dec production . with respect to policy implications , alexander et al . ( 2014b ) note that the provision of establishment grants for decs provides a cost effective ghg emissions abatement policy , and one which would be more effective in encouraging dec uptake than providing higher subsidies to power generation companies . the 50% establishment grant for dec in england closed in august 2013 , without a clear indication of what may replace this ( alexander et al . , 2014a ) ; white et al . ( 2013 ) argue that consistency of policy rules are critical to development of renewable energy . others have argued for area - based payments for the initial years of on - going energy crop production ( lindergaard , 2013 ) . with respect to tenant livestock farmers , policy incentives may need to be targeted towards landlords in order to allow tenants greater freedom in their land use practices and production possibilities . policies developed in the absence of understanding these key issues are argued to be inherently less likely to achieve their desired aims . noting the marginal farmer concept outlined earlier ; a potentially cost effective way to target these farmers would be some form of auction of contracts for the right to receive payments to grow decs . in the context of contracts for agri - environmental policy , latacz - lohman and schillizi ( 2005 ) suggest that auctions work best when the number of bidders is high , contracts are homogeneous and landholders are heterogeneous in their compliance costs . useful experimental research could be conducted to test whether dec contracts , and potential markets in these contracts , meet these criteria . in response to a substantial increase in straw prices , the majority of livestock farmers indicated that they would continue to produce livestock , with lowland grazing livestock farmers being more likely than dairy or lfa grazing livestock farmers to consider reducing livestock production ; the lowland grazing livestock group were also more likely to increase production of their own cereal crops and to consider growing taller varieties . these responses , in part , demonstrate the specialised nature of dairy production in comparison to lowland grazing production , and the more restricted range of production possibilities found in the upland and hill areas of england ( harvey and scott , 2013 ) . lfa grazing livestock farmers would overwhelmingly continue to buy in straw at high prices , while the majority of dairy and lowland grazing livestock farmers would also continue to buy in high price straw . changing bedding or livestock building infrastructure would also occur , but on a smaller proportion of livestock farms . livestock farmers may be more generally reluctant to consider alternatives to straw use or to substitute existing land uses for bioenergy feedstocks . it has been argued that this is because agricultural assets have low values outside of the industry ( the phenomenon of asset fixity , see e.g. boetel et al . , 2007 ) ; existing patterns of production are likely to persist due to low asset resale values . this will particularly be the case where assets have very specific , non - transferable uses , as is often the case in dairy production . ( 2013 ) present evidence that farmers value the option to gain information on future cash flows by deferring disinvestment , even where it would be financially optimal to realise the ( relatively low ) value of the agricultural assets tied up in a particular aspect of the business , or the business as a whole . richards and green ( 2003 ) , in the context of perennial crops , refer to hysteresis , the continuation of a particular economic activity in agriculture in order to gain the small chance of worthwhile returns sometime in the future essentially an attitude of not wanting to miss out. the corollary is that , once established , there would be an option value for continuing in crops such as miscanthus or src production . a straw - based second generation bioenergy plant may lead to an increased amount of straw being made available to the market , as arable farmers increase the volumes of straw baled ( glithero et al . , 2013a ) . however , our findings indicate that bioenergy processors will have to compete , in the market for cereal straw , with livestock farmers . evidence from germany , where large areas of oilseed and maize crops are grown for energy demonstrates that as land available for primary food production decreases , land prices and rents increase , in turn increasing the cost of biomass production ( lupp et al . , 2014 ) . larsen et al . ( 2013 ) note that even with increased production efficiencies in danish agriculture , demand for wheat straw for cpsgb production , would fully utilise wheat straw residues by 2030 , in addition to limiting livestock production expansion . from the evidence presented here , english livestock farmers response to straw price increases would appear to be price inelastic ; while some product substitution may occur , the importance of straw within livestock production would conceivably lead to these livestock farmers outcompeting bioenergy purchasers . the bioenergy sector will need to secure feedstock at a sufficiently competitive price to ensure commercial success ; for livestock farmers , while the cost of straw represents an important production cost , other costs typically outweigh those incurred from purchasing straw . moreover , in order for livestock farmers to achieve large scale reductions in straw use , investment in infrastructure ( e.g. cubicle housing for cattle ) will be required . arguably only when relatively high straw price thresholds are breached , and are forecast to remain high , will farmers seek to make such investments . evidence from the results presented above , combined with previous evidence on straw availability ( copeland and turley , 2008 ; glithero et al . , 2013a ; 2013b ) indicates that under contemporary market , policy and regulatory conditions , wide scale uk straw - based bioenergy production will be constrained by biomass availability and feedstock price pressures . moreover , attitudes towards dec production ( glithero et al . , 2013c ; sherrington et al . , 2008 ) demonstrate a range of farm - level , tenancy and market condition factors that will additionally constrain large scale uk dec biomass production . in addition , marginal land , around which some livestock production systems are based , has been demonstrated to be a constraint to the production of dec , rather than a perceived opportunity which has been frequently assumed ( ajanovic , 2011 ; fischer et al . , 2010 ) . others have also recognised the constraints these geographical areas place on dec production , hence limiting the sustainable supply of biomass ( bindraban et al . , ( 2009 ) ) . however , both straw - based and dec - based bioenergy do offer entrepreneurial opportunities to farmers . recent uk government incentives for renewable energy have led to increased farmer uptake of solar and wind power generation , and use of biomass boilers , in the uk ; farmers have been incentivised by government - guaranteed financial return , yet farmer attitudes play a central role to the uptake of renewable energy opportunities ( tate et al . , currently , desgb production incentives are limited to financial assistance for crop establishment , while no government incentives exist for cpsgb . ( 2013c ) , further incentives will need to be provided to farmers to engage in supplying biomass feedstock on a larger scale . greening activities , within the requirements of the revised common agricultural policy ( cap ) ( eu , 2011 ) . these are required on predominantly arable farms , however , defra s ( 2014 ) implementation of the revised cap means that miscanthus and src will not count as crops that provide environmental benefits under the greening rules . contrasting this outcome , rowe et al . ( 2009 ) find that decs of miscanthus and src can provide positive outcomes to biodiversity , soil properties and ghg mitigation when compared to arable crops , and these outcomes are also noted for miscanthus by wang et al . the financial returns of primary food production have also increased in recent years in part because of the success of biofuel - related policy mechanisms ( wright and cafiero , 2011 ) . these improved food prices reduce the incentive for farmers to adopt new or alternative cropping strategies . while calls for the production of decs on marginal land have been made , much previous research has examined the suitability of land for decs without examining issues related to the economics of agricultural production or farmer decision making on farms . our findings demonstrate limited potential for dec production on farm land in england and reinforce previous estimates of aggregate dec supply potential ( alexander et al . , 2014a ) . moreover , we have identified that livestock farms will typically continue to buy in cereal straw at higher prices , with consequential impacts on the economic viability of any second generation bioenergy sector . this research highlights some key policy messages for those seeking to achieve a positive transition pathway towards the increased use of biomass for bioenergy purposes . moreover , with respect to targeting biomass production policies at particular farm types , our results indicate that livestock farmers are marginally less interested than arable farmers ( glithero et al . , 2013c ) in growing decs . with respect to potential policy implications for increasing biomass supply from farm land in england , our results lead to the following policy suggestions . maintenance grants and on - going area - based payments for decs would , in - part , address issues of the time taken to gain a financial return from these crops . supporting dec - specific extension services would increase farmer awareness of the ease of crop management for decs . de - risking the output market for decs through government - backed output contracts would provide a guaranteed market for the crop addressing a key issue raised . incentivising dec production via eligibility for energy subsidy payments ( e.g. renewable obligations certificates [ roc ] ) would improve the relative profitability of dec in comparison to the current financial returns . targeting both farmers and landlords with respect to policy messages geographic targeting of support within a specific radius of a biomass plant location , or co - supporting biomass plants alongside farm - level biomass production may also be required . in the absence of such policy developments the combination of increased market prices for primary food products , and the policy environment in which farmers currently operate in the uk , in addition , policy makers and the biomass industry must recognise that , in regions within , or in close proximity to , livestock production , farmers will potentially out - compete a biomass plant in the market for cereal straw . hence , policy makers seeking to incentivise dec production arguably need to re - think the current dec support mechanisms to address profitability , market risk , land suitability and land quality concerns , combined with issues of lack of knowledge of dec production amongst farmers in general , and the need for landlord permission on some tenanted farms . policies supporting the production of dec on lower grade agricultural land , or land with lower agricultural or biodiversity potential must acknowledge issues of economic , tenancy and personal objectives if they are to succeed . moreover , if cereal straw is to play a central role in second generation biofuel production , policy makers may need to incentivise changes in livestock building infrastructure , to reduce livestock farmers demand for cereal straw . it is also essential that policy makers ensure that food , fuel and ecosystem services policies are integrated , rather than conflicting , in order to provide coherent and consistent policy signals to farmers .

second generation biofuels utilising agricultural by - products ( e.g. straw ) , or dedicated energy crops ( decs ) produced on marginal land , have been called for . a structured telephone survey of 263 livestock farmers , predominantly located in the west or marginal upland areas of england captured data on attitudes towards straw use and decs . combined with farm physical and business data , the survey results show that 7.2% and 6.3% of farmers would respectively consider growing src and miscanthus , producing respective maximum potential english crop areas of 54,603 ha and 43,859 ha . if higher market prices for straw occurred , most livestock farmers would continue to buy straw . reasons for not being willing to consider growing decs include concerns over land quality , committing land for a long time period , lack of appropriate machinery , profitability , and time to financial return ; a range of moral , land quality , production conflict and lack of crop knowledge factors were also cited . results demonstrate limited potential for the production of decs on livestock farms in england . changes in policy support to address farmer concerns with respect to decs will be required to incentivise farmers to increase energy crop production . policy support for dec production must be cognisant of farm - level economic , tenancy and personal objectives .

familial adenomatous polyposis ( fap ) is an autosomal dominant disease characterized by the presence of numerous adenomatous polyps in the colon . it affects between 1 in 8,300 to 1 in 14,025 live births , affecting both the sexes equally . the polyps inevitably develop into cancer in 10 years to15 years after their appearance , and the only treatment available at present to prevent the cancer is total proctocolectomy . the characteristic of this disease is the young age at presentation with the average age at diagnosis of colorectal cancer being 39 years . in patients diagnosed with fap , prophylactic colectomy the young age of the patient makes cosmesis a significant consideration when choosing the mode of therapy . the aim of this study was to demonstrate the feasibility of laparoscopic restorative proctocolectomy with ileal pouch anal anastomosis ( rpc - ipaa ) for fap and to verify its outcome . a single operating surgeon ( cp ) assisted by a surgical team performed all the surgeries . before embarking on this study , we had developed sufficient experience in laparoscopic colorectal surgery , which has been published elsewhere . all patients are administered intravenous antibiotics in the form of ciprofloxacin 500 mg iv , ornidazole 500 mg iv one hour before surgery , and antithrombotic prophylaxis is administered with low - molecular - weight heparin and elastic stockings . a nasogastric tube and a urinary catheter are inserted before the start of the procedure . basically , the surgery consists of a combination of various phases of mobilization and ligation of pedicles involved in the performance of left , right , and transverse colectomy . the thighs are flexed minimally so as to not to impede the movement of laparoscopic instruments . the importance of a remote - controlled operating table that can be tilted on any axis can not be overemphasized . it is gravity that is most often used as a retractor to keep the bowels away from the operating area . the procedure starts with mobilization of the sigmoid and rectosigmoid , which is continued cranially towards the descending colon . . the surgeon moves between the legs of the patients for mobilization of the splenic flexure and transverse colon . the ascending colon and hepatic flexure are mobilized from the left side of the patient , and the distal ileum is transected using laparoscopic staplers . the surgeon returns to the right side for rectal mobilization and ultra - low resection using staplers . during all the phases of the surgery , the dissection has been medial to lateral . we follow the planes of oncological clearance as these planes are avascular , and secondly , to provide adequate clearance even if the final histopathological examination of the specimen reveals malignancy that was not detected by preoperative sampling of the polyps . thereafter , we deal with the left colic and middle colic pedicles in a similar fashion during the phases of dissection of the left and transverse colon , respectively . during the phase of transverse colon mobilization , the greater omentum is released from the transverse colon and placed in the left upper quadrant . flush ligating the pedicles and use of ultracision for dissection enables a relatively bloodless field and minimal perioperative blood loss . through a minilaparotomy pfannenstiel incision the stapled end of the terminal ileum is delivered into the wound , and a j pouch is fashioned by using the linear stapler . an enterotomy is made at the apex of the pouch , and the blades of the linear stapler ( 7.5 cm ) are introduced into each of the limbs of the pouch and sequentially fired twice to obtain the pouch . the anvil of the circular stapler is inserted into the apex of the pouch through the enterotomy and secured with pursestring sutures . the pouch is delivered into the abdomen , the pfannenstiel incision is closed , and pneumoperitoneum reestablished . after confirming the correct orientation of the pouch , it is anastomosed to the anal canal via a transanally introduced circular stapler ( figure 2 ) . the magnification afforded by the laparoscope allows ultra - low dissection so that the rectal stump is divided at the dentate line itself . in case a proximal division occurs , rectal mucosectomy is carried out transanally prior to pouch anal anastomosis . we perform a protective ileostomy in all patients , which is closed after 6 weeks . we prefer a brooke 's type of everting ileostomy placed in the right iliac fossa . a. 10 mm camera port for mobilization of rectum / sigmoid / hepatic flexure and right half of trans - verse colon ; right - hand working port for mobilization of descending colon , caecum , and ascending colon ; left - hand working port for mobilization of splenic flexure and left half of the transverse colon . b. 10 mm or 5 mm right - hand working port for mobilization of transverse colon ; retraction for mobilization of rectum , sigmoid colon , and descending colon . c. 10 mm camera port for mobilization of descending colon , splenic flexure , left half of transverse colon , caecum , and ascending colon . d. 5 mm left - hand working port for mobilization of rectum , sigmoid colon , descending colon , caecum , ascending colon , hepatic flexure , and right half of transverse colon ; retraction for mobilization of splenic flexure and left half of transverse colon . fifteen fap patients underwent restorative total proctocolectomy with ileal pouch anal anastomosis ( rpc - ipaa ) . in these 4 patients , preoperative colonoscopy and biopsy had revealed adenocarcinoma in rectal polyps . the american joint committee on cancer ( ajcc ) staging of 2 patients was t2n0m0 , and the other 2 patients had t3n0m0 stage malignancy . the tumors were located 8 cm , 12 cm , 13 cm , and 16 cm from the anal verge in these patients . the operating time ranged for 190 minutes to 365 minutes with a median of 225 minutes . the mean blood loss was 60 ml with a range of 30 ml to 180 ml . the average incision length of the mini - pfannenstiel incision was 5 cm ( range , 4 to 6.5 ) . twelve patients resumed a liquid diet on the second postoperative day , while 3 patients did so on the third postoperative day . the malignancy and staging was confirmed in the 4 patients with proven rectal adenocarcinoma . in one patient with pre - operative staging of t3n0m0 , in all the patients , the margins were free from tumors , and the minimum distal margin was 4 cm . the pouch frequency varied between 3 and 6 , with the median being 4 times . though initially , patients complained of liquid stools , by one month postoperatively , they were passing semisolid stools and did not suffer from urgency . one patient developed wound infection that was treated by a change in antibiotic , according to the culture sensitivity report . this patient was an elderly male of 52 years and had an enlarged prostate ( 42 g ) on ultrasonography preoperatively . he was started on tablet tamsulosin 0.4 mg once a day at night , and the catheter was removed on the seventh day . she was kept nil orally with ryle 's tube decompression of the stomach and repeated enemas to clear the pouch . the patient recovered and was started on oral feedings by the fifth day of treatment and was discharged on the seventh day . the patient was taught self - administration of enemas and pouch irrigation . with this , the patient has not experienced any problems after a mean follow - up of 3 years and 10 months . none of the male patients had problems with potency , orgasm sensation , ejaculation , or micturition . in response to a questionnaire , all the patients declared themselves to be highly satisfied with the outcome of the surgery and the cosmesis and would recommend the procedure to other patients . all the patients were called for follow - up after 7 days , 1 month , 6 months , and yearly thereafter , except for the patients with malignancy who were called monthly after the first month and up to 1 year and then yearly thereafter . at every visit , the patient is examined . at the 3-month visit , endoscopic pouch examination with a flexible sigmoidoscope and upper gi endoscopy all patients have maintained follow - up , and the average follow - up has been 3 years and 10 months . there has been no recurrence of malignancy or polyps in any of the patients . moreover , the upper gi endoscopy has revealed the absence of polyps in the duodenum . the patient with t3n1mo malignancy was referred to an oncologist and received adjuvant chemotherapy consisting of irinotecan , 5-fu , and folinic acid . he has tolerated the chemotherapy well and is disease - free after 2 years and 4 months of follow - up . though all the patients ' first - degree relatives have been counseled to undergo screening for familial adenomatous polyposis , only 13 such relatives belonging to 5 patients have come for a colonoscopy . of these familial adenomatous polyposis ( fap ) is a rare autosomal dominant disease caused by a defect in the apc gene of the 5q21 chromosome . all affected untreated patients will die of colorectal adenocarcinomas in the fourth or fifth decades , which was the fate of most patients in the first half of the 20th century . however , thereafter , several national registries were established , and screening programs were started . early detection and treatment of this disease has resulted in a reduced incidence of death from colorectal cancer . it has been demonstrated that establishment of a national registry improves the colectomy rate , reduces the prevalence of colorectal cancer , and improves survival . today , the most frequent causes of death in screening detected patients are duodenal cancer and desmoid tumor . similarly , in a study based on the hong kong registry , the median age of diagnosis was significantly lower in patients detected by screening than those diagnosed by symptomatic presentation ( 29 years vs. 34 years , respectively ) . at the time of diagnosis , 9.7% of the screened patients had malignancy compared with 61% of the unscreened patients . however , india does not have a national registry for fap , nor does it have a screening program . as a result , most of our patients presented late ( mean age being 44.4 years ) , and 4 of the 9 patients had already developed malignancy . of the 9 patients , 5 were diagnosed with fap while being investigated by us for abdominal symptoms , including the 4 patients with malignancy . the remaining patients had siblings or parents suffering from the disease and were referred to us for management of their disease . thus , establishment of national or regional registries is the need of the hour to help early detection and treatment of these patients . though primary chemoprevention of fap has been attempted with sulindac and cyclo - oxygenase inhibitors , it has not proven completely successful . the surgical options available include total proctocolectomy with ileostomy ( tpc ) and restorative proctocolectomy with ileal pouch anal anastomosis ( rpc - ipaa ) , which ensures elimination of all rectal and colonic mucosa . however , tpc mandates a permanent ileostomy , which is not a desirable option in these predominantly young patients . the laparoscopic approach would seem a logical choice in these patients due to the improved cosmetic benefit . however , laparoscopic total colectomy is a most complex and demanding procedure in laparoscopic colorectal surgery , requiring significant experience in advanced laparoscopic surgery . surgeons have to operate in all the quadrants of the abdomen and should have a thorough knowledge of anatomy of these regions before performing this procedure . though the initial reports indicated that laparoscopic colectomy took a significantly prolonged time to accomplish compared with open colectomy , with increasing experience , this difference has been reduced . various studies have shown that laparoscopic surgery is associated with decreased analgesic requirement , earlier return of bowel function , reduced length of hospital stay , earlier return to routine work and improved cosmesis compared with open surgery . restorative proctocolectomy with ileal pouch anal anastomosis ( rpc ipaa ) was first described in 1978 . since then , this procedure has become the procedure of choice in the surgical management of patients with ulcerative colitis ( uc ) and familial adenomatous polyposis ( fap ) . this is the preferred approach , especially in this subset of patients who are usually young and hence appreciate the cosmetic advantage and have a better body image . experience gained in performing partial and segmental colectomies helps in mastering the techniques of total colectomy . it is difficult to judge the impact of laparoscopic surgery in treatment of fap compared with conventional techniques due to a lack of adequate data in the literature . a few controlled studies have compared laparoscopic rpc - ipaa with open procedures for patients with ulcerative colitis . some of these series have also included patients with fap . most of the series have shown prolonged operative times with the laparoscopic approach compared with the open approach , except for a series by araki et al . the latter showed similar operative times in both the arms probably because only the colonic mobilization was being done laparoscopically , while the vessel transection and rectal mobilization was being done by a mini - laparotomy incision . however , with the use of advanced instrumentation like the ligasure ( valleylab , bolder , co , usa ) for vessel tran - section and harmonic scalpel ( ethicon endo - surgery , cincinnati , oh , usa ) for dissection and tissue transection , the speed of surgery increases , which is borne out by the fact that our median operating time of 225 minutes was comparable to open resection times reported in the former series . while the earlier study by schmitt et al reported a higher morbidity of 68% by the laparoscopic approach compared with 35% by the open approach , all of the later series showed lower morbidity by the laparoscopic approach . in our series , we did not encounter any significant complication except for one patient who presented with subacute intestinal obstruction 6 months after the surgery and was managed conservatively . length of hospitalization in our series averaged 8 days , which is similar to that reported by others and slightly less than for the open counterparts . however , this is not a true reflection of the need for hospitalization of these patients because our patients preferred to remain in the hospital until sutures were removed from the mini - pfannenstiel incision , and the final histopathological examination report of the specimen was ready . due to these peculiar geographical and socioeconomic factors , in spite of most ( 80% ) of our patients having started an oral diet by the second postoperative day and all of them being on oral diet by the third day , the length of hospitalization was inordinately long . in fact , cosmetic benefit alone has been shown to be of paramount importance to patients , especially as the young age group of patients is more concerned about body image . denker et al have shown that satisfaction with the cosmetic result of the scar was significantly higher with the laparoscopic rpc - ipaa compared with the open group . we had a similar experience as the cosmetic benefit of a small pfannenstiel incision ( average length of 5 cm ) was appreciated by all our patients in the postoperative surveillance questionnaire , and all of them would recommend this procedure to other patients . all the patients with malignancy underwent total mesorectal excision and after a mean follow - up of 2.1 years are free from recurrence . one of the controversies surrounding rpc - ipaa has been the development of pouch adenomas , the incidence of which has been reported to be between 8% and 60% on long - term follow - up . one of the risk factors for the development of pouch polyps is the retained rectal mucosa as a result of the stapled anastomosis . van duijvendijk et al found that the cumulative risk of developing polyps after rpc and ipaa at the anastomotic site was 31% for the stapled group and 10% for the sutured group . in contrast , polese et al did not find any increase in risk of pouch polyp formation after stapled anastomosis . though our follow - up period has not been long ( range , 1 year to 8 years , median 3.4 ) , we have not encountered pouch polyposis in any of our patients . however , we suggest a regular follow - up regime for our patients for routine screening , which consists of yearly endoscopy of the pouch with biopsy of any suspicious lesions . finally , the contentious issue is the cost factor . though no studies to date have objectively dealt with this issue , we feel that the initial higher instrument cost for laparoscopic surgery can be offset by having a dedicated laparoscopic surgical unit , use of reusable instruments , faster operative times due to availability of advanced instrumentation , and the shorter hospitalization . moreover , the cosmetic benefit and patient satisfaction afforded by the laparoscopic approach have also to be factored in . based on this , we are of the opinion that laparoscopic rpc - ipaa should be the approach of choice in the predominantly young patients afflicted by fap . we have shown that laparoscopic total proctocolectomy with ileal pouch anal anastomosis is a feasible surgery with minimal morbidity . while the laparoscopic approach offers definite cosmetic benefit as well as early return of bowel function , its real worth can only be established by comparative studies with the open approach , which , given the rarity of the condition , may be a difficult proposition . we feel that in the absence of the peculiar geographical and socioeconomic constraints that we face in our country , the length of hospitalization can also be reduced in these patients , further adding to its benefits

background : familial adenomatous polyposis is a hereditary disease characterized by the presence of thousands of colonic adenomas , which , if untreated , invariably undergo malignant transformation . because this disease manifests at a young age , the laparoscopic approach to perform surgery would be desirable due to its cosmetic benefits . we describe our experience with this procedure and review the literature on the topic.methods:this is a case series of 15 patients who underwent restorative proctocolectomy with ileo - anal pouch anastomosis for familial adenomatous polyposis between 2000 and 2007 . the salient operative steps are described.results:there were 9 males and 6 females , 32 to 52 years of age , with an average age of 44.8 years . the median body mass index was 21.5 ( range , 17 to 28 ) . rectal cancer was already present in 4 patients at the time of diagnosis . the median operating time was 225 minutes . mean blood loss was 60ml , with none of the patients requiring perioperative blood transfusion . none of the surgeries required conversion to the open approach . bowel function resumed on the second postoperative day in 12 patients and on the third postoperative day in 3 patients . the median hospital stay was 8 days . postoperatively , there was no mortality and no serious morbidity.conclusion:laparoscopic restorative proctocolectomy with ileal pouch anal anastomosis is a feasible surgery for familial adenomatous polyposis , and considering its cosmetic benefit , is a desirable option for this group of predominantly young patients .

dry eye symptoms are one of the leading reasons why patients seek eye care in the united states . the prevalence of aqueous - deficient disease is far exceeded by that of evaporative dry eye , the primary cause of which is meibomian gland dysfunction ( mgd ) , with mgd present in 86% of all dry eye patients of known cause [ 2 , 3 ] . first - line treatment of mgd usually includes some form of heat therapy , attempting to alleviate terminal duct obstruction by liquefaction of the gland contents . a primary challenge is that mgd alters the molecular composition of the meibum increasing the melting point relative to the normal body temperature . this increase results in the need for higher temperatures within the gland to liquefy obstructive material . previous in vitro and in vivo studies have demonstrated that although the temperatures required to melt the obstructive secretions are reported to range from 32 to 45c , the more severely obstructed glands present in mgd require temperatures > 40c for effective liquefaction . achieving this temperature within the meibomian glands one obstacle is that there is an approximate 5c difference in temperature between heat applied on the external eyelid surfaces and that which reaches the inner surface of the lids ( palpebral conjunctiva ) , where the meibomian glands are located . this difference is due to both dissipation of heat while passing through the lid tissues and to constant movement of blood through vasculature wicking heat away from the lids . therefore , achieving the desired temperature of 40c at the palpebral conjunctiva requires a constant heat of at least 45c be maintained on the outer lid surface , a temperature which may be both uncomfortable and risk causing thermal injury to the eyelid skin . a further conundrum is that while adequate heat is required to liquefy obstructions , the cornea is not designed to withstand temperatures much above body temperature . when subjected to heat > 40c , the corneal tissue becomes more malleable , and therefore more vulnerable to molding when exposed to any external pressure . further , the nature of the obstructive material , particularly when of longstanding , may require a higher temperature than 40c at the palpebral conjunctiva . herein , we describe a case of recalcitrant and symptomatic mgd , where lid warming was deemed necessary but had previously been ineffective , which prompted evaluation and comparison of four different commercially available heat therapies in regard to their effects on the inner surface temperatures of the eyelids . a 28-year - old woman , an optometrist , was self - referred for the evaluation of bilateral eye irritation . she reported symptoms characteristic of dry eye : stinging , foreign body sensation , and tearing . she also reported a decrease in the overall comfort of her contact lenses and decreased contact lens wear time . she was particularly concerned by a nightly burning sensation upon removal of her contact lenses . her score on the standard patient evaluation of eye dryness ( speed ) questionnaire was 11 points of a possible 28 points . she noted that her symptoms first occurred between 1 and 2 years prior to her visit and were increasing in severity . she had previously trialed warm compresses at home without success and was fearful that her dry eye symptoms would worsen and that she would lose the ability to continue wearing contact lenses . she reported no history of systemic disease and denied any current medications . on initial presentation , her eyes appeared white and quiet . fluorescein tear break - up time was 4 s for the right eye and 6 s for the left as measured by the dry eye test method . there was no corneal staining and no significant conjunctival staining detected following the instillation of two drops of sodium fluorescein , the second drop instilled 5 min after the first , followed by evaluation 1 min later . several meibum plugs , minimally protruding and obstructing the gland orifices , were observed on each lower lid . the total number of meibomian glands yielding liquid secretion , as evaluated with the meibomian gland evaluator ( tearscience , morrisville , nc , usa ) , was 4 for the right lower lid and 10 for the left lower lid . prior studies have shown that the application of external heat has limited benefits , and that not all methods are equally efficacious . we therefore questioned whether home therapies in general could achieve the temperature elevation necessary to provide benefit to this patient . as the patient was herself an optometrist and very interested in an evidence - based treatment plan , we elected to compare two commercial methods of home heat therapy for their ability to elevate the temperature at the meibomian glands under carefully controlled conditions , where the authors were present and personally attentive to all procedures . the two home therapies compared were the bruder mask ( bruder healthcare , alpharetta , ga , usa ) and blephasteam ( tha , clermont - ferrand , france ) . additionally , we elected to compare two office procedures for mgd , one with an externally applied heat source : miboflo ( mibo medical , dallas , tx , usa ) , and the second with heat applied to the inner surface of the eyelid : lipiflow ( tearscience , morrisville , nc , usa ) . the purpose of this testing was to evaluate the efficacy of the heat transfer from the device to the meibomian glands . as the meibomian glands are located adjacent to the inner surface of the eyelid , the temperature of the palpebral conjunctiva served as the primary endpoint in determination of which heat therapy offered the best possibility of successful treatment . temperature was measured immediately prior to and immediately after application of each therapy using a non - contact infrared thermometer ( innoo tech , shenzhen , china ) . the temperature of the palpebral conjunctiva was measured by manually everting the eyelid and then taking a reading as quickly as possible ( within 23 s ) to minimize heat loss . to facilitate measurements , only the right upper and lower eyelids each therapy was assessed at a different visit , with visits separated by a minimum of 4 h , to ensure that temperatures would have returned to basal levels and would not be influenced by prior treatments . the devices were tested in order of increasing complexity : bruder mask , blephasteam , miboflo , lipiflow . all therapies were performed following each manufacturer 's instructions precisely . at the first visit , the mask was heated in a microwave for 20 s and applied to the outer surface of the eyelids for a period of 10 min . prior to application , the surface temperature of the mask was variable , ranging from 38 to 48c , with the warmest areas closest to the center of the mask and the coolest at the outer edges . the patient 's eyes remained closed throughout the treatment . at the conclusion of the treatment period , palpebral conjunctival temperatures had increased from 36.7 to 38.3c and from 36.2 to 37.1c for the lower and upper eyelids , respectively . the temperature of the blephasteam itself is not known , but a prior report indicates that the temperature of the outer lid after the application can be as high as 42c . two inserts were moistened with saline solution and placed inside the mask prior to commencing the 10-min treatment . the patient 's eyes were open within the mask , allowing uninhibited blinking throughout the treatment . at the conclusion of the treatment period , palpebral conjunctival temperatures had increased from 37.0 to 39.2c and from 36.5 to 37.6c for the lower and upper eyelids , respectively . these results are consistent with previously published findings [ 11 , 12 ] . at the third visit , miboflo uses an external paddle that is heated to a temperature of 108f ( 42.2c ) . a small amount of ultrasound gel was applied to the instrument 's heated tip prior to gently massaging the outer skin of the upper and lower eyelids for a period of 12 min , as per the recommendation of the manufacturer . the patient 's eyes remained closed throughout the treatment period . at the conclusion of the treatment period , the palpebral conjunctival temperature of the lower eyelid was unchanged compared to pre - treatment measurement , remaining stable at 36.8c . the palpebral conjunctival temperature of the upper eyelid increased very minimally from 36.3 to 36.5c . upon review of the literature , no prior publications could be found describing the effects of miboflo on the temperature of the palpebral conjunctiva , and thus this case appears to be the first providing comparative data relating to the efficacy of heat transfer from the device to the inner surface of the eyelid . at the fourth visit , lipiflow is fundamentally different from the other heat therapies in that it combines heat with directional pressure [ 13 , 14 ] . the effects of this combined technology were not evaluated in this case , but rather only the efficacy of heat transfer to the palpebral conjunctiva . the patient 's eyes remained closed throughout the standard 12-min treatment . at the conclusion of the treatment period , palpebral conjunctival temperatures had increased from 37.0 to 42.0c and from 36.9 to 41.1c for the lower and upper eyelids , respectively . the average changes in palpebral conjunctival temperatures for each treatment option are represented in figure 1 . lipiflow was the only treatment that increased the temperature of the palpebral conjunctiva above 40c . since home therapies did not achieve this temperature , and whereas home treatment had not been effective in the past , the patient elected not to use any home therapy , although she was informed that there could be some additional benefit . at the progress evaluation 3 months later , the patient reported improved comfort with and without contact lenses , decreased foreign body sensation , decreased tearing , and a resolution of the burning sensation she had felt upon nightly removal of contact lenses . her score on the speed questionnaire at this visit was 4 points of a possible 28 points , an improvement compared to her score of 11 points at her initial evaluation . given that the anatomic position of the meibomian glands is adjacent to the palpebral conjunctiva , and therefore closer to the inner eyelid surface than the outer surface , it is reasonable to expect that application of heat to the inner eyelid surface would be more effective in increasing the temperature of the glands . thus , it is not surprising that lipiflow was the only therapy which elevated the temperature above the target value of 40c . the potential problem of heating the inner eyelid surface without raising the temperature of the cornea is achieved by the lipiflow activators in a unidirectional heating surface that simultaneously insulates the ocular surface [ 13 , 14 ] . as illustrated by this case , attempting to heat the inner surface of the eyelids with a heat source applied to the external lid surface has limited efficacy . there may be palliative effects seen from any of these forms of external lid surface heating , but these are likely secondary to factors other than liquefaction of the meibum and amelioration of the obstruction within the gland . while there is evidence to support that bruder mask and blephasteam are capable of elevating the temperature of the palpebral conjunctiva , there is no evidence that the miboflo is capable of doing so [ 11 , 12 ] . there may be some benefit from the use of the bruder mask or blephasteam , as both of these did raise the palpebral conjunctival temperature in this patient , although not to the desired therapeutic level of 40c [ 11 , 12 ] . these therapies should be considered as supplemental home therapy , which can be used daily for 1015 min , as supplements to office treatment to remove meibomian gland obstructions . an inherent limitation of all case reports is the evaluation of a single individual , but in this instance the patient 's interest and willingness to participate in these temperature comparisons has provided an unusually valuable opportunity . a limitation was that measurements of the palpebral conjunctiva required eversion of the target eyelid , which increases evaporation and thereby decreases temperature . ideally , the temperature of both the inner and outer eyelid surfaces would be measured continuously , so as to provide a more comprehensive evaluation of the heat transfer for each therapy . the safety and efficacy of lipiflow has also been studied extensively [ 13 , 14 ] . this case report validates these studies , but is novel in its same - subject comparison and inclusion and evaluation of miboflo .

the effects on the inner surface temperatures of the upper and lower eyelids of four commercial heat therapies were compared for an individual with recalcitrant meibomian gland dysfunction . three therapies ( bruder mask , blephasteam , and miboflo ) involved the application of heat to the external lid surface , and the fourth ( lipiflow ) applied heat to the internal lid surface . only lipiflow was effective in elevating the inner surface temperatures to the reported 40c therapeutic threshold for melting obstructed meibum .

a 38-year - old male patient reported to the department of oral medicine and radiology with complaint of pus discharge from the right lower third of face of 2 - 3 months duration . extra oral examination revealed a draining sinus surrounded by indurated granulation tissue on the right lower border of the mandible 4 cm away from the midline in the molar region [ figure 1 ] . extra oral view showing the extra oral sinus and indurated granulation tissue intraoral examination revealed caries broken right mandibular molar along with pus discharge from the same [ figure 2 ] . an orthopantomograph view was taken that revealed presence of completely developed bilateral supernumerary premolars below the first molars . right supernumerary premolar was placed near the lower border of mandible and was surrounded by intense pericoronal radiolucency of about 5 - 6 mm without sclerotic borders . the right first molar was caries broken and showed periapical radiolucency with both root apices , and track of radiolucency was running down from the apex of mesial root to involve the pericoronal radiolucency of the right supernumerary premolar . on the left side , supernumerary premolar was placed near the apex of the first molar with its follicle almost in close approximation with the root apices of the first molar . considering the clinical and radiographic findings , a provisional diagnosis of supernumerary premolar associated secondarily infected dentigerous cyst with extra oral draining sinus on the right side and supernumerary premolar occurring in close vicinity of the left first molar on the left side and taurodontism of all molars was made . intra oral view showing the carious broken first molar panoramic view showing the bilateral supernumerary premolars and taurodontism with molars the right supernumerary premolar associated with the cystic lining around its crown along with the extra oral chronic indurated tissue was surgically removed extra orally under local anesthesia . showing the surgically removed supernumerary premolars postoperative intra oral view after six weeks postoperative extra oral view after six weeks supernumerary teeth are classified according to the morphology as conical , tuberculate , supplemental , and odontoma , topographically as mesiodens , paramolar , distomolar , parapremolar , chronologically as pre deciduous , similar to permanent teeth and post permanent , or complementary and according to orientation as vertical , inverted , and transverse . the term supplemental tooth refers to extra tooth resembling the tooth of normal series in dentition . supernumerary premolars are usually of normal form and 75% are impacted and generally unerupted . in the present cases , both supernumerary premolars were unerupted and of supplemental type . cases of bilateral supplemental premolar teeth developing later than their counter parts have been reported in literature . various theories exist for different types of supernumerary . the phylogenetic theory of atavism , dichotomy theory , heredity , hyperactivity of dental lamina , aberrations during embryologic formation , progress zone , and unified etiological explanation are various theories for the developmental of supernumerary teeth . there may be no effect with the supernumerary tooth or teeth discovered either as a chance radiographic finding or following their eruption . various complications associated with the presence of supernumerary tooth are failure of permanent teeth eruption , crowding , ectopic eruption displacement , root resorption , dilaceration , loss of vitality of adjacent teeth , dental caries , periodontal abscess , gingival inflammation , sub acute pericoronitis , incomplete space closure during orthodontic treatment , and pathological problems such as dentigerous cyst formation , ameloblastomas , odontomas , and fistulae . they may also interfere in alveolar bone grafting and implant placement . because large percentage of supernumerary premolar remain impacted , unerupted , and are usually asymptomatic and because most cases are diagnosed by chance during inspection of radiograph prior to the commencement of orthodontic treatment . dentigerous cyst and root resorption have been cited in literature as frequent complication associated with supernumerary premolar . kasat , et al . , reported a case of supernumerary premolars associated with odontogenic keratocyst . bhardwaj , et al . , reported a case of bilateral supernumerary premolars present in close vicinity of permanent teeth . in the present case , left supernumerary premolar was present in close vicinity of molar that may cause its root resorption and , on the right side , periapical infection from first molar was moving down to infect the dentigerous cyst associated with supernumerary premolar and giving rise to extra oral draining sinus . although both taurodontism and occurrence of supernumerary premolars are different entity , such association has not been documented earlier . in planning the treatment alternatives for impacted supernumerary premolars , the potential risk of leaving them in situ and hazards of surgical removal of these teeth especially around the lower premolar region , where the teeth are close proximity to inferior dental and mental nerves and blood vessels should be assessed judiciously . the timing of surgical removal of supernumerary premolar is as much debated among clinicians as are treatment methods . whenever these teeth are associated with any pathological formation or when hinder eruption of or give rise to malpositioning of permanent teeth , they should be removed as soon as possible . if left in situ , periodic examination is recommended for their clinical behavior . in the present cases , both the supernumerary premolar were surgically removed because right supernumerary premolar was associated with clinical complication and left supernumerary premolar might cause root resorption as it was in close approximation with the first molar roots . extractions of supernumerary premolars should be performed carefully to prevent the damage to the adjacent permanent teeth . clinician should also be alert during surgical removal of supernumerary premolars to avoid complications of damaging mental nerve and blood vessels . recurrence of supernumerary premolars after being surgically removed has been reported in 8% of cases reviewed . . impacted supernumerary premolars exhibiting the pathological changes seen in the present case should be removed at the earliest . clinician should also be alert during surgical removal of supernumerary premolars to avoid complications of damaging mental nerve and blood vessels . appropriate follow - up with panoramic radiographs are extremely important as the recurrence of supernumerary premolars after being surgically removed has also been reported .

supernumerary teeth are extra teeth in comparison to the normal dentition . their prevalence varies between 0.1% and 3.8% . supernumeraries are more common in permanent dentition and its incidence is higher in maxillary incisor region , followed by maxillary third molar and mandibular molar , premolar , canine , and lateral incisor . the prevalence of supernumerary premolars is between 0.075 - 0.26% , and they may occur in single or multiple numbers bilateral occurrence is uncommon and large percentage of supernumerary premolars remains impacted , unerupted , and usually asymptomatic ; radiograph plays an important role in diagnosis of these . the present paper reports a case of bilaterally impacted completely developed supernumerary premolars associated with common clinical complication in unusual manner along with taurodontism of the upper and lower molars .

idiopathic hypoparathyroidism is an endocrine disorder characterized by hypocalcemia and hyperphosphatemia due to impaired secretion of parathyroid hormone ( pth ) . it may occur as part of a polyglandular autoimmune disorder or as a complex congenital defect , such as in digeorge 's syndrome . in addition , it may occur as a solitary endocrinopathy which is called isolated hypoparathyroidism . isolated hypoparathyroidism may be caused by a mutation in the pth gene , glial cells missing ( gcm)2 gene or calcium - sensing receptor ( casr ) gene ( 1 , 2 ) . gain - of - function mutations in the casr gene cause familial hypocalcemia ( 3 ) . here we report a korean family with two affected siblings and their phenotypically silent father , who were found to carry mutations in the casr gene . a 24-yr - old woman was seen in the endocrinology clinic at samsung medical center because of transient numbness and periodic paralysis . the patient experienced occasional , brief episodes of paralysis during exertion that resolved with rest . mild numbness and tingling of the hands and feet were also present intermittently . on examination , the patient appeared well . her vital signs were normal ; her height was 155 cm , and her weight was 44 kg . laboratory tests revealed hypocalcemia ( 7.3 mg / dl ; reference range 8.4 - 10.2 ) , hyperphosphatemia ( 5.7 mg / dl : reference range 2.5 - 4.5 ) , decreased 1,25-dihydroxycholecalciferol ( [ 1,25(oh)2d ] 13.2 pg / ml : reference range 25.1 - 66.1 ) , and decreased ipth ( 4.9 pg / ml : reference range 10 - 65 ) . 25-hydroxycholecalciferol ( [ 25(oh)d ] 20.2 ng / ml : reference range 11 - 70 ) and 24-hr urinary calcium excretion was normal as were bone densitometry , thyroid functions tests , and radiographs of the kidney , ureter , bladder ( kub ) and skull . the patient was treated with calcium carbonate and alfacalcidol with resolution of symptoms and dosages were adjusted to maintain a serum calcium level within the lower end of the normal reference range . the older brother of the proband had a history of generalized seizures since he was 20-yr - old for which he was seen by a neurologist at an outside hospital . initial evaluation revealed a serum calcium concentration of 7.5 mg / dl ( reference range 8.4 - 10.2 ) , a serum phosphorus concentration of 6.1 mg / dl ( reference range 2.5 - 4.5 ) , and a serum magnesium concentration of 1.9 mg / dl ( reference range 1.9 - 2.5 ) . the serum concentration of ipth level was 6.2 pg / ml ( reference range 10 - 65 ) . he was treated with an antiepileptic medication and calcium carbonate , but seizure activity persisted . the laboratory test on admission showed a serum calcium concentration of 7.1 mg / dl ( reference range 8.4 - 10.2 ) , a serum phosphorus concentration of 5.6 mg / dl ( reference range 2.5 - 4.5 ) , and a serum ionized calcium concentration of 0.92 mm / l ( reference range 1.05 - 1.35 ) . brain magnetic resonance imaging showed non - physiologic calcifications in the basal ganglia , bilateral frontal lobes , and cerebellum . he reported subsequent absence of seizure activity during follow - up . during follow - up the calcium level increased up to 8.3 mg / dl ( reference range 8.4 - 10.2 ) and the ionized calcium level increased up to 1.0 mm / l ( reference range 1.05 - 1.35 ) . after seizure activity subsided , he is followed - up by the physician near the home . although the parents of patients denied symptoms attributable to hypocalcemia , they agreed to evaluation . laboratory examination of their father revealed hypocalcemia , hyperphosphatemia , and an inappropriately low pth level . the remaining members of the family were not included in this study because of inaccessibility ( fig . 1 ) . after we obtained informed consent , the proband and her family members were examined to detect the casr mutations by direct sequencing analysis . genomic dna was extracted from blood using a wizard genomic dna purification kit ( promega , madison , wi , usa ) . each of the 6 exons of the casr gene were amplified by polymerase chain reaction using appropriate intronic primer sets designed by the authors ( table 2 ) . cycle sequencing was performed with a bigdye terminator cycle sequencing ready reaction kit ( applied biosystems , foster city , ca , usa ) on the abi-3100 genetic analyzer ( applied biosystems ) . direct sequencing showed that all affected family members have a c to t transition at nucleotide 662 resulting in a pro221leu missense mutation in exon 3 of the casr gene ( fig . after we obtained informed consent , the proband and her family members were examined to detect the casr mutations by direct sequencing analysis . genomic dna was extracted from blood using a wizard genomic dna purification kit ( promega , madison , wi , usa ) . each of the 6 exons of the casr gene were amplified by polymerase chain reaction using appropriate intronic primer sets designed by the authors ( table 2 ) . cycle sequencing was performed with a bigdye terminator cycle sequencing ready reaction kit ( applied biosystems , foster city , ca , usa ) on the abi-3100 genetic analyzer ( applied biosystems ) . direct sequencing showed that all affected family members have a c to t transition at nucleotide 662 resulting in a pro221leu missense mutation in exon 3 of the casr gene ( fig . in recent years , important advances in endocrinology have resulted from the application of the methods of molecular biology . these advances have helped not only to demonstrate the roles of mutant genes in the etiology of some inherited disorders , but have also helped to identify the chromosomal locations of susceptibility genes that predispose individuals at risk for a particular disorder such as familial hypocalcemia . the results of the studies on the molecular genetics of the hypoparathyroid disorders have helped to elucidate some of the underlying pathways that are involved in calcium homeostasis ( 1 ) . theoretically , hypocalcemic disorders can be caused by deficiency of pth , by a defect in the pth / pthrp receptor , or by insensitivity to pth caused by defects downstream of the pth / pthrp receptor . the mutations in several genes such as casr ( 4 ) , pth ( 5 ) , gcm2 and gata3 ( 6 ) have been shown to cause hypoparathyroidism . among them gata3 mutation is inherited autosomal dominantly , but it usually accompanied by multiple anomalies such as deafness and renal impairment ( 1 , 2 ) . proband and her family member had hypocalcemia in conjunction with low pth level , and a defect in the pth / pthrp receptor and insensitivity to pth could be excluded . the absence of associated anomalies could partly exclude the mutations in gcm2 and gata3 genes . in 1993 , it was discovered that the mutation of the casr gene causes familial hypocalciuric hypercalcemia and severe neonatal hyperparathyroidism . subsequently , it was learned that a loss - of - function mutation causes hypercalcemia and gain - of - function mutation causes hypocalcemia ( 4 ) , which is called autosomal dominant hypocalcemia ( adh ) . the casr gene is located on chromosome 3q13.3-q21 , and it encodes a cell surface protein of 1078 amino acids that is expressed in the pth - producing chief cells of the parathyroid glands and also in cells lining the kidney tubule . the casr belongs to the family of g - protein - coupled receptors ( 3 , 7 , 8) . the casr in the parathyroid gland detects small changes of extracellular calcium concentration and regulates transcription of the pth gene . the casr that is expressed in the distal renal tubule regulates renal calcium excretion ( 1 ) . thus , the casr is vital to maintaining normal serum calcium levels ( 9 ) . loss - of - function mutations in the casr gene are associated with a severe neonatal hyperparathyroidism and familial hypocalciuric hypercalcemia . gain - of - function mutations in the casr gene are associated with a familial hypocalcemia ( 3 ) . since pollack et al . reported an activating mutation in the n - terminal , extracellular domain of the casr gene ( 4 ) , several activating mutations in the casr gene have been reported ( 9 ) . currently , there is a database of mutations of casr available on the internet ( http://www.casrdb.mcgill.ca/ ) which lists all the known casr mutations and their effects ( 10 ) . pro221leu missense mutation was first reported in 2000 , and the mutation is located in a lesion of the extracellular domain of the receptor . replacement of a large , rigid proline with a smaller and more flexible leucine residue , potentially changing the conformation of the calcium - binding region to resemble that of bound calcium or facilitating the binding of calcium ions , resulting in an active mechanism ( 11 ) . it is important to distinguish hypocalcemia due to an activating mutation of casr from isolated hypoparathyroidism because adh patients can develop nephrocalcinosis and renal impairment during treatment with calcium and vitamin d. the distinction between isolated hypoparathyroidism and adh may be difficult to make on based on clinical information alone . therefore , mutational analysis of the casr gene can be considered to assess the risk of nephrocalcinosis during treatment of pth - deficient hypoparathyroidism ( 12 ) . in conclusion , although a case of familial benign hypocalciuric hypercalcemia due to a loss - of - function mutation in the casr gene has been reported in korea ( 13 ) , a gain - of - function mutation of the casr gene that causes familial hypocalcemia has not yet to be reported in korea . here we have described the first korean family with adh with a review of the literature .

hypoparathyroidism is an abnormality of calcium metabolism characterized by low serum levels of parathyroid hormone in spite of hypocalcemia . the causes of hypoparathyroidism are numerous . activating mutations in the calcium - sensing receptor ( casr ) gene are well - known causes of familial isolated hypoparathyroidism , also known as autosomal dominant hypocalcemia ( adh ) . here we describe members of a korean family with a heterozygous pro221leu mutation causing adh . this case is the first report in korea .

, these lesions may remain totally asymptomatic but they frequently lead to symptoms and complications including infection or compression of the airways and/or heart . , most bronchogenic cysts were identified late in childhood either as an incidental finding or following complications . antenatal diagnosis has altered the treatment paradigm by enabling early intervention , but the optimal strategy has not been defined . the purpose of this study was to describe an approach to surgical management of bronchogenic cysts based on the natural course observed from the time of antenatal screening to surgical resection in patients treated at our institution and reported in the literature . this retrospective study included all bronchogenic cysts diagnosed antenatally at the timone university children 's hospital from january 2007 to may 2010 . the clinical history of these children was reviewed including the following items : date of diagnosis , cyst location , size and growth evolution , eventual complications , and indication / date of surgery . cyst size was defined as the maximal diameter along a single axis . for radiological diagnosis , bronchogenic cyst was defined as a single , homogeneous , well - limited , fluid - filled mass located in the mediastinum close to the tracheobronchial tree or esophagus . during the study period , a total of twelve children were treated for bronchogenic cyst including sixin whom diagnosis was achieved antenatally . four children that were treated surgically were excluded because diagnosis was not achieved antenatally and two because subsequent post - natal evaluation demonstrated cystic adenomatoid malformation . one infant in whom antenatal diagnosis was cystic adenomatoid malformation but postnatal diagnosis demonstrated bronchogenic cyst could not be included because complete data was not available . clinical data and management in the six infants included in the study there were five boys and one girl ranging in age from five to thirty - one months . in one patient , antenatal diagnosis was achieved by morphological ultrasound during the second trimester of pregnancy . in the remaining five patients , diagnosis was made during the third trimester . in all cases , antenatal magnetic resonance imaging ( mri ) after birth , computed tomography ( ct ) scan was carried out at one month in all cases and repeated at one year in cases 1 , 2 and 3 and at six months in cases 4 , 5 and 6 . in cases 2 , 4 , 5 and 6 , overall , antenatal screening for pulmonary malformations led to a correct diagnosis in 62.5% ( 5/8 ) of cases ( patients 1 , 2 , 4 , 5 and 6 ) , false - positive diagnosis in two ( patients 7 and 8) , and false - negative diagnosis in one ( patient 3 ) . the bronchogenic cyst was located under the carina in three cases ( patients 1 , 3 and 4 ) , near the left pulmonary artery in one ( patient 2 ) and behind the left superior lobe in two ( patients 5 and 6 ) . cyst diameter at one month ranged from five to twenty - two millimeters . for four children ( patients 1 , 2 , 4 and 5 ) , imaging depicted no change in cyst size between the time of antenatal detection and ct scan at one month of life . conversely , cyst size increased 40% in patient 3 and 150% in patient 6 . during the first year of life , cyst size remained stable in four ( patients 1 , 3 , 5 and 6 ) , doubled in one ( patient 2 ) , and increased by 30% within six months in one ( patient 4 ) [ figure 1 ] . growth of bronchogenic cysts ( in mm ) from antennal diagnosis to the time of surgery the only adverse event observed was pulmonary infection that occurred in only one case ( patient 3 ) at eleven months and was treated at another hospital . no other adverse events occurred in this child , but follow - up examination with pulmonary x - ray at one year depicted stretching of the left bronchus and persistent tracheobronchial tree compression on the ct scan . elective resection of the stable cyst was carried out at twenty two months . in two cases ( patients 5 and 6 ) , surgical resection was indicated at six months for emphysema of the left superior lobe and rapid cyst growth . in two cases ( patients 2 and 4 ) , resection was performed at 16 months for emphysema of the left superior lobe and rapid cyst growth . all specimens exhibited ciliated epithelium associated , in some cases , with a cartilaginous component inside the cyst . the purpose of this study was to describe the natural history of bronchogenic cysts between antenatal diagnosis and postnatal management , as a basis for planning therapeutic strategy . the first finding was a high rate of error in antenatal diagnosis with wrong conclusions being drawn in three out of eight children ( 37.5% ) . reported accurate antenatal ultrasound diagnosis in 77 to 100% of cases with better performance of equipment and interpretation of images . antenatal mri provides better identification of pulmonary malformations including congenital cystic adenomatoid malformation ( ccam ) , pulmonary sequestration , bronchogenic cyst , but reported error rates still range from 6 to 16%.[36 ] bronchogenic cysts appear as well delineated , round , homogeneous , fluid - filled , unilocular masses . in 85% of cases , differential diagnosis is difficult with several entities including ccam , esophageal duplication , emphysema , and neurenteric cyst . in accordance with previous reports , our series confirms the need for postnatal confirmation by ct - scan that allows detailed study of blood supply and connections with tracheobronchial tree . if necessary to confirm diagnosis , mri can also be performed . if respiratory status allows , postnatal imaging to confirm etiology and determine anatomic relations with the tracheal - bronchial tree and adjacent structures can be carried out at one month of life without anesthesia . our approach consists of performing simple pulmonary x - ray at one month followed by ct - scan at one year of life or if complications occur . we agree with previous authors that yearly ct scan in small children is contraindicated due to risks associated with repeated radiation exposure . another interesting finding of this study involved cyst growth during the first year of life . the cyst increased 1.3 to 2 fold in two patients and remained stable in four . considering a cyst as a sphere ( volume = 4/3r ) , a 0.5 fold increase in size would correspond to a 14-fold increase in volume . two patientsunderwent resection due to rapid cyst growth from one and two centimeters antenatally to about threecentimeters at the time of resection . in patient 2 , cyst size increased ( five to twelve centimeters ) but the indication for surgery was presence of emphysema . to our knowledge , no previous studies have provided data on cyst growth . in their series including twenty two children operated on for bronchogenic cyst at ages ranging from one month to nine years , j.l michel et al . only indicated cyst size at the time of the surgery : three to four centimeters at four years , of age , three centimeters at six years and fivecentimeters at nine years . in studies including adults , based on these findings , it can be speculated that cysts have an exponential growth from about one to two centimeters at birth to ten centimeters in adulthood . this suggests that early surgery is indicated and that the best time is during the second year of life . this timing is supported by the facts that early intervention is possible since no parenchymal resection is required and that larger thoracic size in the second year of life greatly facilitates surgery . in this regard , thoracoscopic resection is often possible because larger cyst size decreases the risk of damaging the bronchus . early surgery may reduce the risk of wide resection that led to lobectomy in nine of the thirteen patients in the series of takeda et al . only one developed infectious symptoms at one month old . in the 13-patient series of takeda et al . , only one child who presented mediastinal deviation on the pulmonary x - ray at birth developed symptoms , i.e. , dyspnea , in the first year . the other twelve children who underwent resection at later times ranging from two to ten years did not present repeated infectious symptoms until the last months before surgery . in their comparison of bronchogenic cysts in childhood and adulthood , ribet et al . reported that occurrence of symptoms and compressive complications was more common in children and more strongly correlated with location than size . no case of malignant transformation has been reported in the literature and the dysplasia rate in adult cases has ranged from 15 to 30% . our series is too small to draw any conclusions about the most appropriate follow - up strategy in children that undergo resection for bronchogenic cysts . however , perusal of previous reports on congenital pulmonary malformations highlights a consensus for yearly pulmonary ct - scan . our approach after antenatal diagnosis calls for ct - scan without anesthesia at one month after birth to confirm the malformation followed by chest x - ray every three months to rule out parenchymal complications . based on our experience and perusal of the literature , we have concluded that the growth rate of bronchogenic cysts is slow in the first months of life , but that , even in the absence of complications , size increases constantly at an exponential rate . we recommend complete resection before the age of two in order to prevent complications and facilitate surgery . patients should be kept under regularly surveillance even though most bronchogenic cysts are now diagnosed antenatally .

purpose : the purpose of this study was to describe an approach to surgical management of bronchogenic cysts based on the natural course observed from the time of antenatal screening to surgical resection in patients treated at our institution and reported in the literature.materials and methods : we retrospectively reviewed the clinical features of all children presenting bronchogenic cyst diagnosed antenatally from 2007 to 2010 . a total of six children were included.results:antenatal diagnosis was accurate in 62.5% of cases . in the first year of life , the size of the cyst remained stable in four patients , doubled in one , and increased 30% within six months in one . the indication for surgery was emphysema of the left bronchus in two patients and rapid growth in two patients . one patient is still awaiting surgery.conclusion:bronchogenic cysts grow slowly in the first months of life , but growth is exponential even in the absence of complications . we recommend complete resection before the age of two years to prevent infectious complications and facilitate surgery .

the daiko medical center opened as a restructured clinic succeeding the nagoya university branch hospital in 1996 on the daiko campus . in june , 2004 , preventive medical services not covered by public health insurance began for eradication treatments of helicobacter pylori ( h. pylori ) , smoking cessation support , advice on alcohol consumption , folate - associated diseases and a tumor marker ca19 - 9 with genotype tests , as well as chemoprevention for breast cancer with raloxifene . at that time , we expected that a substantial number of citizens might seek those preventive medical services . since those services are not covered by public health insurance in japan , all the attendant costs have to be paid by patients , whereas only 30% of the costs are charged to patients in cases of medical services covered in japan . six years and 10 months after its start , the center closed in march of 2011 due to the limited number of visitors seeking preventive services . we report the number of patients according to each service , the reason for eradication treatment visits , and the final outcome of eradication treatments . since the reports on uninsured health services in japan were very scarce , some documentation on the center s performance was thought to be meaningful . patients who visited the daiko medical center from june , 2004 to march , 2011 were tracked according to the following medical services received : 1 ) h. pylori diagnosis and eradication treatments , for which cyp2c19 genotyping was introduced from november 2005 ; 2 ) smoking cessation support with genotype tests for cyp1a1 ile462val , gstm1 present / null , gstt1 present / null , and nqo1 pro187ser ; 3 ) advice on alcohol consumption with genotype tests of adh arg47his and aldh2 glu487lys ; 4 ) consultation on folate - associated diseases with a genotype test of mthfr c677 t ; 5 ) consultation on a tumor marker ca19 - 9 with genotype tests of lewis and secretor genes ; and 6 ) raloxifene prescription to prevent breast cancer for high - risk postmenopausal women . fees for insured medical services are fixed throughout japan by the government , with 30% of them being charged to the patient . on the other hand , the fees for uninsured medical services can be set by each medical facility , with patients having to pay 100% of the fees . in our center , the fees for a particular service are arranged to be the same as the corresponding fees when provided as a medical service covered by public health insurance , as described in the fee schedule . accordingly , the actual fees our patients had to pay at the center were about three times ( 100% vs. 30% of the fee ) higher than those under public health insurance . for example , the first visit for an h. pylori infection diagnosis ( a serum anti - h . pylori antibody test and a urea breath test ) cost about 16,000 yen , which would only be about 4,800 yen under public health insurance . in cases where the eradication was successful , the total costs came to about 30,000 yen for three visits ; a diagnosis at the first visit , medication at the second visit , and a test after the medication at the third visit . for other services , about 2,900 yen at the first visit and about 1,300 yen at the second visit or thereafter were charged as the basic rate . patients were not charged for any genetic tests that we ourselves conducted at the center . the percentage differences between two groups were examined by a chi - square test . statistical analysis and tabulation were performed using stata / mp 11 software ( statacorp lp , texas , usa ) . patients who visited the daiko medical center from june , 2004 to march , 2011 were tracked according to the following medical services received : 1 ) h. pylori diagnosis and eradication treatments , for which cyp2c19 genotyping was introduced from november 2005 ; 2 ) smoking cessation support with genotype tests for cyp1a1 ile462val , gstm1 present / null , gstt1 present / null , and nqo1 pro187ser ; 3 ) advice on alcohol consumption with genotype tests of adh arg47his and aldh2 glu487lys ; 4 ) consultation on folate - associated diseases with a genotype test of mthfr c677 t ; 5 ) consultation on a tumor marker ca19 - 9 with genotype tests of lewis and secretor genes ; and 6 ) raloxifene prescription to prevent breast cancer for high - risk postmenopausal women . as a general rule , fees for insured medical services are fixed throughout japan by the government , with 30% of them being charged to the patient . on the other hand , the fees for uninsured medical services can be set by each medical facility , with patients having to pay 100% of the fees . in our center , the fees for a particular service are arranged to be the same as the corresponding fees when provided as a medical service covered by public health insurance , as described in the fee schedule . accordingly , the actual fees our patients had to pay at the center were about three times ( 100% vs. 30% of the fee ) higher than those under public health insurance . for example , the first visit for an h. pylori infection diagnosis ( a serum anti - h . pylori antibody test and a urea breath test ) cost about 16,000 yen , which would only be about 4,800 yen under public health insurance . in cases where the eradication was successful , the total costs came to about 30,000 yen for three visits ; a diagnosis at the first visit , medication at the second visit , and a test after the medication at the third visit . for other services , about 2,900 yen at the first visit and about 1,300 yen at the second visit or thereafter were charged as the basic rate . patients were not charged for any genetic tests that we ourselves conducted at the center . the percentage differences between two groups were examined by a chi - square test . statistical analysis and tabulation were performed using stata / mp 11 software ( statacorp lp , texas , usa ) . from june , 2004 to march , 2011 , 683 subjects ( 293 males and 390 females ) visited the daiko medical center . among them , 26 individuals sought two or more of the six services . table 1 shows the total number of patients according to the preventive service to be 683 . among them , eighty - three percent visited for diagnosis and eradication treatments for h. pylori . number of patients according to preventive services received , who visited the daiko medical center from june , 2004 to march , 2011 the reasons patients visited for h. pylori diagnosis and eradication treatments are shown in table 2 . the most frequent reason was h. pylori infection diagnosed at other facilities ( 45.1% ) , followed by gastrointestinal symptoms ( 36.5% ) , family history of gastric cancer ( 20.3% ) , and anxiety aroused by tv programs featuring h. pylori ( 18.2% ) . significant differences in the percentage between males and females were observed for items regarding h. pylori infection diagnosed at other facilities ( =5.84 , p=0.016 ) , past diagnosis of gastric ulcer and/or duodenal ulcer ( =4.44 , p=0.035 ) , and urticaria ( =4.36 , p=0.037 ) . reasons for visits for h. pylori diagnosis and eradication treatments ( listed by percentage ) * two or more reasons for visits were allowed . * * significant difference between males and females ( p<0.05 ) figure 1 shows the number of patients visiting for h. pylori diagnosis and eradication treatments between june , 2004 and march , 2011 . around 2004 , public interest in h. pylori infection was generally high ; tv programs , newspaper articles , and public lectures on h. pylori were relatively common . the opening of our h. pylori clinic was reported in the press on july 16 , 2004 . however , patients at the center decreased , possibly due to reduced public interest and/or the increased opportunity for h. pylori eradication under public health insurance . between june , 2009 and may , 2010 , we enrolled 5,172 participants in a cohort study at the daiko medical center . in that study , a free urinary h. , 112 sought h. pylori eradication at the center , making 2009 the patient peak ( fig . there were no incentives to encourage people to seek h. pylori diagnosis and treatments at the center , resulting in a drastic reduction in 2011 . number of patients who visited the center for h. pylori diagnosis and eradication treatments between june , 2004 and march 2011 we previously reported the h. pylori eradication rate for first - time treatments during the period before the introduction of a routine cyp2c19 genotype test and during the period after its introduction . before the introduction , a first - line regimen ( lansoprazole , clarithromycin , and amoxicilline ) after the introduction , a second - line regimen ( rabeprazole , metronidazole , and amoxicilline ) was prescribed as a first - time treatment for those with cyp2c19 * 1 * 1 ( rapid metabolizers ) , and a first - line regimen for the others . the final eradication rate was 80.0% ( n=90 ) for the period before the test introduction , and 88.9% ( n=144 ) for the period after it ; the difference was marginally significant ( =3.52 , p=0.06 ) , which was similar to a previous result ( p=0.08 ) . the eradication rate was 100% ( n=53 ) among those with cyp2c19 * 1 * 1 treated by the second - line regimen ( table 3 ) . eradication rate during periods without ( june , 2004 to october , 2005 ) and with ( november , 2005 to march , 2011 ) a routine genetic test for cyp2c19 * untested for cyp2c19 before prescription * * examined for h. pylori infection * * * the parencethese shows rates excluding those unevaluated the second most frequent patients ( 6.4% ) were 43 smokers ( and one former smoker ) who sought cessation support , including a nicotine patch or genotyping related to their susceptibility to carcinogens . from may 31 , 2008 , when the nicotine patch became available at pharmacies without a prescription , the role of the center diminished as a result . moreover , smoking cessation support ( including the prescription of a novel medicine , varenicline ) under public health insurance started in may 8 , 2008 . among 44 patients , 35 requested genotyping . in the follow - up of 25 smokers who agreed with a questionnaire survey three months after their first visit , nine participants out of 16 responders ( 36.0% out of 25 smokers ) reported quitting at the time of their response . the demand for genotyping of alcohol metabolism was small ; most were from those who can not drink alcoholic beverages to confirm their genotype of aldh2 glu487lys . the great majority of those who sought mthfr c677 t genotyping were patients and their families with oral clefts , or parents with a spina bifida child . in those cases , a specialist ( teaching staff ) together with a licensed midwife attended them throughout the process . those who visited the center for lewis and secretor genotyping were patients with elevated ca19 - 9 , all of whom had le allele and sese genotype . two of them actually sought raloxifene after an explanation of its effect , but the prescription was given for only one time . in japan , medical services not covered by public health insurance are occasionally provided for cosmetic surgery , normal childbirth , private special consultation , some dental services , etc . uninsured health services are often regarded as inappropriate , and are only provided to make money off of ineffective or unevaluated treatments . however , h. pylori eradication was one of the exceptions . the japanese society for helicobacter research approved uninsured medications for h. pylori eradication , and listed the medical facilities providing them on their homepage ( http://www.jshr.jp/ ) . although the number of patients treated in this framework has not been reported , the number at the daiko medical center seemed to our knowledge to be one of the highest in japan . the reasons to visit the center were mainly the past - positive tests of h. pylori without subsequent eradication treatment , and gastrointestinal symptoms with no occasion for h. pylori tests . at that time , h. pylori diagnosis and eradication were covered by public health insurance only for those with current peptic ulcers . accordingly , hospitals or clinics could not usually provide such services for those without current peptic ulcers under the public health insurance system . in addition , uninsured medical service in hospitals / clinics providing insured medical service might result in a mixed practice ( kongou shinryou , in japanese ) , which is prohibited in japan under financial penalties . since the daiko medical center did not provide insured medical services , we were free from any criticism or penalties regarding so - called mixed practice . the improvement of the eradication success rate for first - time treatment through a genetic test for cyp2c19 at the center was reported in our previous paper . since the report was on those who visited the center until august 2010 although the improved eradication success rate was still marginally significant ( p=0.06 ) , there seemed to be a possibility that the introduction of a genetic test would improve the success rate of those receiving eradication treatments for the first time . however , in our trial , we found no problems related to genetic testing ; no cases needed genetic counseling , as experienced in previous studies . the responses of those who seek genetic tests for reasons unrelated to disease severity might differ from responses of those seeking help for severe diseases . a distinction in the approach between genetic tests of useful polymorphisms and those for severe hereditary diseases seemed to be necessary . we expected that there would continue to be a large demand for the six services at the center . however , even for h. pylori eradiation services , which were relatively popular among people , the demand did not continue . the center s services might have been expensive for ordinary persons , although there were no available data on this issue . a lingering challenge is that preventive services are not covered by public health insurance in japan . other trials will be needed to find optimal conditions for medical / preventive services not covered , and to provide novel medical services which people can seek even if they are not covered .

abstractpreventive medical services not covered by public health insurance started in the daiko medical center of nagoya university in june , 2004 . those services included : 1 ) helicobacter pylori ( h. pylori ) diagnosis and eradication treatments , for which cyp2c19 genotyping was introduced in november 2005 ; 2 ) smoking cessation support with genotype tests of cyp1a1 ile462val , gstm1 present / null , gstt1 present / null , and nqo1 pro187ser ; 3 ) advice on alcohol consumption with genotype tests of adh arg47his and aldh2 glu487lys ; 4 ) advice on folate - associated diseases with a genotype test of mthfr c677 t ; 5 ) advice on a tumor marker ca19 - 9 with genotype tests of lewis and secretor genes ; and 6 ) raloxifene prescription aimed to prevent breast cancer for high - risk postmenopausal women . a total of 683 patients visited the center until it closed in march 2011 . those given diagnoses and eradication treatments for h. pylori numbered 567 , followed by 44 for smoking cessation support , 35 for advice on folate - associated diseases , 26 for advice on alcohol consumption , 8 for ca19 - 9 , and 3 for raloxifene prescription . around 2004 , public interest in h. pylori was relatively high , but thereafter patient numbers dropped markedly . the center closed in march 2011 due to the reduction in patient visits . our unique trial showed that continuing to provide uninsured preventive services at a clinic was difficult in japan without the affiliation of hospitals / clinics providing medical services covered by public health insurance .

allergic conjunctivitis is the most common type i ige - mediated hypersensitivity reaction to environmental allergens involving the ocular surface , affecting approximately 25% of the general population and 30% of allergy - predisposed children.1 symptoms of allergic conjunctivitis include ocular pruritus , tearing , conjunctival edema , hyperemia , watery discharge , burning , and photophobia.2 treatment of allergic conjunctivitis involves topical and sometimes oral therapy as well as allergen avoidance . the current literature is limited in terms of the association between sinus disease and chronic allergic conjunctivitis.3,4 while in other ophthalmic symptomatology ( proptosis , epiphora , deep orbital pain , double vision),58 physicians often look to the adjacent sinus for the etiology , such an analysis is often overlooked in the patient with severe , unremitting allergic conjunctivitis refractory to traditional medical management . in particular , paranasal sinus mucocele has not been well documented as a possible cause of conjunctivitis , particularly in children . to our knowledge , there is only one case reported of a mucocele leading to chronic blepharoconjunctivitis and this was in an adult patient ( the adult population is more likely to have mucoceles than the pediatric population).8 more common ophthalmic symptoms of these benign , epithelial - lined , mucus filled sacs include proptosis , visual disturbances , motility defects , and pain due to mass effect.38 mucoceles have been documented in association with systemic diseases , such as cystic fibrosis ( cf ) , with a prevalence of 16% in cases of cf with symptoms of chronic upper airway disease . in addition , patients with cf generally have a large number of bacteria in their sputum and nasal secretions9 and therefore have a higher risk of infection ( mucopyocele).10 in cases of infection , drainage of the mucopyocele is indicated to ensure eradication of the organism that can have significant pulmonary consequences . identification and diagnosis of the mucocele is critical in helping maintain the pulmonary health of such patients , which can be difficult in atypical cases without symptoms of nasal obstruction . we present herein a case of a 3-year - old cf patient misdiagnosed for 2 years with chronic allergic conjunctivitis whose symptoms were caused by an ethmoidal mucopyocele infected with escherichia coli . a 3-year - old boy under pediatric pulmonary care for cf presented to the bascom palmer eye institute , university of miami , with a 2-year history of chronic bilateral conjunctival inflammation , mild epiphora , and irritation . despite bilateral disease , the patient was evaluated in multiple subspecialty ophthalmology clinics both in the united states and internationally and in each , he was diagnosed with allergic conjunctivitis . his parents denied symptoms of nasal obstruction , other than snoring , which had been present prior to the onset of the eye symptoms . treatment regimens , including emergency room treatment , involved the use of topical anti - allergy drops such as olopatadine ( patanol ) which improved the swelling only temporarily . he was referred to us for further evaluation . clinical examination disclosed erythema in the medial aspect of the orbit , lower lid greater than upper eyelid , a palpable prominence under the medial canthal tendon and in the region of the nasolacrimal sac , and telecanthus on the right ( figure 1 ) . he had a normal tear lake and no mucopurulent reflex with compression of the nasolacrimal sac . past medical history was notable for cf ; pancreatic insufficiency had been appropriately managed with pancreatic enzymes . additionally , he was noted to have chronic airway ( deep throat culture based ) colonization with e. coli , an organism seen infrequently in young children with cf . despite multiple attempts to eradicate it with nebulized tobramycin and oral antibiotics , the organism persisted . a decision was made against long - term antibiotics , as e. coli is not considered to be a gram - negative organism that predicts an adverse pulmonary outcome in patients with cf . magnetic resonance imaging ( mri ) of the brain was obtained to further evaluate the medial canthal prominence . in the orbits , an area of mass effect was seen in the canthal region of the right orbit . this area extended inferiorly to the level of the inferior turbinate and involved a large area of the ethmoid sinus on the right side . the mass had high signal intensity on t1 and low signal on t2 weighted images , and there was ring - like enhancement ( figure 2a c ) , most consistent with an ethmoidal mucocele . the patient underwent functional endoscopic sinus surgery at the university of miami with marsupialization of the right ethmoidal mucocele ( figure 3a and b ) , which was later confirmed by culture to be infected with e. coli . after post - operative follow - up and continuous pulmonary care , the patient showed no symptoms of allergic conjunctivitis , the telecanthus and medial canthal mass resolved , and the upper airway has remained free of e. coli . the association between chronic conjunctivitis and mucocele in cf patients has not been well established in the literature but is an important consideration for the ophthalmologist , otolaryngologist , and pulmonologist managing these patients . disease of the nose and paranasal sinuses occurs in almost all cf patients ; most patients develop chronic sinusitis , and often bacterial infection thereof early in the course of their disease . while there is little recognition in the cf community of the presence of mucoceles , paranasal mucocele have in fact been documented to have a prevalence as high as 16% in patients with cf with symptoms of chronic upper airway disease who underwent otolaryngology evaluation.10 interestingly , ocular or orbital findings are relatively rare in this population . only one patient in this series presented with hyperemic conjunctiva , and this patient also had nasal symptoms including persistent purulent rhinorrhea and nasal obstruction.10 other case reports document mucoceles in the pediatric cf patients with the more typical findings of nasal obstruction.11 thus , in the common cf practice , conjunctival disease is not viewed as part of the presentation of a mucocele , and its presence would not alert the cf expert to a potential underlying contiguous infectious focus . more specifically , sinus disease is often not considered in a patient who presents with symptoms of periorbital or ocular allergic disease . our patient developed chronic , refractory , and asymmetric allergic conjunctivitis as well as recurrent e. coli colonization of the respiratory tract , both of which were manifestations of an ethmoidal mucopyocele . both the recurrent airway e. coli colonization and symptoms of allergic conjunctivitis resolved post - functional endoscopic sinus surgery and he has remained stable . we hypothesize that the chronic low grade exposure to e. coli created an atypical allergic conjunctivitis . this hypothesis is supported by previous documentation of allergic conjunctivitis secondary to stimulus of the nasal mucosa , thought to be potentially related to various neuropeptides , cytokines , chemokines , chemotactic , and other factors.12 it is also possible that he in fact had a low - grade bacterial conjunctivitis given the significantly worse symptoms and signs on the eye ipsilateral to the mucocele , but this can not be confirmed as cultures of the ocular surface were not performed . e. coli is a rare cause of ophthalmic infection , most commonly in the neonate causing infectious conjunctivitis or in immunosuppressed adults and is rarely found in the normal conjunctival flora.13 the important clinical factor is that his symptoms and exam , which were most consistent with allergic conjunctivitis , resolved completely after eradication of the mucocele . it is always important to consider the adjacent sinuses when managing a patient with chronic periocular symptomatology , particularly allergic disease , even in pediatric cases . of further important note is that in the specific condition of cf , mucoceles are more common and are more likely to be infected ( ie , mucopyoceles ) , while in otherwise normal patients they are almost uniformly sterile . this is an important distinction for the patient with cf , since a chronic source of bacterial infection in the upper airway can become the source of infection spread to the lower airway , the latter infections being the major determinant of long - term outcome in such patients . our case report highlights that chronic conjunctivitis in cf patients that is refractory to standard management should lead to a thorough evaluation including bacterial cultures and in some cases neuroimaging to determine and properly treat a putative underlying cause .

this case report details an association of chronic allergic conjunctivitis and respiratory tract colonization in a cystic fibrosis ( cf ) patient due to an ethmoidal mucocele infected with escherichia coli . a 3-year - old cf patient presented for evaluation with complaints of chronic periocular erythema , conjunctival injection , and irritation for 2 years . he was treated for presumed allergic conjunctivitis with no improvement and continued to have overall worsening of symptoms on the right greater than the left eye in a waxing and waning pattern . on presentation to the bascom palmer eye institute , he was noted to have telecanthus and prominent erythema in the region of the medial canthus . orbital imaging disclosed a mucocele in the right ethmoid sinus . the patient underwent functional endoscopic sinus surgery , with successful marsupialization of the ethmoidal mucocele , which was found on culture to be infected with e. coli . post - operatively with continuous pulmonary care , the patient remains free of allergic conjunctivitis and e. coli colonization of the upper airway . this case highlights the importance of analyzing the adjacent sinus in patients with chronic , relapsing allergic conjunctivitis refractory to medical management , particularly in patients with underlying systemic diseases such as cf .

spinal anesthesia is regularly performed by anesthesiologists , as well as by obstetricians and gynecologists . several complications of spinal anesthesia have been described , including hypotension , nausea , and vomiting 1 ; however , cardiac arrests during this procedure are very rare 2 . we present a case of unexpected cardiac arrest during spinal anesthesia in a young , healthy woman . a 31yearold woman ( height , 136 cm , weight , 45.6 kg ) , gravida 0 , was scheduled for cervical conization due to cervical intraepithelial neoplasia 3 . all preoperative assessments , including blood biochemistry , chest and abdominal radiography , and 12lead electrocardiogram ( ecg ) were normal . an ecg monitor , an automated blood pressure cuff , and pulse oximetry were applied in the operating room . before spinal anesthesia , the patient 's blood pressure was 118/65 mmhg and heart rate ( hr ) was 58 beats / min . with the patient in the lateral position , hyperbaric bupivacaine ( 10 mg ) was injected into the subarachnoid space at the l3l4 interspace . at 5 min , maximum sensory block up to t10 was noted . in the first 15 min , after disinfection of the vagina in preparation for cervical conization , the patient complained of nausea . cardiac arrest then occurred without her developing bradycardia . immediately , cardiopulmonary resuscitation ( cpr ) was initiated and epinephrine ( 1 mg ) was administered . approximately 2 min after the initiation of cpr , sinus rhythm was restored and the patient recovered consciousness . however , she subsequently lost consciousness again , and pulseless ventricular tachycardia ( vt ) occurred . because the patient 's ecg changed immediately from pulseless vt to cardiac arrest , we restarted cpr and administered epinephrine ( 1 mg ) . the patient was successfully resuscitated after 2 min of cpr . we canceled the operation and consulted cardiologists , who performed coronary angiography because the 12lead ecg after the revival of the patient showed cardiac infarction of the lateral wall ( fig 1 ) . however , no coronary artery disease was found , and the patient was diagnosed with coronary spastic angina . the incidence of cardiac arrest during spinal anesthesia varies from 1.3 in 10,000 to 18 in 10,000 patients 2 . according to previous reports , young and healthy patients may experience cardiac arrest in the operating room during spinal anesthesia 3 , 4 , 5 , 6 . unintentional total spinal anesthesia , myocardial infarction or conduction disorders , and reduction of venous return due to sudden hemorrhage or imbalance with background vagal dominance have been suggested as causative factors in previous reports 4 , 7 . in this patient , because cardiac arrest occurred in the setting of oxygen saturation readings of 99% , it is unlikely that unintentional total spinal anesthesia occurred . baron et al . found that cardiac vagal tone is enhanced by reduced venous return 8 , and young patients typically have a strong vagal tone 7 . thus , possible causes for cardiac arrest in this patient include a significant decrease in venous return to the heart due to the inhibition of sympathetic efferents during spinal anesthesia , enhanced vagal tone caused by a decrease in venous return , and vasovagal reflection due to nervous strain . risk factors for cardiac arrest during spinal anesthesia have been reported as follows : age < 50 years , baseline heart rate < 60 beats / min , american society of anesthesiologists ( asa ) physical status i and ii , use of beta blockers , sensory blockade above t6 level , and prolonged pr interval 7 . pollard reported that patients may be considered at high risk for cardiac arrest during spinal anesthesia when two or more of these factors are present 7 . the patient in the present case could be classified as having a greater tendency for cardiac arrest because she met three risk factors ( i.e. , 31 years old , baseline heart rate of 58 beats / min , and asa status i ) . however , patients who meet two or more of the aforementioned risk factors are not uncommon . for example , asa i or ii denotes a normal healthy patient or a patient with mild systemic disease ; therefore , many young women who are scheduled to receive spinal anesthesia for gynecological surgery or cesarean section will fall into this category . thus , medical professionals who perform spinal anesthesia should always be aware of this possibility . with regard to specific strategies for addressing the possibility of cardiac arrest during spinal anesthesia , the appropriateness of spinal anesthesia in highrisk patients should be evaluated carefully . although multiple factors may lead to cardiac arrest during spinal anesthesia , evidence suggests that a decrease in venous return is one of the most important 3 , 4 , 5 , 6 , 7 , 9 . when spinal anesthesia has been selected for a patient , maintaining an adequate preload is the key to decreasing the risk of cardiac arrest . intravascular fluid administration of mixed and agonists has also been advocated to decrease the frequency of cardiac arrest during spinal anesthesia 7 . furthermore , excessive anxiety and fear may cause vasovagal reflection , which applies to the present patient . appropriate sedation might reduce this risk 9 . worsening bradycardia ( hr of 3060 beats / min ) often precedes the onset of cardiac arrest during spinal anesthesia ; intravenous administration of atropine or ephedrine has been recommended as a treatment for this condition 4 , 10 . the effectiveness of epinephrine for cardiac arrest during spinal anesthesia has previously been reported 3 , 4 , 5 , 6 , 9 , 10 , and in our patient , the administration of epinephrine immediately after cardiac arrest restored the sinus rhythm and allowed the patient to regain consciousness . many obstetricians and gynecologists have low exposure to perioperative cardiac arrest . in remote rural areas , cesarean section is often carried out in a small clinic ; this necessitates that the treating physicians be prepared to deal with intraoperative complications such as cardiac arrest , which has been reported after spinal anesthesia for this procedure 3 , 9 . although the perioperative mortality is almost 0.001% in the world 11 , the survival rate of cardiac arrest during spinal anesthesia is reported to range from 20% to 77% 12 , 13 . cardiacarrest mortality between spinal anesthesia and other anesthesia methods in japan 14 . moreover , charuluxananan et al . 15 reported that a surgeon performing spinal anesthesia was a significant factor associated with cardiac arrest ( odds ratio , 23.5 ) . therefore , cardiac arrest is one of the complications that obstetricians and gynecologists should be most aware of during spinal anesthesia . we report a case of cardiac arrest during spinal anesthesia a complication that is not uncommon among young , healthy women . obstetricians , gynecologists , and other medical professionals who perform this procedure should be aware of the potential for this complication , and be prepared to administer treatment . in the present patient , we conclude that when making the decision to use spinal anesthesia , judicious patient selection , adequate preventive measures , and strict monitoring are very important .

key clinical messagespinal anesthesia is regularly performed worldwide and is an integral part of the modern day anesthesia practice . although unexpected cardiac arrests during this procedure are very rare , medical professionals should be aware of the potential for this complication . in making the decision to use spinal anesthesia , judicious patient selection , adequate preventive measures , and strict monitoring are important .

the severe shortage of human donor organs has stimulated interest in the potential use of animal organs for transplantation into humans . xenotransplantation , as such , would not only offer an unlimited supply of organs and tissues for transplantation , but might also provide an opportunity to carry out transplantation without the problems associated with infections or disease conditions , which would inevitably occur in human transplants . on the other hand , one essential question in xenotransplantation is whether , and to what extent , an animal organ or tissue can provide a physiologic replacement for a human organ or tissue . foreign organs and tissues might function poorly compared with their human counterparts . however , it is equally intuitive that the optimal functioning of an organ transplant is not often achieved and is rarely necessary . most of the physiological incompatibilities and species - specific differences between mammal species are unknown . targeted investigations suggest that even individuals of one species or strain may demonstrate slight genetically - based metabolic differences . this is particularly true for widely divergent species , which show significant and multiple incompatibilities due to their evolutionary development . this is not restricted to differences in molecules , hormones , or enzymes and their receptors , but also to species - specific products that will prove antigenic . therefore , the question as to whether xenogeneic donor organs will be physiologically compatible with human hosts needs to be addressed , since even the suboptimal functioning of one or several systems may cause life - threatening problems to the xenografted patient . the cardiac outputs of porcine and human hearts of similar size have been found to be similar , and their action potentials are also similar . however , a recent comparison of the anatomy of porcine and human hearts has identified several differences , many of which are due to the different stances ( walking on four legs rather than two ; unguligrade versus orthograde ) and the effect of gravity on the development of the heart . despite these differences , the transplanted organs appear to adapt very well to new hosts , for example pig hearts in monkey hosts . however , there is limited data on the functioning of the porcine heart in primates . recently , bhatti et al . reported that porcine hearts could provide physiological support in non - human primates for up to three months . the echocardiographic examination has been the cornerstone of cardiac diagnosis in a variety of cardiovascular disorders for over two decades . two - dimensional and doppler echocardiographic modalities provide valuable information on the cardiac structures , chamber function , and hemodynamic derangements . this report is the first to show that echocardiography is a much more sensitive method for assessing micropig cardiac functions for xenografts in nonhuman primates than palpation . all animal experiments were carried out in accordance with the standard operation procedures of the institutional animal care and use committe , seoul national university , korea . the studies were performed with mixed - breed , conditioned yucatan micropigs and landrace breed conventional pigs , which were purchased from pwg genetics ( korea ) . prior to their purchase , the pigs were physically examined and confirmed to be healthy . the pigs were individually housed indoors in cages , fed dry pig food , and provided with water ad libitum . the mean age of the experimental animals was 80 days for the conventional pigs and 360 days for the micropigs . the mean body weight was 30.50 0.45 kg in the conventional pigs and 32.10 1.29 kg in the micropigs ( table 1 ) . blood pressure was measured five times for each individual animal with cardell bp monitor ( sharn veterinary , usa ) in the premedicated condition with atropine ( 0.04 mg / kg i m ) , xylazine ( 2.2 mg / kg i m ) , and a zolazepam / tiletamine cocktail ( 4.4 mg / kg i m ) prior to echocardiographic examination . the animals were fasted overnight and premedicated with atropine , xylazine , and a zolazepam / tiletamine cocktail , intubated , mechanically ventilated , and maintained with isoflurane ( 1% to 5% ) inhalational general anesthesia . there is a good correlation between the body length and heart size regardless of the chest conformation . because of this relationship , the vertebral heart score ( vhs ) can be used to both determine the presence and quantify the degree of cardiomegaly in animals . the vhs was evaluated by transposing the long - axis ( l ) and short - axis ( s ) heart dimensions onto the vertebral column , and recording the number of vertebra , beginning with the cranial edge of the fourth thoracic vertebra . the vhs was calculated using the following formula : x - ray fluoroscopy was carried out to observe the cardiac blood flow of the right circumflex artery , left anterior descending artery , and left circumflex artery . an 8fr arterial sheath was then inserted through the carotid artery after local anesthesia with 2% lidocaine , and a cutdown was made . the 7 or 8fr coronary guiding catheter and guide - wire assembly in the coronary ostium was engaged under fluoroscopic guidance using a phillips c - arm system ( bv-25 gold ) . the long guide wire was used to introduce a pigtail infusion catheter , and the pigtail catheter was positioned in the left ventricle to infuse iohexol as a contrast medium . echocardiographic examinations were carried out using a cardiac ultrasound system equipped with a 5.0 - 7.5 mhz transducer ( ge logiq 7 ; ge medical systems , usa ) . the echocardiographic images were obtained in the right parasternal long- and short - axis view . the anatomy and function were measured in various cardiac regions including four cardiac chambers , valves , and great vessels by b - mode , m - mode , and spectral doppler echocardiographic tracing . the dimensions and thicknesses of the cardiac structures were measured using m - mode echocardiography according to the american society of echocardiography guidelines . the blood flow velocity , amounts of blood flow , pressure gradients across the valves , and stroke volume were measured using pulsed or continuous wave doppler echocardiography . the cardiac output was calculated by multiplying the stroke volume by the heart rate . the left ventricular ( lv ) systolic function was evaluated by measuring the ejection fraction ( ef ) and fractional shortening ( % fs ) by m - mode echocardiography according to the following formulas : ef = ( end - diastolic volume - end - systolic volume)/end - diastolic volume 100 , % fs = ( lv end - diastolic dimension - lv end - systolic dimension)/lv end - diastolic dimension 100 . all measured values used in the final calculations for each pig are the mean value from three to five sequential measurements regarded as being of the optimal quality . all of the measured values are expressed as a mean sd . the difference between two mean values was analyzed by the wilcoxon test . all animal experiments were carried out in accordance with the standard operation procedures of the institutional animal care and use committe , seoul national university , korea . the studies were performed with mixed - breed , conditioned yucatan micropigs and landrace breed conventional pigs , which were purchased from pwg genetics ( korea ) . prior to their purchase , the pigs were physically examined and confirmed to be healthy . the pigs were individually housed indoors in cages , fed dry pig food , and provided with water ad libitum . the mean age of the experimental animals was 80 days for the conventional pigs and 360 days for the micropigs . the mean body weight was 30.50 0.45 kg in the conventional pigs and 32.10 1.29 kg in the micropigs ( table 1 ) . blood pressure was measured five times for each individual animal with cardell bp monitor ( sharn veterinary , usa ) in the premedicated condition with atropine ( 0.04 mg / kg i m ) , xylazine ( 2.2 mg / kg i m ) , and a zolazepam / tiletamine cocktail ( 4.4 mg / kg i m ) prior to echocardiographic examination . the animals were fasted overnight and premedicated with atropine , xylazine , and a zolazepam / tiletamine cocktail , intubated , mechanically ventilated , and maintained with isoflurane ( 1% to 5% ) inhalational general anesthesia . there is a good correlation between the body length and heart size regardless of the chest conformation . because of this relationship , the vertebral heart score ( vhs ) can be used to both determine the presence and quantify the degree of cardiomegaly in animals . the vhs was evaluated by transposing the long - axis ( l ) and short - axis ( s ) heart dimensions onto the vertebral column , and recording the number of vertebra , beginning with the cranial edge of the fourth thoracic vertebra . the vhs was calculated using the following formula : x - ray fluoroscopy was carried out to observe the cardiac blood flow of the right circumflex artery , left anterior descending artery , and left circumflex artery . an 8fr arterial sheath was then inserted through the carotid artery after local anesthesia with 2% lidocaine , and a cutdown was made . the 7 or 8fr coronary guiding catheter and guide - wire assembly in the coronary ostium was engaged under fluoroscopic guidance using a phillips c - arm system ( bv-25 gold ) . the long guide wire was used to introduce a pigtail infusion catheter , and the pigtail catheter was positioned in the left ventricle to infuse iohexol as a contrast medium . echocardiographic examinations were carried out using a cardiac ultrasound system equipped with a 5.0 - 7.5 mhz transducer ( ge logiq 7 ; ge medical systems , usa ) . the echocardiographic images were obtained in the right parasternal long- and short - axis view . the anatomy and function were measured in various cardiac regions including four cardiac chambers , valves , and great vessels by b - mode , m - mode , and spectral doppler echocardiographic tracing . the dimensions and thicknesses of the cardiac structures were measured using m - mode echocardiography according to the american society of echocardiography guidelines . the blood flow velocity , amounts of blood flow , pressure gradients across the valves , and stroke volume were measured using pulsed or continuous wave doppler echocardiography . the cardiac output was calculated by multiplying the stroke volume by the heart rate . the left ventricular ( lv ) systolic function was evaluated by measuring the ejection fraction ( ef ) and fractional shortening ( % fs ) by m - mode echocardiography according to the following formulas : ef = ( end - diastolic volume - end - systolic volume)/end - diastolic volume 100 , % fs = ( lv end - diastolic dimension - lv end - systolic dimension)/lv end - diastolic dimension 100 . all measured values used in the final calculations for each pig are the mean value from three to five sequential measurements regarded as being of the optimal quality . all of the measured values are expressed as a mean sd . the difference between two mean values was analyzed by the wilcoxon test . echocardiography was performed on the micropigs while they were under general anesthesia and mechanical ventilation . as compared to the human or small laboratory animals ( rat , mouse ) , the pig heart long axis is " rotated " posteriorly in the thorax ; hence , the right ventricle appears posterior to the left ventricle . while imaging from the long - axis view , the atrioventicular valves and the left ventricle outflow tract were imaged to obtain flow measurements through the aortic valve . table 1 shows the clinical and laboratory characteristics of the study population . there were no significant differences between the vital signs of the conventional pigs and micropigs . the mean systolic arterial pressure was 122.15 3.50 mmhg in the conventional pigs and 121.79 3.64 mmhg in the micropigs under a premedicated condition . the morphological differences between the conventional pig and micropig were compared using radiological examinations . as shown fig . the vhs can be used to determine the range of normal cardiac dimensions in micropigs . no significant difference was observed in the length of the long axis , but there was a difference in the length of the short axis . in the micropig , the cardiac length of the short axis was shorter than in the conventional pig , but a significant difference in the vhs was not observed ( table 2 ) . x - ray fluoroscopy was also performed to assess the blood flow and morphologic characteristics of the cardiac blood vessels . there were no significant differences observed between the conventional pigs and micropigs for nearly every measured parameter , except in the lv trace . the lv outflow track velocity , gradient , and lv fraction shortening were significantly lower in the micropigs than in the conventional pigs . in addition , we compared cardiac function of the micropig with that of the human . as shown tables 6 - 8 , there were similarities in the echocardiographic values between human and micropigs . highlights from numerous presentations on the use of echocardiography have led to the conclusion that this technique is becoming a commodity . from the considerable number of presentations , ubiquitous use is clearly the direction , not only in the noninvasive imaging section , but throughout the program , be it in novel percutaneous procedures such as valve implantation or in established procedures such as cardiac resynchronization therapy . however , this is far from the reality given the plethora of new advances being made in the field of echocardiography . in this study , we performed echocardiography in pigs using an open chest model , and transesophageal and transthoracic echocardiography ( personal experience ) . the thorax is more oval - shaped in the anterior - posterior direction , and the long axis of the heart follows an anterior - posterior direction . these differences between humans and pigs are related to the basic differences in body orientation , with the pig having an unguligrade posture and the human having an upright posture . finally , the animal is usually mechanically ventilated under general anesthesia , which makes transthoracic echocardiographic imaging difficult . an evaluation of the right ventricle , through an assessment of the right ventricle free wall motion , and left ventricular function from a parasternal view has been described . parasternal short - axis views have been described as requiring the two - dimensional short - axis image to be " visually approximated by the closest fitting ellipse at the end - diastole and end - systole " . this study assessed the physiological characteristics of the heart of a micropig and compared the data with that of a conventional pig . the vertebral heart scores ( vhss ) of each group were not significantly different , but the cardiac long axis : short axis ratio was larger in the micropigs than in the conventional pigs ( fig . 1 , this suggests that the cardiac shape of the micropigs was slightly different from that of conventional pigs . the echocardiographic study showed that there were no the significant differences between the conventional pigs and micropigs , except for several values of the mitral valve and left ventricle . however , the general aspects of the echocardiographic values observed for the micropigs were lower than those of the conventional pigs . these aspects may not only be due to differences in cardiac shape , but also to the different sizes of the hearts . it was previously reported that the weight of the heart in micropigs was lower than that in conventional pigs with an identical body weight . in addition , the fractional shortening results of micropigs in this study showed lower values than those measured in conventional pigs . the most common clinical methods for assessing the systolic ventricular function are the lv ejection phase indices . instead , they are measures of the global left ventricular performance . as such , they are as easily altered by changes in the preload and afterload as they are by the contractility . several indices of the lv function can be calculated from the lv dimensions measured from the m - mode or two - dimensional echocardiogram . the lv fractional shortening is the most commonly used one - dimensional assessment , and is the simplest and most often used index of the lv systolic function . moreover , the left ventricular velocities and gradients of the micropigs were lower than those of the conventional pigs . therefore , the differences in the lv function , including those of fractional shortening , between two groups might be due to differences in the systolic function . the differences in these aspects may be result from the decreased development of the cardiac function due to the breeding environments of the micropigs , including limited movement . physiological differences may also pose problems . while the handling of sodium and potassium by pig and human kidneys is similar , there are differences in the normal serum calcium and phosphate concentrations . these simple metabolic incompatibilities may not be of great significance , but multicellular organisms also require cells to be able to communicate with each other through hormones and other molecules . the question as to whether the pig heart is functionally and anatomically similar to its human counterpart was investigated . in this study , we could observe that there are similarities in the echocardiographic values between humans and micropigs . the cardiac outputs of porcine and human hearts of similar size were found to be comparable , and their action potentials were also similar . however , the innervation and overall morphology of the atrioventricular node in pigs was significantly different from that in humans . this may create problems terms of the control of the heart rate and contractility in a human patient . however , the differences could be amenable to other treatments , and may not be an insurmountable barrier . in this study , two limitations should be noted . first , we used different age groups to compare the heart function of micropigs with another group having identical body weights . several reports showed that there were not significant differences between different groups with identical body weights . second , we used the chemical restraining method to control the movement of pigs , and maintained animals general anesthesia using isoflurane ( 1% to 5% ) . isoflurane is known to be an inhalable anesthetic drug that maintains the cardiovascular function well in pigs , and does not induce disarrythmia by epinephrine compared with other inhalable anesthetic drugs such as halothane or enflurane . in conclusion , echocardiography imaging of the micropig is possible , and has potential utility for laboratory investigators . in particular , the parasternal images are readily obtainable for assessing the lv function , atria , and atrioventricular valves , as well as flow measurements through the left ventricle outflow .

the production of miniature animals has been suggested for use in organ transplantation . at present , many of the studies about application of animal organs to human have been focused on pigs because of the number of advantages involved and due to their similarities with human . however , a physiological analysis of the organs to be transplanted has not yet been carried out . therefore , this study analyzed whether or not there were physiological and morphological differences in the hearts of conventionally - reared pigs and micropigs . in this study , the morphological and physiological functions of the heart were examined using radiographic and echocardiographic equipment . in the lateral radiographic view , the heart of the micropig has a larger cardiac long axis : short axis ratio than does the conventional pig , but the difference in the vertebral heart score was not significant . in addition , there were no morphological differences on the x - ray fluoroscopic view . there were no differences in echocardiographic values , except for several values in the left ventricle traces . overall , it is expected that the values measured in this study will contribute to understanding of the physiological characteristics of micropigs .

however , they are relatively more often seen in children . most often , these arise from the lateral ventricular walls . meningiomas of the velum interpositum are extremely unusual and difficult to diagnose . these arise from the arachnoid cap cells within the potential space of the velum interpositum and lack a dural attachment . we encountered one such case where the massive size obscured all anatomical information and the site of origin was only established at surgery . a 3-year - old girl presented with history of difficulty in walking since 2 months . she had a normal perinatal history and developmental milestones until the current symptoms . on examination , the computed tomography ( ct ) head [ figure 1 ] revealed a large hyperdense uniformly enhancing lesion occupying the intraventricular space ( bilateral lateral as well as third ventricles ) with a thin rim of calcification . on magnetic resonance ( mr ) imaging [ figure 2 ] , the lesion appeared isointense on t1 , mildly hypointense on t2 and showed uniform enhancement . the internal cerebral veins ( icv ) appeared stretched out and displaced laterally and the cerebral peduncles were splayed [ figure 3 ] . because of the asymmetric extension into the right frontal horn , a transcortical , transventricular approach was taken . the tumor was identified in the medial wall of the right lateral ventricle under the septal wall , which was stretched over it . gradual internal debulking revealed the location of the tumor entirely within the third ventricle , displacing the internal cerebral veins around it laterally . these were carefully dissected and preserved . within the third ventricle ( which was grossly dilated ) , the tumor lobules could be delivered free from all the recesses except in the region of the pineal recess where there was dense adherence to the confluence of the great veins extending into what would have been the potential space of the velum interpositum . in the absence of any other site of attachment , this was most likely the site of origin of the tumor . the drain cerebrospinal fluid revealed growth of acinetobacter baumannii and , on commencing antibiotic therapy with meropenem , she improved remarkably . there was a small area of ischemia in the anterior commissure on the right side . she subsequently had an uneventful course and was discharged on the 14 postoperative day after completing a 10-day course of antibiotics , with minimal residual left hemiparesis . the histopathology revealed a transitional meningioma ( who grade i ) and she was advised a regular clinical follow - up . preoperative computed tomography ( plain a and b ; contrast enhanced c and f ) showing a hyperdense intraventricular tumor with uniform and brilliant contrast enhancement preoperative gadolinium - enhanced t1-weighted magnetic resonance images ( axial a d ; coronal e ; sagittal f ) showing the enhancing tumor occupying the third ventricle with lobules extending into its various recesses preoperative t2-weighted ( axial a ; coronal b ) images showing the tumor in the pineal recess splaying the internal cerebral veins and their confluence ( black arrows ) postoperative magnetic resonance ( t1 weighted a and b ; postgadolinium t1 weighted c and d ) showing complete excision postoperative t2-weighted images ( coronal ) showing the preserved internal cerebral veins ( black arrows ) although meningiomas are common in adults , they constitute less than 5% of childhood central nervous system tumors . among the intracranial meningiomas , often , due to the large size of these tumors , it becomes very difficult to ascertain the exact site of origin . these tumors are divided into anterior third ventricular meningiomas without dural attachment , which may arise from the tela choroidea in the roof or rarely from the floor of the third ventricle , and posterior third ventricular tumors arising from the posterior tela choroidea and the tenia fornicis in the velum interpositum . meningiomas of the pineal region are again rare and often include tumors having a dural base along the falcotentorial junction . true meningiomas of the velum interpositum ( without any dural involvement ) are indeed exceedingly uncommon . radiologically , they may mimic any third ventricular or pineal region tumor depending on the exact site of origin and direction of growth . giant intraventricular meningiomas are often seen in childhood , just as in our case , and this may obscure all anatomical imaging clues as to the site of origin . of these , only three were in children , all more than 5 years of age.[79 ] our case is probably the youngest such case reported . although lozier et al . describe radiological imaging features ( especially the course of the internal cerebral veins ) in preoperatively differentiating these tumors , the information may not always be forthcoming and , even when available , may not always help differentiate them from other intraventricular meningiomas . what is however critical is to appreciate the venous anatomy ( which may be grossly distorted ) and preserve the veins during surgery . we selected the transcortical transventricular transchoroidal approach to the tumor as has been described for safe entry into velum interpositum , because the tumor had extended significantly into the lateral ventricles . although we were not sure of the site of origin a priori , following the anatomy and gradually debulking the tumor led us to the site . we were able to identify and preserve the internal cerebral veins as was apparent in the postoperative mri as well as trace the origin of the tumor to the dense fibrous tissue at the confluence of the great veins . although challenging , complete surgical excision is curative for the grade i lesions , and should be attempted . recovery is often uneventful ( and satisfying ) if the deep venous structures are preserved .

intraventricular meningiomas are rare , but are relatively more often seen in children . large size at presentation often obscures anatomical details . a particular subset of such tumors arising from the velum interpositum pose a significant surgical challenge . thorough preoperative imaging , especially with respect to the course of the deep venous structures , provides useful evidence as to the origin . preservation of venous anatomy at surgery is vital . we describe a 3-year - old girl with a giant velum interpositum meningioma that was completely excised with excellent outcome . this is probably the youngest such case reported .

we examined 15 consecutive neonates and infants who were undergoing congenital cardiac surgery between 1 may 2009 and 31 october 2009 using an 8-fr acunav ivuc probe . the characteristics of the patients , including the type of surgery , are presented in table 1 . anesthesia was induced with midazolam ( 0.2 mg / kg ) , fentanyl ( 10 g / kg ) , and sevoflurane . for maintenance of anesthesia , electrocardiography , invasive arterial blood pressure , central venous pressure , pulse oximetry , temperature , capnography , and cerebral oximetry were monitored throughout the operation . with the patient in the supine position , a lubricated ivuc probe was inserted through the mouth and passed into the esophagus without any complications . because the probe does not have scale marks , the depth of placement was guided by echocardiography imaging . n = 2 ) or ventricular septal defects ( vsds ; n = 11 ) , and immediately after surgical repair ( asd , direct closure ; vsd , patch closure ) , we confirmed if there was leakage from the direct or patch closure site and evaluated de - aeration in the cardiac chamber ( fig . 2 ) . after total correction for tetralogy of fallot ( tof ) , it is important to examine the right ventricular outflow tract ( rvot ) , including blood flow to the pulmonary artery , pressure gradient , velocity , and vsd . 3 shows how to check the postoperative pulmonary artery pressure gradient and velocity using an ice probe . in this patient , we clearly evaluated the rvot , leakage of the patch closure site , the main pulmonary artery , and the pressure gradient between the pulmonary valves . during the period of study , there was a patient with hypoplastic left heart syndrome ( hlhs ) who underwent a norwood procedure . we evaluated the neoaorta ( conduit from the right ventricle to the pulmonary artery ) after completion of the procedure ; blood flow through the neoaorta was measured , as shown in fig . 4 . there were no patients with complications related to introduction of the probe or its use during the surgical procedure . heart surgery for congenital heart disease in very small infants is often carried out , but it has been difficult to use conventional pediatric omniplane tee probes in such patients . as a result , we could not previously assess the information about the anatomy , surgical repair , and heart function in patients weighing under 5 kg . recently , the ivuc probe has been applied to animals and neonates as a tee , proving it useful and safe . we also experienced the feasibility and usefulness of the ivuc probe based on 15 cases in our hospital . the acunav ivuc probe is reasonable in small infants weighing under 5 kg , but it has several limitations . because the acunav ivuc probe has monoplane features , it is difficult to fully appreciate the three - dimensional nature of the complex cardiac anatomy and the ivuc probe requires significant manipulation , which can be challenging in small patients . in cases involving simple vsd , asd , and tof , the defects and the surgical repairs were easily demonstrated . however , more complex congenital diseases , like anomalous origins of the right pulmonary artery ( rpa ; table 1 ) , could not be easily assessed . the probe utilized in this study is not steerable and has only one imaging plane . even though the probe has a four - way head articulation , it was difficult to obtain standard long - axis two- or four - chamber images of the heart on a case - by - case , and it was not easy to gain transgastric images comparable to tee with a conventional probe . moreover , the current study did not include patients with oropharyngeal anomalies in whom the conventional tee probe could not be passed and for which the ivuc probe may be a useful tool . mart et al . reported successful tee evaluation in a 1.4 kg infant with complex congenital heart disease . although intraoperative tee can be safely performed , even in small infants , it frequently causes mild mucosal injuries to the esophagus . we suggest that an ivuc for tee in small infants eliminates any complications in patients previously thought to be too small for intra - operative tee . the ivuc probe is designed for single use when it is applicated to intravascular access . ivuc costs about three million won per probe , it is very expensive for single use . but in the case of transesophageal use , it can be reusable like other conventional tee probe . considering that ivuc has the term of validity ( 2 months ) , we could use 2 probes for 15 cases with sterilization . we obtained good image quality at every case and its cost per patient was similar as conventional tee . finally , as though it is difficult to get obvious images in some cases , anyone who is familiar with conventional tee can use this ivuc with only several attempts . in conclusion , although the ivuc probe is disadvantageous based on its monoplane features and not being steerable , we found the ivuc probe useful for tee in small infants and safe without any complications , such as hemodynamic instability , difficulty in ventilation , or esophageal injury .

transesophageal echocardiography ( tee ) has an important role during congenital heart surgery . tee in small infants is associated with complications , including an inability to pass the tee probe , esophageal trauma , airway compression , aortic compression , and interference with ventilation . recently , a monoplane intravascular ultrasound catheter ( ivuc ) has been developed for intracardiac echocardiography . the efficacy of ivuc for transesophageal use has been shown in numerous animal studies and several human studies , but there have been few reports involving small infants using an ivuc probe . we examined 15 pediatric patients undergoing congenital cardiac surgery using an 8-fr acunav ivuc probe . we checked the cardiac anatomy , cardiac function pre - operatively and de - aeration before weaning from cpb ; the surgical repairs were evaluated post - operatively . although the ivuc probe has limitations associated with the monoplane , we found the ivuc probe to be useful in small infants and safer than the tee .

even when return of spontaneous circulation ( rosc ) is achieved , mortality related to cardiac arrest due to pte is high . in the 2010 american heart association and the european resuscitation council guidelines , although surgical embolectomy is considered the most effective method to treat pte , emergency surgery after pte - related cardiac arrest is associated with high mortality . we report two successfully resuscitated cases of cardiac arrest due to massive pte by using recombinant tissue - type plasminogen activator ( rtpa ) and therapeutic hypothermia . a 52-year - old female was transferred to our emergency department ( ed ) from a local orthopedic clinic , presenting with cardiac arrest . she previously had a right ankle fracture and lumbar vertebral compression fracture due to a traffic accident seven days prior to cardiac arrest . she was found unconscious with a pulse in the bathroom of a local clinic after complaining of chest discomfort and dyspnea . on arrival in the ed , her initial rhythm was pulseless electrical activity . the estimated no - flow time and low - flow time were unknown and three minutes , respectively . after achieving rosc , blood pressure ( bp ) was 49/35 mmhg and heart rate was 135 bpm . after using norepinephrine ( 120 g / min ) and dopamine ( 30 g / kg / min ) , bp was stabilized at 129/93 mmhg . computed tomographic pulmonary angiography showed heavy burden of pulmonary embolism in the bilateral pulmonary arteries and branches ( fig . on echocardiography , a d - shaped left ventricle was observed during systole and diastole . within two hours of arrival at the ed , she was admitted to the ed intensive care unit , and we started therapeutic hypothermia using a commercial temperature regulation system ( arctic sun temperature management system ; medivance , louisville , co , usa ) with our hospital protocol ( rapid cooling to 33c , 24-hour maintenance , and 0.15c / hr rewarming to 36.5c ) . we also administrated 100 mg of rtpa over two hours , with a loading dose of 60 unit / kg followed by a continuous intravenous infusion of unfractionated heparin ( 12 unit / kg / hr ) . while conducting therapeutic hypothermia , no bleeding complications were observed . on her third hospitalized day ( hd3 ) , she was able to follow simple commands and vital signs were stable without inotropes . the follow - up computed tomographic angiography at hd3 showed that the burden of pulmonary embolism remained in the right main pulmonary artery ( fig . right ventricular systolic pressure , estimated by the maximum velocity of tricuspid regurgitation and right atrium pressure , was normalized ( from > 58 to 24.1 mmhg ) on echocardiography . she was transferred to the thoracic surgery department to undergo a pulmonary artery embolectomy . at hd28 , she was discharged with a cerebral performance categories scale score of 1 , without neurological deficits . a 62-year - old female was transferred to our ed from a local tertiary hospital after achieving rosc after cardiac arrest . she had had a radical hysterectomy and bilateral salpingo - oophorectomy due to cervical cancer 20 days prior to the cardiac arrest . when she arrived in our ed four hours after cardiac arrest , her bp and heart rate were 129/66 mmhg and 120 bpm with norepinephrine ( 20 g / min ) , but she was comatose . 2a ) , with a d - shaped left ventricle during systole and diastole visible on echocardiography . after admission to the ed intensive care unit within two hours of arrival , we started therapeutic hypothermia using a conventional method ( a commercial cold blanket , as well as 4c cold saline bladder irrigation or traditional ice packs placed on the patient s groin , armpits , and neck and head ) as per our hospital protocol . we administered 100 mg of rtpa over two hours , with a loading dose of 60 unit / kg followed by a continuous intravenous infusion of unfractionated heparin ( 12 unit / kg / hr ) . at two hours after administration of rtpa , massive bleeding was observed via nasogastric ( ng)-tube . right ventricular systolic pressure was normalized ( from 41.72 to 29 mmhg ) on echocardiography . at hd7 , she was extubated and transferred to the cardiology department for an inferior vena cava filter insertion . at hd27 , she was discharged with a cerebral performance categories scale score of 1 . a 52-year - old female was transferred to our emergency department ( ed ) from a local orthopedic clinic , presenting with cardiac arrest . she previously had a right ankle fracture and lumbar vertebral compression fracture due to a traffic accident seven days prior to cardiac arrest . she was found unconscious with a pulse in the bathroom of a local clinic after complaining of chest discomfort and dyspnea . on arrival in the ed , her initial rhythm was pulseless electrical activity . the estimated no - flow time and low - flow time were unknown and three minutes , respectively . after achieving rosc , blood pressure ( bp ) was 49/35 mmhg and heart rate was 135 bpm . after using norepinephrine ( 120 g / min ) and dopamine ( 30 g / kg / min ) , bp was stabilized at 129/93 mmhg . computed tomographic pulmonary angiography showed heavy burden of pulmonary embolism in the bilateral pulmonary arteries and branches ( fig . on echocardiography , a d - shaped left ventricle was observed during systole and diastole . within two hours of arrival at the ed , she was admitted to the ed intensive care unit , and we started therapeutic hypothermia using a commercial temperature regulation system ( arctic sun temperature management system ; medivance , louisville , co , usa ) with our hospital protocol ( rapid cooling to 33c , 24-hour maintenance , and 0.15c / hr rewarming to 36.5c ) . we also administrated 100 mg of rtpa over two hours , with a loading dose of 60 unit / kg followed by a continuous intravenous infusion of unfractionated heparin ( 12 unit / kg / hr ) . while conducting therapeutic hypothermia , no bleeding complications were observed . on her third hospitalized day ( hd3 ) , she was able to follow simple commands and vital signs were stable without inotropes . the follow - up computed tomographic angiography at hd3 showed that the burden of pulmonary embolism remained in the right main pulmonary artery ( fig . right ventricular systolic pressure , estimated by the maximum velocity of tricuspid regurgitation and right atrium pressure , was normalized ( from > 58 to 24.1 mmhg ) on echocardiography . she was transferred to the thoracic surgery department to undergo a pulmonary artery embolectomy . at hd28 , she was discharged with a cerebral performance categories scale score of 1 , without neurological deficits . a 62-year - old female was transferred to our ed from a local tertiary hospital after achieving rosc after cardiac arrest . she had had a radical hysterectomy and bilateral salpingo - oophorectomy due to cervical cancer 20 days prior to the cardiac arrest . when she arrived in our ed four hours after cardiac arrest , her bp and heart rate were 129/66 mmhg and 120 bpm with norepinephrine ( 20 g / min ) , but she was comatose . 2a ) , with a d - shaped left ventricle during systole and diastole visible on echocardiography . after admission to the ed intensive care unit within two hours of arrival , we started therapeutic hypothermia using a conventional method ( a commercial cold blanket , as well as 4c cold saline bladder irrigation or traditional ice packs placed on the patient s groin , armpits , and neck and head ) as per our hospital protocol . we administered 100 mg of rtpa over two hours , with a loading dose of 60 unit / kg followed by a continuous intravenous infusion of unfractionated heparin ( 12 unit / kg / hr ) . at two hours after administration of rtpa , massive bleeding was observed via nasogastric ( ng)-tube . right ventricular systolic pressure was normalized ( from 41.72 to 29 mmhg ) on echocardiography . at hd7 , she was extubated and transferred to the cardiology department for an inferior vena cava filter insertion . at hd27 massive pulmonary embolism ( mpe ) with hemodynamic instability is a clinical condition with a poor prognosis and a mortality rate greater than 50% . current guidelines recommend aggressive treatment with systemic fibrinolytic therapy , catheter embolectomy , or surgical embolectomy , depending on the local situation . fibrinolytic therapy remains the most commonly used treatment in hypotensive patients presenting with a mpe in the ed . catheter embolectomy or surgical embolectomy can be used as an alternative mpe treatment method when fibrinolytic treatment is contraindicated or fails . when cardiac arrest due to acute mpe occurs , the risk of death increases up to 70% . in spite of achieving rosc , cardiac arrest due to mpe is often fatal because it results from mechanical obstruction of the pulmonary arterial tree by thrombi . although fibrinolytic therapy is not recommended for undifferentiated cardiac arrest , it should be considered for cardiac arrest patients diagnosed with mpe . rtpa has been preferred as a fibrinolytic agent . according to one small case series reported in 2014 , surgical embolectomy with percutaneous cardiopulmonary support is considered the preferred method for mpe - induced cardiac arrest . however , overall mortality remained high in patients who underwent cardiopulmonary resuscitation ( cpr ) for more than 15 minutes . there are no randomized trials guiding the use of fibrinolysis with hypothermia in cardiac arrest survivors following mpe . it has been reported that a hypothermia - induced coagulation disorder could increase the risk of serious adverse events , but it is unclear if bleeding complications occur more often in cardiac arrest patients treated with therapeutic hypothermia . in a recent systematic review and meta - analysis , the risk of bleeding was not significantly higher for patients treated with therapeutic hypothermia . relative contraindications to fibrinolytic therapy include prolonged cpr ( > 10 minutes ) and major surgery within three weeks prior to cardiac arrest . however , current guidelines recommend that physicians weigh the relative benefits of fibrinolysis on a case - by - case basis . in our cases , the cardiac arrest survivor with prolonged cpr was diagnosed with mpe and then had fibrinolytic therapy using rtpa with therapeutic hypothermia . although gastric ulcer bleeding was observed for the first few hours , it is unclear if this bleeding was made worse by fibrinolytic therapy or therapeutic hypothermia . in conclusion , we describe two cases of cardiac arrest due to mpe in which fibrinolytic therapy was successfully combined with therapeutic hypothermia . this report suggests that fibrinolysis with hypothermia are feasible in the treatment of mpe with cardiac arrest . however , larger scale and randomized trials are necessary to establish standard therapy for mpe with cardiac arrest .

massive pulmonary embolism ( mpe ) with hemodynamic instability is a clinical condition with a poor prognosis and high mortality rates . there are no definitive treatment options for cardiac arrest due to mpe . a 52-year - old female presented at our emergency department with cardiac arrest , and a 62-year - old female presented after achieving return of spontaneous circulation of cardiac arrest from a local hospital , respectively . in each case , computed tomographic pulmonary angiography after return of spontaneous circulation demonstrated heavy burdens of pulmonary embolism in the pulmonary arteries . we immediately started therapeutic hypothermia and fibrinolytic therapy . they were transferred to the thoracic surgery and cardiology departments respectively , and then discharged with a cerebral performance categories scale score of 1 . in summary , we report two cases of out - of - hospital cardiac arrest due to mpe in which fibrinolytic therapy was successfully combined with therapeutic hypothermia .

type 2 diabetes mellitus ( t2 dm ) is a hyperglycemic endocrine and metabolic disorder caused by insulin resistance and pancreatic -cell dysfunction . it is a complicated , multifaceted disease induced by the combined actions of both genetic and environmental factors . with rapid changes in lifestyle , such as overnutrition and lack of exercise , and aging of the population , the prevalence of diabetes for all age groups worldwide estimated to be 2.8% in 2000 recent human epidemiological data show over 92 million diabetes patients in china , 90% of which suffer from t2 dm . as a polygenic hereditary disease , first , there is usually more than one gene involved in the onset and development of t2 dm with each of the genes exerting different functions and intrinsic interactions . second , t2 dm susceptibility genes vary within the population and individuals . a group of people without diabetes might carry the disease susceptibility genes but with a tiny amount . third , each susceptibility gene plays a role in various metabolic processes of t2 dm . fourth , environmental factors also play an important role in the pathogenesis of t2 dm , which suggests that diabetes cases not only demonstrate erratic gene patterns but also show defects in gene expression and post - gene - expressive modification . additional information is required to fully understand the etiology of t2 dm . in recent years , there has been significant research on the relationship between protein kinase c isoforms ( protein kinase c epsilon zeta , prkcz , or pkc ) and diabetes . these studies have shown that prkcz is a significant contributing factor to t2 dm as it is involved in the pathogenesis of t2 dm by participating in the impulse transmission of insulin , which is closely related to the function of the pancreas in excreting insulin . the incidence of t2 dm involves many factors , such as heredity , environment , and diet . thus , it is important to study the correlation between methylation of the prkcz gene promoter and the pathogenesis and treatment of t2 dm . the t2md group : utilizing the 1999 who diagnostic standard , 152 patients , 63 males and 89 females , were diagnosed with t2 dm . the cases were excluded from other types of diabetes or endocrine disorders based on clinical examination and family history . the normal control ( nc ) group : the control group consisted of 120 healthy unrelated to one another individuals , 55 males and 65 females , with a fasting blood glucose ( fbg ) < 5.6 mmol / l and a postprandial blood glucose ( pbg ) < 7.7 mmol / l . analysis by genome - wide methylation chip was conducted on the extracted dna ( completed by capital bio - corporation co. , ltd . ) . the entire chromosomal gene sequences of the t2 dm and the nc groups were analyzed by medip ; the analysis included 22532 gene promoters , fragments from upstream 2.44 kb to downstream 610 bp , and methylation detection of 27728 cpg islands . based on the chip analysis results , the prkcz gene , the susceptibility gene of t2 dm , was identified as the candidate gene as well as the object of study for this research . the bisulfite sequencing pcr ( bsp ) primer was designed according to the cpg islands of the prkcz gene promoter using meth primer online software and was synthesized by shanghai sangon biological technology services co. ltd . a 204-bp target fragment was amplified in 35 pcr cycles and sequenced by shanghai invitrogen biotechnology co. ltd . primer sequence : forward primer , 5-tttttgtttaggttggagtgtagtg-3 reverse primer , 5-ccacttaaaatcaaaaatttaaaacc-3. forward primer , 5-tttttgtttaggttggagtgtagtg-3 reverse primer , 5-ccacttaaaatcaaaaatttaaaacc-3. the serum from the t2 dm and nc groups was collected . serum of t2 dm and normal control were collected , and the serum proteins with sds page electrophoresis were separated ; electrophoresis result was shifted to nitrocellulose membrane ( nc ) ; nc membrane was soaked the first anti-(1 : 500 ) of rabbit anti - human prkcz antibody which was directly diluted with 5% nonfat dry milk , shaked in room temperature 4 hours or 4c slow foster overnight , and washed membrane three times with tbst . added hrp secondary antibody ( l : 1000 ) , slowly shaked at room temperature for 2 hours , washed the membrane with tbst four times , each time 8 min . the relevant hypermethylated , disease - causing gene of t2 dm was determined using medip . the hypermethylated gene was found to be located on chromosome 1:2036289 - 2036725 . this gene had a log ratio of 0.215 and a peak score of 2.21 ; log - ratio is the value of the probe and peak score refers to the significance of methylation ( figure 1 ) . the prkcz gene promoter is situated at this same site ; therefore , the prkcz gene promoter was hypothesized to be the hypermethylated gene in t2 dm patients . in contrast , the prkcz gene promoter showed no obvious methylation in the nc group . a cpg island - intensive area of 204 bp , a gc content > 50% , and an obs / exp value > 0.6 were found , and this fragment was chosen as the target fragment of bsp . target fragments of the prkcz gene promoter from 272 subjects were amplified by bsp to study the promoter 's methylation status . the working principle of bsp is that sodium bisulfite can effectively transform cytosine ( c ) into uracil ( u ) in a single strand of dna . after pcr amplification , uracil ( u ) in the template is further transformed into thymine ( t ) , while methyl cytosine ( c ) is not affected by the sodium bisulfite and exists in the unchanged form of deoxidized cytosine ( c ) . when the differences have been amplified by bsp , target fragments can then be obtained . finally , the pcr products are subjected to sequencing and sequence comparisons after modification with sodium hydrogen sulfite , and the methylated cpg sites can be determined . the bsp products from both groups were sent to shanghai yingjun corporation to be sequenced . the data from the two groups were then analyzed to compare the primary gene sequence with the sequence generated after modification by sodium bisulfite . the target fragment of the prkcz gene promoter was found to be 204 bp in a length and contain 9 cpg sites ( figure 2 ) . when the detected base sequence of the methylation cpg site remained in the state of c , the site was methylated . after comparing the differences between the base sequences , the degree of methylation could be calculated by c / cpg . the comparison between the base sequences in the t2 dm and nc groups showed that seven cpg sites were methylated in the t2 dm group ( 7/9 100% = 77.7% ) . cpg sites 2 , 3 , 6 , and 7 showed a high probability of methylation in t2 dm patients , whereas only one cpg site ( cpg site 4 ) was methylated in the nc patients ( 1/9 100% = 11.1% ) ( table 1 ) . the protein expression levels of prkcz in the serum of the t2 dm and nc groups were analyzed by western blotting . the expression level of prkcz in t2 dm patients decreased significantly when compared with the level in the nc group ( p < 0.05 ) , suggesting that the prkcz gene plays a major role in the initiation of t2 dm . it plays an essential role in the maintenance of chromosome structure , x chromosome inactivation , and the expression of genomic imprinting and tumor disease . dna methylation occurs when a dna methytransferase ( dnmit ) catalyzes the conversion of cytosine into 5-methyl cytosine with s - adenosylmethionine acting as a methyl donor [ 7 , 8 ] . the modification occurs mainly at the cytosine residue in the two - nucleotide cpg islands . some research has already shown that dna methylation is one of the pathogenic factors of t2 dm , and it is assumed that dna methylation plays a role in the initiation and progression of t2 dm . prkcz is also an important regulatory molecule that affects the signaling pathway of insulin through epigenetic changes . in t2 dm patients , there is a disruption of the glucose transport stimulated by insulin , which regulates the transport of blood glucose through interaction with the insulin substrate receptor protein ( isr-1/2 ) , and prkcz participates in the occurrence and development of t2 dm through insulin signaling pathway mechanisms . recent studies have identified prkcz as a susceptibility gene of t2 dm using human genome scanning and fine localization . the hypermethylated genes of t2 dm patients were determined in this study through high - flux screening of the methylated chip . after screening and analysis , prkcz was chosen as the target gene for the study . there was no indication of prkcz methylation in the normal control group . our findings demonstrate that methylation can be observed in peripheral blood leukocyte dna from both the t2 dm and normal control groups but that the prevalence of methylation of the prkcz promoter in peripheral blood leukocytes is significantly higher in the t2 dm group ( 77% ) than in the normal control group ( 7% ) ( = 36.42 , p < 0.01 ) . the fact that methylation has also been detected in the normal control group might indicate that gene methylation not only exists in the development of t2 dm but is also an early event of t2 dm . based on previous studies , it was generally hypothesized that the hypermethylation of dna was correlated with gene silencing , whereas low methylation or demethylation was generally related to gene activation . currently , a collection of research [ 11 , 12 ] indicates that the variation in epigenetics plays a vital role in the initiation and progression of t2 dm . research by fujiki et al . revealed that high methylation of peroxisome proliferator - activated receptor gamma ( ppar- ) inhibited the gene expression of t2 dm , which was regulated by the methylation of its promoter . research by ling et al . reported the involvement of methylated peroxisome proliferator activated receptor gamma , coactivator 1 , alpha ( ppargcia ) promoter in the development of t2 dm . the research showed that the methylation of ppargcia promoter in patients with t2 dm was twice as much as that in the normal group with the mrna expression of reduced by 39% and the secretion of islet declined by 34% , which contributed to the reduction of the gene expression , functional disorder of islet and the declined secretion of islet . the methylation of ppargcia was closely correlated with t2 dm . the expression of ppargcia displayed a positive correlation with the secretion of human insulin stimulated by glucose . the above - mentioned studies demonstrate how epigenetic mechanisms might regulate gene expression , such as insulin secretion in human beings . moreover , the research suggests that hypermethylation of the glucose transporter protein 2 ( glut2 ) promoter suppresses its gene expression and thus leads to the reduced consumption of glucose . expression of the insulin gene is closely related to the level of methylation at its promoter . these studies indicate that dna methylation correlates with gene silencing and highly consistent with our study in prkcz gene . in our study , the prkcz gene promoter in t2 dm patients was hypermethylated while prkcz protein expression level in the serum was decreased , suggesting a negative correlation between prkcz gene expression and methylation level of the cpg islands . hypermethylation of the prkcz genetic promoter may lead to the reduction of prkcz gene expression , which in turn induces the incidence of t2 dm via insulin signaling pathways . first , methylation leads to structural changes in the genome , such as changes in repetitive sequences and thus suppresses the expression of certain genes . second , methylation may affect chromosomal structure by shrinking the nucleosome and denying access to the transcription factor , thus leading to instability of the chromosomal structure . sequencing results demonstrated that the methylation frequency varied by site ; the methylation frequencies for the 9 sites were different . the results suggest that the methylation of these sites is probably related to the incidence of t2 dm , especially as the frequency of methylation at cpg sites 2 , 3 , 6 , and 7 is equal to or greater than 65% . the findings suggest that the nearby sites might play a role in the regulation of prkcz gene expression . the study indicates that methylation can serve as a potential diagnostic index of t2 dm . as a certain positive rate also occurs in normal tissues , the index could be used as an accessory examination . higher specificity genes might include some non - detected genes or other genes related to t2 dm . therefore , analysis of these genes could be conducted together with tests of the biopsy samples and peripheral blood of patients , and the occurrence of methylation could be a useful omnidirectional reference for the early diagnosis , prognosis , and clinical therapy of t2 dm . it is of great significance in theory and in practice for the primary prevention of t2 dm and the screening of a high - risk population .

objectives . to study the correlation between the methylation of protein kinase c epsilon zeta ( prkcz ) gene promoters and type 2 diabetes mellitus ( t2 dm ) . methods . the case - control method was implemented in 272 unrelated to one another cases in shiyan people 's hospital . of those , 152 were diagnosed as t2 dm cases , and the other 120 cases were healthy individuals visiting the hospital for a physical examination . the subjects were first divided into two groups : the t2 dm group and the normal control ( nc ) group . next , methylated dna immunoprecipitation chip ( medip - chip ) was used for detection . bisulfite sequencing pcr ( bsp ) and gene sequencing were then performed to detect and analyze the correlation between prkcz gene promoter methylation and t2 dm . finally , western blotting was applied to determine the serum level of prkcz . the data were then analyzed with the statistics analyzing software spss 17.0 . results . in contrast with cases in nc , t2 dm patients showed a high level of methylation , with 7 of 9 cpg sites were shown to be methylated , whereas , in the control group , only one cpg site was found to be methylated . the methylated cpg sites for the two groups showed marked differences ( p < 0.01 ) . additionally , the level of prkcz was decreased in t2 dm subjects , and the difference between the two groups was statistically significant ( p < 0.05 ) . discussion . this study suggests that the prkcz gene is the hypermethylated gene of t2 dm and the hypermethylation prkcz gene may be involved in the pathogenesis of t2 dm .

the term palliative is of latin origin and comes from the word pallium , which means robe , cover , veil , raincoat . indeed , in palliative care symptoms coats , cover with various treatments in order to improve the comfort of patients , or to eliminate discomfort , such as overcoat protects the body from rain and cold ( 1 - 4 ) . the word hospice is often used in relation to palliative care and , although it is in use with multiple meanings ( sometimes it s the building st . christopher s hospice , sometimes a group of employees team of hospice , and sometimes the program - hospice service ) , a hospice is primarily elaboration of the philosophy that the association for hospice of south australia can be summarized as follows : hospice care accepts death as a normal process and is understood as the last phase of life of the person who is dying , as a special time for integration and reconciliation ( 5 ) . furthermore , acceptance of the need of dying persons to live fully , proudly and comfortably until they die , and do not hasten nor postpone death . finally , it provides support to grieving family and friends ( 1 , 2 ) . interestingly , palliative care and hospice are mainly related to malignant tumors ( in the public perception , perception of laic , but also in perception of health professionals in general , and in professional and scientific literature ) , although many other ( non - malignant ) , internal or neurological diseases can and should be treated with quality of life is a wide term that is used in many natural and social sciences . it includes aspects of environmental protection in which we live as a social geographical aspects . when this term refers to health it is common to use the term quality of life related to health . from a health perspective , quality of life refers to the social , emotional and physical well - being of patient after treatment , reflecting the definition of health of world health organization ( who ) . quality of life presents functional effect of the disease and the effect of therapy on patients , as observed by patient itself . there are four major areas that contribute to the overall quality of life : physical and working ability , psychological status , social contacts . nordenfelt ( 5 ) defined the quality of life as the evaluation of their own life situation of the individual , equated it with the pleasure of life . starting from the fact that in tuzla , within the university clinical center , department of palliative care existing for several years now and daily positive experiences in working with people suffering from lung cancer who have used this service , and a relatively small number of studies in this area , in this work was made an attempt to establish the effect of palliative care on the quality of life of patients with lung cancer . a prospective study was conducted at the department of palliative care clinic for pulmonary diseases and tuberculosis of university clinical center tuzla . the study group included 40 patients diagnosed with advanced stage lung cancer at an advanced that were selected consecutively and were treated at the department of palliative care . the control group was formed of the same number of patients with approximately the same age and sex distribution , that are after a diagnosis of lung cancer were treated at home by the authorized doctor from corresponding health center . tests in both groups were carried out using the test sf-36 scale for assessing quality of life in the space of two weeks . a total of 36 questions in the test sf-36 were divided into two groups , one referred to the total physical health , and the other to the total mental health . the result is obtained when the 36 questions , that carry a certain number of points , are grouped into eight sub - groups : physical function , physical limitation , emotional limitation , energy , emotional well - being , social function , pain and general health ( 6 ) . two groups were formed out of these eight groups : total physical and mental health , and of these two groups overall quality of life . value to 0.25 implies to poor quality of life , 0:26 to 0:50 medium good , 0.51 to 0.75 is good , and over 0.75 points as the excellent quality of life . statistical analysis was performed using standard methods , descriptive and inference statistics . when testing statistical hypotheses significance level of 5% ( p<0.05 ) was used . quality of life was tested in study and in the control group before and after treatment . since these were the same patients , paired test , called t - test was applied for paired samples . quality of life was checked ( tested ) in the space of two weeks in both groups ( experimental and control group ) . the first test was performed immediately after the lung cancer was diagnosed , and the second after two weeks . all parameters of life quality ( general , physical and emotional ) were better in the second test , in patients who were treated in the department of palliative care ( study group ) ( p < 0.0001 ) . but in the control group ( patients who were not treated at the department of palliative care ) improvement of life quality was not recorded ( table 1 ) . distribution of patients who stayed in the department of palliative care according to two tests of quality of life . x average value , sd standard deviation , t - test value , p statistical significance in the analysis of life quality changes in the area of physical health ( physical function , physical limitations , pain , general health ) , in patients who have resided in the department of palliative care ( study group ) , showed a statistically significant improvement in all the aspects ( p < 0.0001 ) ( table 2 ) . this was not the case in patients who have not resided in the department of palliative care . in these patients , there was a small improvement in quality of life of a field of physical function ( p=0.002 ) and the physical limitations ( p = 0.183 ) , while the pain ( p=0.193 ) and general health ( p<0.0001 ) ( table 3 ) deteriorates after two weeks . distribution of respondents who have stayed in the department of palliative care according to changes in the area of physical health . x- average value , sd- standrd deviation , t - test value , p- statistical significance distribution of respondents who did not resided in the department of palliative care accoring tochanges in the area of physical health . x- average value , sd- standard deviation , t - test value , p - statistical significance in the study patients ( who resided in the department of palliative care ) all aspects of mental health were statistically significantly improved after two weeks stay in the department ( p<0.0001 ) ( table 4 ) . distribution of respondents who have stayed in the department of palliative care according to certain aspects of mental health . x- average value , sd- standard deviatoin , t - test value , p - statistical significance in the area of mental health in the control group of patients ( who have not resided in the department of palliative care ) in the second test , after two weeks , also showed improvement in emotional limitations and the same value for a social function , but statistically significantly less than in the study group , while energy ( p=0.001 ) and emotional satisfaction ( p<0.005 ) showed worse results ( table 5 ) . comparing these two groups for this aspect of quality of life was found significantly more pronounced improvement in the study group compared to the control group ( t=9.9 , p<0.0001 ) . distribution of respondents who did not stay in the department of palliative care according to certain aspects of mental health . x- average value , sd- standard deviation , t - test value , p - statistical significance the studies that have been conducted show the value of improving the quality of life in patients treated at the department of palliative care compared to those who were not treated at the department . forty - three studies were performed in the uk that have examined the location of care of patients suffering from lung cancer . a study conducted at the department of oncology , mcgill university on department of palliative care determined the quality of life of patients treated in hospital conditions and ambulatory and received results of improvement in physical function and psychological . reduced anxiety and depression , as well as improving the overall quality of life of the treated patients in the department . the study was conducted on 88 patients and evaluated with life quality test at the reception and after 8 days . a study that was conducted on 58 patients with advanced cancer who was tested the first day and after two weeks , measuring the quality of life has shown a significant improvement of the physical and mental condition , along with rehabilitation of disease symptoms ( 8) . lung cancer is the most common cancer in men population , as well as a cancer with worse five years survival ( 9 ) . due to specificity of lungs as an organ and vicinity of other important organs as heart , esophagus , large blood and lymphatic vessels leads very quickly to developing of symptoms that threaten the life of the patient ( 10 ) . the family , as an essential factor , suffers because it is in a situation where have no control over things that happening ( 11 ) . the suffering and pain of the patient , lead them in a situation to seek for help , but due to the specific organization of hospitals and health centers adequate assistance to the patient and family is not possible ( 12 ) . only specific departments for care and therapy of patients and care of the family called hospices can provide appropriate treatment with the participation of a multidisciplinary team consisting of : doctor , nurse , social worker , physical therapist , specialist in other specialties , religious official and volunteers ( 13 ) . thus , our results confirm this claim because the value of the overall quality of life in hospitalized patients are much higher , while outpatient have the same value at the beginning and after two weeks of testing . the values of the physical quality of life were higher after treatment in the department , while in the ambulance patients is slightly worse . the emotional quality of life for hospitalized patients was higher , while the ambulance is slightly worse . physical health with all the elements increases in hospitalized patients while in the patients treated in ambulances is imperceptibly different . patients who were hospitalized have lower lever of anxiety and depression after two weeks being in the department , and patients who were not hospitalized have values that are approximately the same at the beginning and after two weeks of testing . all this tell us about the necessity for opening specialized institutions for treatment and care of advanced cancers , as it is the case in the world where the majority of western european countries have hundreds of such hospitals already ( 14 ) . treatment of patients with lung cancer in the department of palliative care leads to a significant improvement in overall quality of life . all general aspects of quality of life in patients with lung cancer were significantly better after two weeks of treatment in the department of palliative care . all aspects of the physical health of patients with lung cancer were statistically significantly better after a two - week treatment in the department of palliative care . total mental health in patients with lung cancer was significantly better after a two - week treatment in the department of palliative care .

objective : objective of this work is to determine influence of palliative care on the quality of life in patients with lung cancer.subjects and methods : our study group included 40 patients , consecutively selected , which is determined by symptomatic treatment and hospitalized at the department of palliative care of university clinical center tuzla . the control group consisted of 40 patients who had a diagnosis of lung cancer treated at home by an authorized ambulance health center tuzla . tests in both groups were carried out using the test sf-36 scale for assessing quality of life in period of two weeks . two - stage test was performed , initially , immediately after disease was diagnosed , and two weeks later.results:all life quality parameters ( general , physical and emotional ) were better in second test , in patients who were situated in the department of palliative care ( study group ) ( p<0.0001 ) . in the area of physical health ( physical function , physical limitations , pain , general health ) , in patients who have resided in the department of palliative care ( study group ) , showed a statistically significant improvement in all the aspects ( p<0.0001 ) . in the study patients all aspects of mental health were statistically significantly improved after two weeks being in department ( p<0.0001).conclusion : two weeks treatment of patients with lung cancer in the department of palliative care significantly improve all general aspects of quality of life .

medical records were retrospectively reviewed for cats with nasal lymphoma treated with radiation therapy at the rakuno gakuen university veterinary teaching hospital . the 30 cats included in this study were treated between april 2004 and december 2011 . all cats had biopsies taken under general anesthesia and were diagnosed with lymphoma before any treatments . pretreatment evaluation included complete blood counts , serum chemistry , three - view thoracic radiographs , abdominal ultrasounds , ct scans of head , chest and abdomen , and aspiration cytology of lymph nodes or organs . follow - up information was obtained from the medical records or by contacting the referring veterinarians . radiation therapy was performed by use of an orthovoltage x - ray machine ( titan-450s , ge , tokyo , japan ) with a half - value layer of 4.8 mm of cu at 450 kv and 10 ma . the exposure rate was 1.68 gy / min with a filter of 1.0 mm of al , 0.3 mm of cu and 0.5 mm of sn . the distance from the x - ray source to the skin was 60 cm . radiation was delivered via single dorsal portal directed to the tip line of the nose to the top of the head . the response was assessed based on ct images at one month after finishing the radiation therapy . complete response ( cr ) was determined , if the tumor disappeared from ct images . partial response ( pr ) was defined as a decrease of 30% or greater in the sum of the longest diameters . stable disease ( sd ) was defined as between a decrease of 30% and an increase of 20% in the sum of the longest diameter . progressive disease ( pd ) was defined as an increase of 20% or greater in the sum of the longest diameters . apoptotic and proliferative activities in pretreatment biopsy samples in feline nasal lymphoma were detected . a terminal deoxynucleotidyl transferase dutp nick end - labeling ( tunel ) assay was carried out using an apoptag in situ apoptosis detection kit ( s7100 , chemicon merck millipore , billerica , ma , u.s.a . ) to detect apoptotic cells . all 3-m sections from paraffin - embedded tissues that were taken before radiation therapy were cut . the samples were pretreated with proteinase k ( 20 g / ml , dako , glostrup , denmark ) . working - strength tdt solution was then added to the slides at 37c for 1 hr . the reaction was stopped by addition of working - strength wash / stop buffer for 10 min . anti - digoxigenin peroxidase was applied , and the tissue sections were then incubated for 30 min at room temperature . 3 - 3-diaminobenzidine ( kanto chemical co. , tokyo , japan ) solution was used for color developing , and then , mayer s hematoxylin was used for counterstaining . . the antigen was retrieved by autoclaving at 121c for 15 min with 0.1 m citrate buffer ( ph 6.0 ) . all sections were blocked for endogenous peroxidase activity and non - specific binding with 3% hydrogen peroxide and 1% horse serum in phosphate - buffered saline ( pbs ) . sections were then incubated with a mouse anti - ki-67 monoclonal antibody ( clone mib-1 , dako ) at a 1:100 dilution in pbs for 60 min at room temperature . the secondary antibody dilution and avidin - biotin complex solution ( vectastain abc kit , vector , burling , ca , u.s.a . ) were applied to the sections and incubated for 30 min each at room temperature . 3 - 3-diaminobenzidine solution and mayer s hematoxylin were used as chromogen and counterstain , respectively . the apoptotic index ( ai ) was determined as the percentage of apoptotic positive tumor cells that stained brown in pretreatment biopsy samples . the ki-67 labeling index , defined as the percentage of ki-67-positive tumor cells , was counted in five random fields . the ki-67 index was divided into two groups ( high and low ki-67 ) with the level of 40% as the cut point . for statistical analysis , 30 samples were divided into two groups based on the clinical findings ( age , sex , presence of cribriform plate destruction and response to radiation ) . we compared the two indices ( apoptosis and ki-67 ) and analyzed the differences between these groups using t - test for independent samples . the correlation between the ai and ki-67 indices was analyzed according to spearman s correlation method . the cats that had a stage i disease ( single organ affected ) and treated with radiation alone were studied with survival time . survival time of the patient was defined from the starting date of radiation therapy to the time of death . the correlation between the msts and the indices was estimated using the kaplan meier method , and statistically significant differences between survival curves were analyzed using a log - rank method . a p value of < 0.05 ( two - sides ) was considered statistically significant . thirteen of the enrolled cats were neutered males , thirteen were spayed females , two were intact males , and two were intact females . the age of the cats that started treatment with radiation therapy ranged from 4 to 17 years with mean and median of 10.7 and 11 years , respectively . mean weight was 4.2 kg ( range of 2.5 to 8.7 kg , median of 4.7 kg ) . only one pure breed of persian cat was represented in this study , and the others were mixed - breed cats ( n=29 ) . the most common clinical sign at the time of diagnosis was purulent or mucous nasal discharge ( 73.3% ) followed by facial deformity , nose bleeding , sneezing and exophthalmos . fourteen cats ( 46.7% ) were found to have cribriform plate destruction due to the tumor on pre - treatment ct images . the mean of the total skin surface radiation dose was 34.4 gy ( median of 36 gy ; range of 28 to 42 gy ) . thirteen cats also received chemotherapy after radiation therapy , because of having remaining nasal mass or evidence of systemic disease . fourteen cats died owing to distant disease ( kidney , n=12 ; spleen , n=1 ; and brain , n=1 ) , four cats died from local recurrence and brain involvement , three cats died from renal failure but no evidence of renal lymphoma , two cats died of causes other than tumor , three cats died of unknown causes , and four cats were still alive at the time of data analysis . the ai and ki-67-positive rates are shown in table 1table 1.comparison of the apoptotic and ki-67 indices according to clinical findingsnumber of samplesindices ( mean sd ) ( % ) aiki-67age11 years160.92 0.7240.61 13.22>11 years140.98 0.6136.39 15.46genderfemale150.87 0.7035.58 15.39male151.02 0.6341.37 12.42destruction of cribriform plateyes141.05 0.7039.84 15.15no160.87 0.6337.54 13.89response to treatmentcr161.22 0.7744.43 10.76pr90.77 0.2439.64 12.68sd50.44 0.4316.30 9.41significant differences were calculated with the t - test . a ) p<0.05 .. the ai of all pre - treatment biopsy samples in feline nasal lymphoma ranged from 0 to 2.4% , with a median of 0.85% and mean of 0.94% . the mean ai for cr samples was 1.22% , and the mean ais for the pr and sd samples were 0.77% and 0.46% , respectively . a statistically significant difference was observed between the samples from cats with cr and from cats with sd ( p=0.045 ) . the percentage of ki-67-positive tumor cells in all samples ranged from 7.5 to 57.1% ( median of 41.6% and mean of 38.9% ) . the samples from the cats with cr and pr had mean ki-67 index of 44.4% and 39.6% . the ki-67 index in both the cr and pr samples was significantly higher than in the sd samples ( p<0.001 and p=0.008 ) . however , a significant difference was not observed between the samples from cats with cr and pr ( p=0.334 ) . in all samples of feline nasal lymphoma , a significant positive correlation was found between ai and ki-67 index ( r=0.509 ; p=0.005 ) ( fig . 1fig . 1.correlation between apoptotic and ki-67 indices in cats with nasal lymphoma ( r=0.509 ; correlation between apoptotic and ki-67 indices in cats with nasal lymphoma ( r=0.509 ; p=0.005 ) . seventeen cats diagnosed with stage i disease at first diagnosis day and only treated with radiation therapy were estimated with overall survival times . the cut - off levels of the apoptotic and ki-67 indices were 0.9% and 40% , respectively . the respective msts for cats with high ai and low ai were 402.5 days and 135 days , respectively , but there was no statistical difference between the cats with high and low ai ( p=0.202 ; fig . 2.kaplanmeier curves for overall survival time in 17 cats comparing with different levels of apoptotic and ki-67 indices . ( a ) the mst in cats with high ai and low ai was 402.5 days and 135 days , respectively ( p=0.202 ) . ( b ) the mst in cats with high ki-67 and low ki-67 was 582 days and 77 days , respectively ( p=0.015 ) . ) . on the other hand , the cats with a high ki-67 index had significantly longer survival time compared with those with a low ki-67 tumor ( 582 days vs. 77 days ; p=0.015 ; fig . meier curves for overall survival time in 17 cats comparing with different levels of apoptotic and ki-67 indices . ( a ) the mst in cats with high ai and low ai was 402.5 days and 135 days , respectively ( p=0.202 ) . ( b ) the mst in cats with high ki-67 and low ki-67 was 582 days and 77 days , respectively ( p=0.015 ) . nasal lymphoma is the most common nasal tumor in cats [ 4 , 13 , 20 , 23 ] . chemotherapy should be considered as a top priority of treatment in feline nasal lymphoma , because it is generally a systemic disease . previous larger studies of cats with nasal lymphoma that were treated with chemotherapy alone reported to have msts ranged from 98 to 456 days [ 12 , 13 , 30 , 31 , 33 ] . owing to the nature of lymphomas being sensitive to radiation , radiation therapy is also thought to be treatment choice for localized form of lymphomas [ 12 , 28 , 37 ] . a large retrospective study by haney et al . of 97 cats with nasal lymphoma showed that the mst for radiation therapy alone was 456 days . additionally , the nasal lymphoma cats treated with a combination of local radiation therapy and systemic chemotherapy had a mst from 174 to 955 days [ 12 , 28 ] . a small number of studies showed prognostic indicators for feline nasal lymphomas , including anemia , irradiation dose , response and destruction of the cribriform plate [ 12 , 28 ] . to our knowledge , this study is the first report to correlate the rate of apoptosis and cell proliferation in pretreatment biopsy samples , and the outcome of radiation therapy in feline nasal lymphoma . in veterinary medicine , immunohistochemistry has not been widely used to assess the response to irradiation at the cellular level and has not been used to indicate outcome to radiation [ 11 , 21 ] . ionizing radiation may cause dna damage . the cells occur death when the damage of dna is irreparable , which triggers the cellular pathways . mitotic death is the cells failing to mitosis completely and then death , whereas interphase cell death is the irradiated cells undergoing cell death through apoptosis before they enter mitosis , particularly soon after treatment . previous studies suggested that radiation - induced apoptosis closely correlates with radiosensitivity [ 1 , 9 , 22 ] . the tumor with a high spontaneous apoptotic rate would occur apoptosis easily after irradiation . the relationship between apoptosis and outcome in radiation has been studied in several human cancers . a high apoptotic rate is strongly associated with good prognosis in cancer patients treated with radiation therapy [ 1 , 29 , 35 ] . however , there were few studies focused on spontaneous apoptotic rate for a prognostic predictor in lymphomas . apoptosis - induced death is considered major reason in lymphoid cells compared to other type cells after radiation . although it was unsignificant , the spontaneous apoptosis trended to be predictive to response to in vivo irradiation in a study of human non - hodgkin s lymphoma . moreover , one study of feline lymphoma cell lines also suggested that the more radioresistent cell line was prone to a lower ratio of spontaneous apoptosis . the tunel method is a common technique used for detecting dna fragmentation associated with apoptosis . tunel was used in this study , and the ai was considered as a response predictor for feline nasal lymphomas . the tumor achieving cr showed significantly higher rate of apoptosis than which with sd ( mean of 1.22% vs. 0.46% ; p=0.045 ) . although no significant difference was observed , the cats with a high spontaneous apoptosis rate in the pretreated biopsy sample trended to a longer survival time ( mst of 402.5 vs. 135 days ) . some proteins also have been linked to radiosensitivity , such as p53 , bcl-2 and survivin . p53 gene contributed to the delay of induction of apoptosis in the more radioresistant human lymphoid cell lines . cell nuclei were more likely to appear swelling and multi - nucleated after irradiation in the feline lymphocyte cell line that appeared to be resistant to radiation - induced apoptosis . it is possible that some lymphoma cells may die via mitotic death after radiation . some human cancers have been shown to have higher ki-67 tumors and were linked to better outcomes with radiation therapy [ 2 , 7 , 10 , 18 ] . a study on feline squamous cell carcinoma also found that cats with higher ki-67 had markedly favorable prognosis than cats with lower ki-67 . nevertheless , no significant difference was observed in canine nasal carcinomas . cell cycle of g0 phase is also considered to be a radioresistant period in cells . thus , a low level of ki-67 in tumor cells is believed to reflect hypoxia and radioresistance [ 7 , 16 ] . ki-67 expression detects the proliferating cells , which has also been reported in feline and canine lymphomas [ 8 , 17 ] . the rapidly proliferating tissues may have more radiosensitivity than those growing slowly . in our study , cats with a good response had a higher ki-67 index than cats with a poor response , by a statistically significant margin ( cr and pr vs. sd ) . moreover , cats with a higher level of ki-67 treated with radiation therapy had significantly longer mst than cats with a low ki-67 ( 582 vs. 77 days , respectively ) . despite the relation between proliferation and prognosis being suggested , the reason of this relationship is necessary to be clarified by a further study in lymphomas . in our study , a significant positive correlation was observed between the rate of spontaneous apoptosis and ki-67-positive cells in feline nasal lymphomas ( r=0.509 ; p=0.005 ) . high apoptotic factors have been associated with high proliferation indices in canine lymphomas and human non - hodgkin s lymphomas [ 19 , 24 ] . many of the genes that control cell death also regulate proteins involved in cell division , such as p53 . therefore , cell apoptosis and proliferation control appear to be closely linked with each other [ 15 , 24 , 34 ] . faster proliferating tumors usually indicate better response to irradiation , and higher apoptosis levels may also reflect rapidly proliferating tumors . our results show that the spontaneous apoptotic and ki-67 indices are both strong predictors of response to radiation therapy in feline nasal lymphomas . in conclusion , although our study was retrospective and the study was limited in population size , the response predictive power of measuring apoptosis and proliferation was strong . assessment of apoptosis and proliferation before radiation therapy could be helpful for decision making to decide an appropriate protocol of treatment . because there are many regulators of apoptosis and proliferation in tumor cells , and because radiosensitivity of cancer treatment is not clearly known , additional larger studies would be necessary in order to increase the benefit of treatment .

nasal lymphoma is the most common nasal tumor in cats and is generally a solitary and radiosensitive tumor . we retrospectively evaluated the response to radiation and survival time in relation to apoptosis and ki-67 indices in feline nasal lymphomas treated with radiation therapy . the apoptotic and ki-67 indices were evaluated with tunel and immunohistochemical staining in 30 biopsy tissues that were taken before any treatment . these two indices were compared , and differences between different treatment response groups were analyzed . the correlation between the median survival times ( mst ) and the indices was estimated using the kaplan meier method , and statistical differences between survival curves were analyzed using a log - rank method . with regard to apoptotic index , a statistical difference was observed between the samples taken from cats with complete response and stable disease ( 1.22% vs. 0.45% ; p=0.045 ) . the ki-67 index in cats with both complete response and partial response was significantly higher than in cats with stable disease ( 44.4% and 39.6% vs. 16.3% ; p<0.001 and p=0.008 , respectively ) . the cats with a high level of apoptosis ( > 0.9% ) nasal lymphoma were not significantly prolonged msts ( p=0.202 ) , however , high ki-67-positive ( > 40% ) cats experienced a statistically significant relationship with longer survival time ( p=0.015 ) . our results indicate that spontaneous apoptotic and ki-67 indices are strong predictors for response to radiation therapy in feline nasal lymphomas .

the online version of this article ( doi:10.1007/s00251 - 012 - 0640 - 2 ) contains supplementary material , which is available to authorized users . ( docx 27 kb ) peptide spot array sequences of the three rhesus macaque kir3d proteins . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

killer immunoglobulin - like receptors ( kirs ) represent a highly polymorphic and diverse gene family in rhesus macaques . analyses of the respective gene products have been hampered until now due to non - availability of specific monoclonal antibodies and failure of cross - reactivity of anti - human kir antibodies . we utilised one activating ( kir3dsw08 ) and two inhibitory ( kir3dlw03 and kir3dl05 ) rhesus macaque kir - fc fusion proteins for generation of monoclonal antibodies in mice . besides broadly reacting ones , we obtained anti - rhesus macaque kir antibodies with intermediate and with single specificity . these monoclonal antibodies were tested for binding to a panel of rhesus macaque kir proteins after heterologous expression on transiently transfected cells . epitope mapping identified two polymorphic regions that are located next to each other in the mature kir proteins . the availability of monoclonal antibodies against rhesus macaque kir proteins will enable future studies on kir at the protein level in rhesus macaques as important animal models of human infectious diseases.electronic supplementary materialthe online version of this article ( doi:10.1007/s00251 - 012 - 0640 - 2 ) contains supplementary material , which is available to authorized users .

between march 2007 and june 2010 , 108 overweight and obese subjects without signs and symptoms of cardiac , vascular , renal , metabolic , and gastrointestinal diseases completed a 6-month hypocaloric diet ( 30% energy intake ) with either reduced carbohydrate or reduced fat content . we excluded subjects reporting more than 2 h of physical activity per week , subjects consuming > 20 g / day of alcohol , and pregnant or nursing women . all subjects who completed the study were invited to participate in a long - term follow - up . our institutional review board approved the study and written informed consent was obtained before entry . the follow - up investigation presented here is part of the b - smart study ( clinical trial reg . nct00956566 , clinicaltrials.gov ) , which compared weight loss and changes of associated metabolic and cardiovascular markers in hypocaloric diets with reduced carbohydrates and reduced fat . the randomized 6-month dietary intervention consisted of individual and group dietary counseling with the goals of reducing energy intake by 30% with a minimum of 1,200 kcal / day and adherence to one diet or the other . the reduced carbohydrate diet contained 90 g carbohydrates , 0.8 g protein / kg body weight , and 30% fat . the reduced - fat diet contained 20% fat , 0.8 g protein / kg body weight , and the remaining energy content comprised carbohydrates . all subjects who completed the 6-month diet the only contacts during follow - up were occasional telephone calls to monitor weight changes and to invite subjects to the final examination . the follow - up visit occurred between 17 and 36 months after completion of the supervised dietary intervention ( supplementary fig . all anthropometric , metabolic , and magnetic resonance imaging studies were conducted in an academic clinical research center with the same equipment , scientific staff , and protocols as applied during the baseline and postdiet measurements ( 11 ) . we measured body weight , waist circumference , and height in a standardized manner after an overnight fast . we obtained blood samples at baseline and at 15 , 30 , 45 , 60 , 90 , and 120 min after glucose ingestion ( 75 g glucose/300 ml ; oral glucose tolerance test [ ogtt ] ) to measure glucose and insulin . participants provided a 7-day food protocol that was analyzed for energy intake and macronutrient content using optidiet ( v3.1.0.004 ; goe , linden , germany ) , an analysis software based on nutritional content of food as provided by the german national food key . a clinical 1.5-t magnetic resonance scanner ( sonata and avanto ; siemens medical solution ag , erlangen , germany ) was used to measure abdominal subcutaneous and visceral fat mass as well as liver fat content as previously described ( 14 ) . briefly , we applied a t1-weighted , water - suppressed , gradient echo technique ( repetition time , 80 ms ; echo time , 6.11 ms ; 512 512 matrix ; field of view , 500 500 mm ; slice thickness , 10 mm ; interslice gap , 10 mm ) to image abdominal fat during repetitive breath - holds . we quantified visceral and subcutaneous adipose tissue by semi - automated image segmentation software using a contour - following algorithm ( vitom ) . in addition , we measured intrahepatic lipids by respiratory - gated h spectrometry ( spin - echo : repetition time according to respiratory cycle ( > 5 s ) ; echo time , 30 ms ) . unsuppressed spectra were acquired in end - expiration from a single 30- 30- 20-mm voxel located at liver segment 7 ( 24 averages ) . intrahepatic lipid content was quantified using peak areas and expressed ( as percent ) as fat ( fat + water ) . glucose ( mmol / l ) , insulin ( u / ml ) , lipoproteins , alanine aminotransferase ( u / l ) , aspartate aminotransferase ( u / l ) , and -glutamyltransferase ( u / l ) were determined by standard methods in a certified clinical chemistry laboratory . homeostasis model assessment of insulin resistance was calculated from fasting insulin and glucose by the following equation : ( insulin [ u / ml ] glucose [ mmol / l ] ) 22.5 ) . whole - body insulin sensitivity was calculated by the composite insulin sensitivity index ( 15 ) . composite insulin sensitivity index = 10,000 [(fasting plasma glucose fasting plasma insulin ) ( glucose insulin ) ] ; fasting plasma glucose was expressed as mg / dl and fasting plasma insulin was expressed as u / ml , and glucose ( mg / dl ) and insulin ( u / ml ) were the mean glucose and mean insulin concentrations during the glucose load . this approach has been validated in nondiabetic subjects by using euglycemic insulin clamp testing including labeled glucose administration ( 16 ) . data were first tested for normal distribution and variance homogeneity with kolmogorov - smirnov test and the levene test , respectively . differences between time points ( baseline , after diet , follow - up ) were analyzed using anova for repeated measures with bonferroni post hoc test . univariate associations between parameters were described by pearson correlation coefficient . to test for interactions between diet groups or weight loss groups over time , we used two - way anova for repeated measures and bonferroni post hoc test . to identify independent predictors of hepatic fat content at baseline and of long - term reduction in hepatic fat after the dietary intervention , we conducted multivariate linear regression analyses . all statistical analyses were performed with spss 18 ( spss , chicago , il ) . unless otherwise stated , values are given as mean sd . post hoc power analysis was calculated with g*power 3.1.7 ( http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3 ) . between march 2007 and june 2010 , 108 overweight and obese subjects without signs and symptoms of cardiac , vascular , renal , metabolic , and gastrointestinal diseases completed a 6-month hypocaloric diet ( 30% energy intake ) with either reduced carbohydrate or reduced fat content . we excluded subjects reporting more than 2 h of physical activity per week , subjects consuming > 20 g / day of alcohol , and pregnant or nursing women . all subjects who completed the study were invited to participate in a long - term follow - up . our institutional review board approved the study and written informed consent was obtained before entry . the follow - up investigation presented here is part of the b - smart study ( clinical trial reg . nct00956566 , clinicaltrials.gov ) , which compared weight loss and changes of associated metabolic and cardiovascular markers in hypocaloric diets with reduced carbohydrates and reduced fat . the randomized 6-month dietary intervention consisted of individual and group dietary counseling with the goals of reducing energy intake by 30% with a minimum of 1,200 kcal / day and adherence to one diet or the other . the reduced carbohydrate diet contained 90 g carbohydrates , 0.8 g protein / kg body weight , and 30% fat . the reduced - fat diet contained 20% fat , 0.8 g protein / kg body weight , and the remaining energy content comprised carbohydrates . all subjects who completed the 6-month diet the only contacts during follow - up were occasional telephone calls to monitor weight changes and to invite subjects to the final examination . the follow - up visit occurred between 17 and 36 months after completion of the supervised dietary intervention ( supplementary fig . all anthropometric , metabolic , and magnetic resonance imaging studies were conducted in an academic clinical research center with the same equipment , scientific staff , and protocols as applied during the baseline and postdiet measurements ( 11 ) . we measured body weight , waist circumference , and height in a standardized manner after an overnight fast . we obtained blood samples at baseline and at 15 , 30 , 45 , 60 , 90 , and 120 min after glucose ingestion ( 75 g glucose/300 ml ; oral glucose tolerance test [ ogtt ] ) to measure glucose and insulin . participants provided a 7-day food protocol that was analyzed for energy intake and macronutrient content using optidiet ( v3.1.0.004 ; goe , linden , germany ) , an analysis software based on nutritional content of food as provided by the german national food key . a clinical 1.5-t magnetic resonance scanner ( sonata and avanto ; siemens medical solution ag , erlangen , germany ) was used to measure abdominal subcutaneous and visceral fat mass as well as liver fat content as previously described ( 14 ) . briefly , we applied a t1-weighted , water - suppressed , gradient echo technique ( repetition time , 80 ms ; echo time , 6.11 ms ; 512 512 matrix ; field of view , 500 500 mm ; slice thickness , 10 mm ; interslice gap , 10 mm ) to image abdominal fat during repetitive breath - holds . we quantified visceral and subcutaneous adipose tissue by semi - automated image segmentation software using a contour - following algorithm ( vitom ) . in addition , we measured intrahepatic lipids by respiratory - gated h spectrometry ( spin - echo : repetition time according to respiratory cycle ( > 5 s ) ; echo time , 30 ms ) . unsuppressed spectra were acquired in end - expiration from a single 30- 30- 20-mm voxel located at liver segment 7 ( 24 averages ) . intrahepatic lipid content was quantified using peak areas and expressed ( as percent ) as fat ( fat + water ) . glucose ( mmol / l ) , insulin ( u / ml ) , lipoproteins , alanine aminotransferase ( u / l ) , aspartate aminotransferase ( u / l ) , and -glutamyltransferase ( u / l ) were determined by standard methods in a certified clinical chemistry laboratory . homeostasis model assessment of insulin resistance was calculated from fasting insulin and glucose by the following equation : ( insulin [ u / ml ] glucose [ mmol / l ] ) 22.5 ) . whole - body insulin sensitivity was calculated by the composite insulin sensitivity index ( 15 ) . composite insulin sensitivity index = 10,000 [(fasting plasma glucose fasting plasma insulin ) ( glucose insulin ) ] ; fasting plasma glucose was expressed as mg / dl and fasting plasma insulin was expressed as u / ml , and glucose ( mg / dl ) and insulin ( u / ml ) were the mean glucose and mean insulin concentrations during the glucose load . this approach has been validated in nondiabetic subjects by using euglycemic insulin clamp testing including labeled glucose administration ( 16 ) . data were first tested for normal distribution and variance homogeneity with kolmogorov - smirnov test and the levene test , respectively . differences between time points ( baseline , after diet , follow - up ) were analyzed using anova for repeated measures with bonferroni post hoc test . univariate associations between parameters were described by pearson correlation coefficient . to test for interactions between diet groups or weight loss groups over time , we used two - way anova for repeated measures and bonferroni post hoc test . to identify independent predictors of hepatic fat content at baseline and of long - term reduction in hepatic fat after the dietary intervention , we conducted multivariate linear regression analyses . all statistical analyses were performed with spss 18 ( spss , chicago , il ) . values are given as mean sd . post hoc power analysis was calculated with g*power 3.1.7 ( http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3 ) . fifty subjects had complete data sets at follow - up and were included in the analysis ( table 1 ) . the time between completion of diet and follow - up visit was 24 6 months ( range , 1736 ) . the long noninterventional follow - up period was associated with a substantial discontinuation rate ( 54 of 108 eligible subjects participated in the follow - up visit ) . approximately half of the subjects changed housing and contact details without notifying the investigators ( lost to contact ) . the other subjects withdrew consent to further participation because of time constraints or disinterest in further scientific evaluations . to test the possibility that the high discontinuation rate introduced a substantial bias , we compared participants and nonparticipants with respect to baseline data and changes at 1 , 3 , and 6 months after diet initiation ( supplementary table 1 ) . subjects who discontinued the follow - up period had similar age , baseline bmi , and improvements in anthropometric and metabolic parameters during the active intervention period compared with those who participated in the follow - up evaluation . anthropometric and metabolic variables after the 6-month diet intervention and at follow - up 1736 months after diet total energy intake was reduced during the dietary intervention period ( p < 0.01 ) and remained reduced at follow - up ( baseline : 2,245 619 kcal / d ; end of diet : 1,720 450 kcal / d ; follow - up : 1,836 468 , we conducted a multivariate regression analysis with age , sex , bmi , insulin sensitivity , total cholesterol , ldls , hdls , triglycerides , free fatty acids , visceral and subcutaneous abdominal fat mass , cardiorespiratory fitness ( vo2max ) , total energy intake , and percentage of fat and carbohydrate intake relative to total energy intake . the analysis revealed only visceral fat mass ( = 0.64 ; p = 0.006 ) as an independent predictor of baseline hepatic fat content . we calculated weight regain as the difference between body weight at follow - up and body weight after the active dietary intervention period at 6 months . after successful reduction of body weight and bmi with dietary intervention , we observed a significant weight regain at follow - up ( table 1 and fig . 1 ) . the concomitant increase in visceral and subcutaneous abdominal fat mass followed a parallel trend ( fig . , intrahepatic lipids decreased during the dietary intervention but remained reduced at follow - up ( fig . body weight regain was correlated with a regain in visceral ( r = 0.70 ; p < 0.001 ) or subcutaneous abdominal adipose tissue ( r = 0.90 ; p < 0.001 ) , but less so with increased hepatic fat content ( r = 0.30 ; p < 0.05 ) . long - term reduction of hepatic lipids was associated with sustained improvements in serum alanine aminotransferase , aspartate aminotransferase , and -glutamyltransferase activities ( table 1 ) . all measured data and calculated variables improved with the active diet and remained so at follow - up ( table 1 and fig . when analyzed separately for subjects initially randomized to reduced - carbohydrate or reduced - fat diets , all analyzed variables changed similarly in both groups over time . bmi , abdominal fat , and intrahepatic fat after 6-month diet and at follow - up . changes in bmi and subcutaneous abdominal adipose tissue ( at ) ( upper panel ) and visceral at and intrahepatic lipids ( lower panel ) in 50 overweight and obese subjects . magnetic resonance studies were performed at baseline , after a 6-month hypocaloric diet , and at long - term follow - up . * p < 0.01 compared with baseline . p < 0.01 compared with end of diet . group comparison by one - way anova for repeated measures with bonferroni post hoc test . data are mean sem . glucose and insulin concentrations during ogtt after 6-month diet and at follow - up time course and areas under the curve ( auc ) for glucose ( upper panel ) and insulin ( lower panel ) during a 2-h ogtt ( n = 46 ) . the test was performed at baseline , after a 6-month hypocaloric diet , and at long - term follow - up . * p < 0.01 compared with baseline . group comparison by one - way anova for repeated measures and bonferroni post hoc test . hepatic fat reduction is particularly relevant in subjects with nonalcoholic fatty liver disease ( nafld ) . we stratified subjects into a group with nafld and a group without nafld at baseline ( hepatic fat > 5.6% or < 5.6% ; table 2 ) . both groups lost similar amounts of body weight and visceral and subcutaneous abdominal fat mass with the same total energy intake reduction during dietary intervention . subjects with nafld showed sustained improvement in hepatic fat content and liver function despite modest weight regain during follow - up . changes with 6-month diet and at long - term follow - up in subjects stratified for the presence of nafld at baseline we conducted a multivariate regression analysis using age , sex , baseline bmi , type of diet , changes in body weight with diet , changes in visceral and subcutaneous fat mass with diet , changes in hepatic fat with diet , changes in total cholesterol with diet , and changes in insulin sensitivity with diet as independent variables ; changes from baseline to long - term follow - up of liver fat was used as the dependent variable . only changes in total body weight with diet ( = 0.31 ; p = 0.02 ) and changes in hepatic fat with diet ( = 0.86 ; p < 0.001 ) predicted long - term intrahepatic fat loss . the model explained 68% of the total variation in the observed long - term liver fat reduction . the important and novel finding of our study is a sustained improvement in hepatic fat content , liver function tests , and insulin resistance over > 2 years after a 6-month hypocaloric diet in overweight and obese subjects . all improvements occurred despite regain of body weight , abdominal visceral adipose tissue , and subcutaneous adipose tissue mass during follow - up . our findings highlight the beneficial long - term effects of a well - controlled dietary lifestyle intervention on hepatic fat content and metabolism . furthermore , our findings suggest that the benefit of dietary interventions should not be judged solely by anthropometric measurements . in fact , reductions in caloric intake may have weight - independent effects on liver fat and metabolism . various types of caloric restriction are effective in decreasing body weight ( 13 ) and fat mass ( 17 ) . whereas short - term reductions of body weight and fat mass can be impressive , most studies with long - term follow - up were discouraging and reported at least partial body weight regain within 12 years ( 13 ) . data regarding changes of visceral adipose tissue and ectopic fat storage during long - term follow - up are scarce . the issue is relevant because visceral adipose tissue is independently associated with cardiovascular and metabolic risk ( 1 ) . furthermore , ectopic fat storage in liver , skeletal muscle , pancreas , and the heart adversely affects organ function through a mechanism often referred to as lipotoxicity . intrahepatic fat content is an important predictor of whole - body insulin resistance , increased secretion rates of vldls , and progression to type 2 diabetes ( 2,4,18 ) . type 2 diabetes , in turn , increases the risk of progressive liver disease ( e.g. , nonalcoholic steatohepatitis , cirrhosis , and hepatocellular carcinoma ) ( 1921 ) . our study is the first to assess abdominal fat mass and liver fat several months after diet - induced weight loss and has scientific as well as clinical implications . abdominal visceral adipose tissue drains into the portal vein and the liver is exposed to large amounts of free fatty acids derived from this metabolically active fat depot . in our study , hepatic fat at baseline was strongly related to visceral fat mass . yet , visceral adipose tissue mass may not be the sole determinant of how hepatic fat responds to dietary interventions ( 22 ) . short - term studies suggested that hepatic fat accumulation is , at least in part , regulated independently of body weight and abdominal visceral fat mass . in obese subjects , caloric restriction decreased hepatic fat by 1030% within 48 h ( 23 ) . furthermore , < 5% weight loss decreased hepatic fat by 2840% ( 2426 ) . finally , aerobic exercise training ameliorated hepatic fat content while body weight ( 27 ) and visceral fat mass ( 28 ) remained stable . our study extends these observations and suggests that differential regulation of adipose tissue mass and hepatic fat stores may not be restricted to the acute weight loss period . moreover , our observations further support the idea that intrahepatic fat is independently associated with metabolic risk ( 4,5,29 ) . there is an ongoing debate whether hepatic insulin resistance is primarily mediated through fatty acid and adipokine release from expanded and dysfunctional visceral adipose tissue or through generation of intrahepatic lipid mediators , such as diacylglycerols and ceramides that directly interfere with insulin signaling ( 30 ) . our finding that insulin sensitivity remained improved during follow - up despite the weight and fat mass regain strengthens the notion that intrahepatic lipids and their metabolites are mediators of hepatic insulin resistance . we observed a constantly reduced energy intake during follow - up but regain of body weight and fat mass . a possible explanation is that weight loss and caloric restriction led to compensatory reductions in metabolic rate ( 31 ) . approximately 80% of liver fatty acids originate from circulating fatty acids ( 32 ) that are mobilized predominantly from adipose tissue . improved insulin resistance with weight loss attenuates adipose tissue lipolysis and enhances fatty acid storage within the large adipose tissue depots . fatty acids are taken - up into liver and adipose tissue through fatty acid transport proteins and fat / cd36 ( 33 ) . in obese subjects , intrahepatic lipid content determined the expression of fatty acid transporters and fat / cd36 in liver and adipose tissue . fatty acid transporters were expressed to a lesser extent in the liver and to a higher extent in adipose tissue when subjects had normal liver fat content , and this was reversed in subjects with high liver fat content , suggesting that fatty acid flux from adipose tissue to the liver determines liver fat content ( 33 ) . overall , our study suggests that successful weight loss achieved through hypocaloric dieting improves fatty acid flux from the liver toward the primary storage site in the long - term . the same pattern , rerouting of free fatty acids from the liver toward adipose tissue together with improved insulin sensitivity , also has been observed with thiazolidinedione treatment ( 34 ) . our clinically important observation is that a controlled 6-month intervention elicited sustained lifestyle changes in a surprisingly large proportion of our subjects . thus , important lifestyle improvements may be obscured if the focus is on weight changes only . we also observed that similar sustained improvements in hepatic fat , markers of liver function , and insulin resistance occurred in subjects initially assigned to carbohydrate - reduced or fat - reduced diets . this finding extends our results observed at the end of the 6-month diet period ( 11 ) and suggests that changes in energy balance may be more important than changes in dietary composition for sustained hepatic fat reduction . also clinically important is the observation that subjects fulfilling diagnostic criteria of nafld had a particularly pronounced reduction in hepatic fat over time . nafld affects up to 30% of adults in developed countries ( 35 ) and is related to the ongoing obesity epidemic ( 3 ) . the condition is an independent risk factor for cardiovascular disease and type 2 diabetes , predisposes to nonalcoholic steatohepatitis , and may progress to cirrhosis and hepatic cancer ( 6 ) . the 45% reduction of hepatic fat with hypocaloric diets observed in this study is a particular benefit in subjects with nafld . our findings suggest that this important improvement in hepatic liver fat can be maintained in the long - term even when body weight is partly regained . the major limitation of our study is that 50% of the subjects who had finished the active intervention period discontinued participation during the follow - up period . discontinuation rates ranged between 33 and 50% in two recent trials reporting 1-year treatment data for food and drug administration approved weight loss drugs ( 36,37 ) . in these studies , discontinuation occurred during the active treatment period , whereas our participants discontinued during an observational period after the active intervention while contact was maintained through occasional telephone calls only . clearly , more close contact may have increased the retention rate ; however , the long - term value of our weight loss program for the influence on hepatic steatosis could not have been assessed as planned because close and regular contact represents an intervention by itself . instead , we wanted to determine the impact of the intervention with the least interaction with subjects as possible . this study design , however , resulted in the problem of losing contact with approximately half of the subjects who discontinued participation . the other half discontinued by withdrawing consent ; however , if we were informed correctly by these subjects , withdrawn consent was not related to unsuccessful weight loss or any other undesired developments during follow - up . based on mathematical models estimating discontinuation rates in dietary intervention studies ( 38,39 ) , however , subjects more successful at losing weight and maintaining their diets could be more motivated to participate in follow - up investigations . as shown in supplementary table 1 , subjects who discontinued participation did not differ from participants included in the follow - up analysis in terms of baseline anthropometric measurements , metabolism , or early and sustained weight loss success during the intervention period . furthermore , we observed no differences in sex distribution between participants and those who discontinued study participation . the analysis suggests that discontinuation was not limited to a specific patient population , thus rendering a major bias less likely . to test whether the sample size was sufficiently large to assess the reduction in intrahepatic fat from baseline to 24-month follow - up , we conducted a post hoc power calculation . with a statistical power of 80% , a sample size of 33 would have been sufficient to detect the observed difference in liver fat of 3.1 6.1% with a one - sided -error of 0.025 . given the inclusion of 50 subjects in our study , the probability of the detected difference with a one - sided -error of 0.025 was 94% . nevertheless , losing 50% of participants could have introduced bias ; therefore , results should be interpreted with caution . thus , we can not prove that the sustained improvement in hepatic fat in our study was associated with beneficial changes in hepatic inflammation and fibrosis . in contrast to steatosis , advanced liver fibrosis is less amenable to therapeutic interventions ( 35 ) . second , we are aware that hyperinsulinemic euglycemic clamping would have provided more direct information regarding insulin sensitivity compared with ogtt . because of the possibility of underreporting , the magnitude of the reduced caloric intake after diet and at follow - up needs to be interpreted with caution ( 40 ) . despite these limitations , particularly the high discontinuation rate , we conclude that hypocaloric dietary interventions have a long - lasting effect on intrahepatic lipid accumulation despite weight regain . because obesity management programs commonly focus on body weight and anthropometric measurements , beneficial long - term effects of dietary interventions on human metabolism may be underestimated . we suggest that the beneficial response to reduced caloric intake in our study may be driven in large part by changes in liver metabolism .

objectiveweight loss reduces abdominal and intrahepatic fat , thereby improving metabolic and cardiovascular risk . yet , many patients regain weight after successful diet - induced weight loss . long - term changes in abdominal and liver fat , along with liver test results and insulin resistance , are not known.research design and methodswe analyzed 50 overweight to obese subjects ( 46 9 years of age ; bmi , 32.5 3.3 kg / m2 ; women , 77% ) who had participated in a 6-month hypocaloric diet and were randomized to either reduced carbohydrates or reduced fat content . before , directly after diet , and at an average of 24 ( range , 1736 ) months follow - up , we assessed body fat distribution by magnetic resonance imaging and markers of liver function and insulin resistance.resultsbody weight decreased with diet but had increased again at follow - up . subjects also partially regained abdominal subcutaneous and visceral adipose tissue . in contrast , intrahepatic fat decreased with diet and remained reduced at follow - up ( 7.8 9.8% [ baseline ] , 4.5 5.9% [ 6 months ] , and 4.7 5.9% [ follow - up ] ) . similar patterns were observed for markers of liver function , whole - body insulin sensitivity , and hepatic insulin resistance . changes in intrahepatic fat und intrahepatic function were independent of macronutrient composition during intervention and were most effective in subjects with nonalcoholic fatty liver disease at baseline.conclusionsa 6-month hypocaloric diet induced improvements in hepatic fat , liver test results , and insulin resistance despite regaining of weight up to 2 years after the active intervention . body weight and adiposity measurements may underestimate beneficial long - term effects of dietary interventions .

together with the ability of generating a large amount of data per experiment , high - throughput technologies also brought the challenge of translating such data into a better understanding of the underlying biological phenomena . first released in 2001 , onto - tools is a freely available web - accessible software suite that addresses some of these challenges . the onto - tools suite includes : ( i ) onto - express used to translate lists of differentially regulated genes into a better understanding of the underlying biological phenomena ( 15 ) ; ( ii ) onto - design used to select the best set of genes to be included on a custom microarray designed for the study of a given biological phenomenon ( 2,4 ) ; ( iii ) onto - compare used to analyze the functional bias of various focused commercial microarrays and select the one that is most appropriate for a given biological hypothesis ( 2,6 ) ; ( iv ) onto - translate used to translate lists of genes from one reference system to another ( e.g. from genbank accession numbers to unigene cluster ids to affymetrix probe ids , etc . ) ( 2,5,7,8 ) ; ( v ) onto - miner provides a unified access point and an application programming interface ( api ) allowing queries for various information such as the gene name , official symbol , reference accession number , coded protein , etc . ( 4 ) ; ( vi ) promoter - express which allows the users to find condition - specific transcription factor binding sites ( tfbss ) ( 7,9 ) and ( vii ) nssnpcounter which allows analysis of synonymous and non - synonymous codon substitutions in protein coding genes ( 7 ) . previous publications have described in detail the motivation , implementation and validation of these tools . the logical workflow between the onto - tools applications has also been previously explained ( 2,4 ) . this article describes two new tools added to the ensemble and discusses other enhancements recently made to the existing tools . onto - express ( oe ) is a web - based tool in the onto - tools suite that performs automated function profiling for a list of differentially expressed genes . however , onto - express does not support functional profiling for the organisms that do not have annotations in public domain , or use of custom ( i.e. user - defined ) ontologies . this limitation is also true for most of the other existing tools for functional profiling ( 10 ) , which means that researchers working with uncommon organisms and/or new annotations or ontologies may be forced to construct such profiles manually . onto - express - to - go ( oe2go ) is a new tool added to the onto - tools ensemble to address these issues . , the users now have an option to use either the onto - tools database as a source of functional annotations or provide their own annotations in a separate file ( figure 1 ) . a go - formatted annotation file has 15 tab - delimited columns that contain a database name , a unique i d for an entity being annotated , its corresponding symbol , one or more references supporting an annotation , type of evidence , date of annotation , type of entity ( e.g. a gene or a protein ) , etc . the annotation files for approximately 40 organisms in go format are available for download from go ftp site that can be directly used with oe2go ( ftp://ftp.geneontology.org/pub/go/gene-associations/ ) . figure 1.oe2go input interface for using custom annotations . when the user selects my own annotations , oe2go allows the user to specify the name of the annotation file in go format and the ontology file in obo format . when using custom annotations , oe2go assumes that the identifiers in the input file are of the same type as those used in the annotations file . my own annotations , oe2go allows the user to specify the name of the annotation file in go format and the ontology file in obo format . when using custom annotations , oe2go assumes that the identifiers in the input file are of the same type as those used in the annotations file . as shown in figure 1 , when using custom annotations , the user must also specify the ontology file in obo format . an ontology file in obo format contains a header section and a number of stanzas consisting of a set of tag - value pairs . a tag - value pair consists of a tag name , a colon and a tag value . the header section must be before any stanzas , and contains meta - information about the ontology such as its creation date , default namespace , remarks , format version , etc . typedef , where a term stanza describes an ontology term , and a typedef stanza describes a type of relationship between two terms in the ontology . oe2go uses java code from an open - source tool obo - edit in order to parse the ontology file in obo format . the gene ontology consortium provides an ontology file in obo format that can be directly used with oe2go ( www.geneontology.org/ontology/gene_ontology_edit.obo ) . figure 2.an example obo formatted ontology file that contains a header , a term stanza and a typedef stanza . the stanzas contain a set of tag - value pairs in < tag>:<value > format . the complete list of allowed tags for each type of stanza and the header are described on go web site . an example obo formatted ontology file that contains a header , a term stanza and a typedef stanza . the header and the stanzas contain a set of tag - value pairs in < tag>:<value > format . the complete list of allowed tags for each type of stanza and the header are described on go web site . for each gene in the input file , hence , the functional profiles created by oe2go depend on the information present in the annotation file . if the file does not contain the annotations that are otherwise well known , they are not provided in the oe2go results . the strength of oe2go is that it enables the researchers to use the functional annotations that are not yet in the public domain , by allowing them to be included in the annotation file . another advantage of oe2go is that it enables the researchers to avoid annotation bias ( 10 ) by allowing them to remove the biological processes that are more studied than the others from the annotation file . the automated functional profiling approach , first proposed by onto - express in 2001 , has now become the de facto standard in the second stage analysis of gene expression data ( 10 ) . although this approach is widely adopted , it considers each biological process independent of the others , and ignores dependencies and interactions among them ( 10 ) . at the same time , a number of pathway databases available in public domain describe how genes interact with each other in metabolic and signaling pathways ( e.g. kegg ( 11 ) , biocarta ( www.biocarta.com ) , reactome ( 12 ) , etc . ) . several tools that allow researchers to reveal the pathways associated with a given set of differentially expressed genes already exist ( 1323 ) . pathway - express ( pe ) is a tool in the onto - tools ensemble that is designed to perform a pathway analysis . when a user submits a list of genes , pe searches the onto - tools database and builds a list of all associated pathways . however , pe can analyze any collection of pathways described in sbml ( 24 ) . pe performs a classical enrichment analysis based on a hypergeometric distribution in order to identify those pathways that contain a proportion of differentially expressed genes that is significantly different from what is expected just by chance . this analysis produces a set of p - values that characterize the significance of the pathway from this statistical perspective ( a lower p - value corresponds to a higher significance ) . this perturbation factor takes into consideration the ( i ) normalized fold change of the gene and ( ii ) the number and amount of perturbation of genes upstream of it ( i.e. its position on a pathway ) . advanced options button to specify different weights for different types of interactions between genes on the pathways ( figure 3 ) . as shown in figure 3 , pe uses negative weights for inhibition and repression. this gene perturbation factor reflects the relative importance of each differentially expressed gene on the pathway . the impact factor of the entire pathway is calculated using a probabilistic term that takes into consideration the proportion of differentially expressed genes on the pathway and gene perturbation factors of all genes in the pathway . more details about the gene perturbation factors and pathway impact factors , a comparison with the existing methods , and a full discussion of the advantages and disadvantages of these methods are described elsewhere . pathway - express allows the users to choose advanced options such as the distribution to use for calculating significance of pathways , the type of correction to apply for multiple hypotheses , whether to use p - values or corrected p - values for calculating impact factor , etc . it also allows a user to specify different weights for different types of interactions ( e.g. activation , binding , repression , phosphorylation , etc . ) between genes on the pathways . pathway - express allows the users to choose advanced options such as the distribution to use for calculating significance of pathways , the type of correction to apply for multiple hypotheses , whether to use p - values or corrected p - values for calculating impact factor , etc . it also allows a user to specify different weights for different types of interactions ( e.g. activation , binding , repression , phosphorylation , etc . ) between genes on the pathways . when a user submits a list of input ids , pe converts the list into a list of gene ids using the onto - tools database . the onto - tools database integrates a number of public databases including genbank , dbest , unigene , entrez gene , refseq , kegg , etc . after creating a list of gene ids , pe searches the kegg pathways in the onto - tools database for each input gene , and builds a list of pathways containing at least one input gene . if a gene is known to be involved in a pathway , but is not annotated as such in kegg , pe does not return the pathway in its output . the top left panel displays detailed results for each pathway including : number of input genes and total number of genes on a pathway , probability of obtaining the same number of genes on a given pathway by random chance , impact factor and probability of obtaining the impact factor by random chance for a given pathway , etc . the bar graph in panel a can be sorted in increasing or decreasing order of any column by clicking on the corresponding column header . the bar graph in panel a , pathway details in panel b and input details in panel c are synchronized with each other . for instance , in figure 4 , selecting the apoptosis pathway in panel b , highlights the corresponding bar in red color in panel a and also selects the input genes in panel c ( i.e. genes fadd and rela ) panel b provides details about each pathway including : ( i ) number of genes on the pathway , ( ii ) number of genes found in the reference array that are on the pathway , ( iii ) number of input genes found on the pathway , ( iv ) its impact factor , ( v ) database identifier of the pathway , etc . panel c provides input details including the input i d , its corresponding gene name and symbol , number of pathways the gene is found on , and the fold change as provided by the user . double - clicking a pathway name in panel b provides a list of genes on that pathway in panel d along with their perturbation factor . panel b provides details about each pathway including : ( i ) number of genes on the pathway , ( ii ) number of genes found in the reference array that are on the pathway , ( iii ) number of input genes found on the pathway , ( iv ) its impact factor , ( v ) database identifier of the pathway , etc . panel c provides input details including the input i d , its corresponding gene name and symbol , number of pathways the gene is found on , and the fold change as provided by the user . double - clicking a pathway name in panel b provides a list of genes on that pathway in panel d along with their perturbation factor . right - clicking a mouse in the pe output window brings up a context - sensitive popup menu ( figure 4 ) . for instance , the menu displayed by clicking a mouse on a pathway name allows the user to view the corresponding kegg pathway diagram , download and specify a gml viewer to use with pe , etc . the kegg pathway diagram that corresponds to the selected pathway can also be viewed by double - clicking the pathway name in either panel a or panel b. pe highlights the input genes in red ( up - regulated genes ) or blue ( down - regulated ) in the kegg diagram ( figure 5 ) . the popup menu also allows to save a pathway in gml format that can be viewed in any gml viewer . if a program able to read gml files ( e.g. yed ( www.yed.com ) or cytoscape ( 21 ) ) is already installed on the local machine , the user can specify its location by selecting set gml viewer command from the popup menu . after specifying the gml viewer , a user can select show pathway graph to access gml representation of the pathway from within pe . all tables in the pe output can be saved as a tab - delimited text file by selecting figure 5.the apoptosis pathway as described by kegg ( panel a ) and its internal representation , as constructed by pathway - express ( panel b ) . pe can save this internal representation in a gml file that can be exported to any other visualization application . in this internal representation , each node corresponds to a gene , and each edge represents an interaction between two genes . in this example , the up - regulated input gene fadd is highlighted with red color and down - regulated gene rela is highlighted with blue color in both panels . the internal graph also shows how pe propagates the perturbation through the pathway , where the direction of an arrow represents the direction of propagation . note that in some cases , a gene in kegg diagram may represent a group of genes . for instance , gene pi3k in the kegg diagram in fact corresponds to a group of eight nodes in pe 's internal representation : pik3r5 , pik3ca , pik3cb , pik3cd , pik3cg , pik3r1 , pik3r2 and pik3r3 . the apoptosis pathway as described by kegg ( panel a ) and its internal representation , as constructed by pathway - express ( panel b ) . pe can save this internal representation in a gml file that can be exported to any other visualization application . in this internal representation , each node corresponds to a gene , and each edge represents an interaction between two genes . in this example , the up - regulated input gene fadd is highlighted with red color and down - regulated gene rela is highlighted with blue color in both panels . the internal graph also shows how pe propagates the perturbation through the pathway , where the direction of an arrow represents the direction of propagation . note that in some cases , a gene in kegg diagram may represent a group of genes . for instance , gene pi3k in the kegg diagram in fact corresponds to a group of eight nodes in pe 's internal representation : pik3r5 , pik3ca , pik3cb , pik3cd , pik3cg , pik3r1 , pik3r2 and pik3r3 . figure 5 also shows the internal representation of the apoptosis pathway ( read from the gml file ) with the two input genes represented by elliptic nodes . pe 's internal representation also shows how perturbation introduced by the genes fadd and rela is propagated throughout the pathway in the given condition . the perturbed genes on the apoptosis pathway are shown with colored background , whereas the unperturbed genes are shown with white background , and the direction of the propagation is indicated by an arrow between two genes . in figure 5 , notice that only the area of the pathway downstream of the input genes is perturbed , while the rest of the pathway is unperturbed . over the past year , onto - miner ( om ) has been reimplemented as a java tool to make its interface user friendly and consistent with the onto - tools suite . the previous html input interface of om is replaced by a new java interface that is easier to use ( figure 6 ) . unlike previous version of om that required the user to manually download the results from the server , om now automatically downloads the result file from the onto - tools server and saves it in the location specified by the user . om automatically retrieves the results from the onto - tools server and saves them into the output file specified by the user . om automatically retrieves the results from the onto - tools server and saves them into the output file specified by the user .

onto - tools is a freely available web - accessible software suite , composed of an annotation database and nine complementary data - mining tools . this article describes a new tool , onto - express - to - go ( oe2go ) , as well as some new features implemented in pathway - express and onto - miner over the past year . pathway - express ( pe ) has been enhanced to identify significantly perturbed pathways in a given condition using the differentially expressed genes in the input . oe2go is a tool for functional profiling using custom annotations . the development of this tool was aimed at the researchers working with organisms for which annotations are not yet available in the public domain . oe2go allows researchers to use either annotation data from the onto - tools database , or their own custom annotations . by removing the necessity to use any specific database , oe2go makes the functional profiling available for all organisms , with annotations using any ontology . the onto - tools are freely available at http://vortex.cs.wayne.edu/projects.htm .

health care and old - age care in the western world are facing demographical and financial challenges [ 13 ] . an increasing proportion of elderly people in the population , improved conditions of living , and medical technological advances during the 20 century have raised the demands and expectations on the services provided by the health care system [ 46 ] . medical advances have made it possible to move health care partly from hospital to primary care , and often directly into patients ' private homes [ 7 , 8 ] . today both rehabilitation and advanced palliative care can be provided in patients ' private homes . the boundary between health care institutions and homes has been displaced and sometimes almost erased . the transition from hospital to community care is a general international trend [ 7 , 10 , 11 ] . policy makers in europe recommend home care and , wherever possible , a reduction in the number of residential homes . home care consists of two different kinds of activities : home care services and home help services . home care services means provision of medical treatment by trained medical and nursing personnel ( or delegated to home helpers ) in patients ' private homes . home help services , on the other hand , entails provision of social services , and involves domestic work and personal services . previously , the old - age care structure was similar in many countries : medical care was provided in hospitals and social care in the community . the deinstitutionalisation has raised the expectations of overcoming the traditional problems of integration of medical and social aspects of care . attempts have been made , and are still in progress , to integrate health and social care in several countries [ 1419 ] . the purpose of integrated care is to provide care without service gaps , fragmentation or lack of cooperation . ageing is connected with complex and often interrelated problems comprising physical , psychological and social health . this complexity calls for collaboration between and integration of health and social care [ 20 , 21 ] . identify three different approaches to integrated care : structure , process , and outcome . of these structural integration is mainly a matter of organizational structure ( e.g. to bring together staff and resources in one single organization under a unified hierarchical structure ) . sweden is by some researchers regarded to be among the most advanced countries with respect to structural integration , mainly due to the introduction of a major organizational reform in 1992 [ 22 , 23 ] . the swedish public sector consists of three administrative levels ( central government , county councils and municipalities ) , but only two hierarchical ones . county councils ( 20 in total , consisting of 18 traditional county councils and two recently formed regions , each consisting of a number of former county councils1 ) and municipalities ( 290 in total2 ) are at the same hierarchical level but are both subordinate to the central government . county councils and municipalities are responsible for the provision of health and social care in sweden . traditionally , swedish county councils have been responsible for health and medical services , while social services have been the responsibility of municipalities at the local level . home care services have been a part of county councils ' health and medical services generally , whereas municipalities have provided home - help service . generally , the 1990s was a decade of reorganization of the public sector [ 2528 ] . one of the most far - reaching swedish organizational reforms was the care of the elderly reform ( del reform ) of 1992 , whereby the organization of old - age care was radically changed . one important aim of the reform was to find more efficient ways to organize old - age care under one authority ( i.e. to increase the integration of health and social care ) . the essence of the del reform was that municipalities were given total responsibility for elderly ( 65 years ) and old - age care outside of hospitals . as a result of the reform , swedish old - age care outside of hospitals is currently mainly provided by municipalities and takes place either in special housing for the elderly or in private homes . in special housing for the elderly , most elements of the reform were compulsory , unilaterally decided by central government , and imposed on county councils3 and municipalities . however , the taking over of the responsibility for home care services by municipalities was voluntary . the swedish government gave county councils and municipalities a free hand to negotiate the organization of home care services . the freedom to negotiate organizational forms locally was intended to result in variation among municipalities , based on local conditions , regarding the responsibility for home care services . in other words , the reform consisted of a combination of two coordinating mechanisms , namely , central steering from the swedish government , and incentives to create networks at the regional and local levels . in terms of a common distinction between three ideal typical coordinating mechanisms hierarchy , market and network the reform was implemented mainly through the use of hierarchy and , specifically regarding home care services , through the use of networks . the swedish political system , including its organizations , institutions and culture , can be viewed as a historically evolved configuration , and such national configurations shape the steering processes . sweden has a large public sector and a strong central state , and political reforms are often carried through by means of using hierarchy . during the last few decades elements of market mechanisms have been introduced in swedish public sector , and decentralisation from national to sub - national levels has been a common trend . however , deliberate attempts from national government to initiate implementation networks at sub - national levels is a more recent phenomenon in sweden , and the voluntary elements of the del reform is an early example of this new political style . networks are often viewed in a positive way as a flexible and non - traditional method for solving complex and cross - cutting issues or wicked problems . integrated care is often regarded to be such a cross - cutting issue , and implementation networks may therefore be expected to be an appropriate form of organization . though initiated at the national level , the networks of the swedish home care organization are actually structured through negotiations between county councils and municipalities . two aspects of network structuring are central in this study , namely , the responsibility and provision of home care services . home care services may be provided by county council , municipality and other actors ( e.g. private companies and cooperatives ) . different solutions regarding these two aspects result in different local organizational models of home care . therefore , the terms network and organizational model will henceforth be used inter changeably . if the design of networks were determined only by unique local circumstances it would be difficult to find any clear patterns regarding the distribution of local organizational models among swedish municipalities . even if the introduction of an organizational model is voluntary its dissemination may still form inter - organizational patterns , due to e.g. imitation and fashion - following . moreover , the adoption of a new organizational form is not always voluntary . it may also be more or less coercively imposed by strong organizations on weaker ones , or it may be the result of inter - organizational negotiations [ 3538 ] . in all those cases the distribution of organizational models may be expected to form inter - municipal patterns . the aim of this study was to study a structural aspect of integrated care , namely , the design and distribution of different organizational solutions regarding the responsibility for and provision of home care for elderly in swedish municipalities . the aim of this study was to study a structural aspect of integrated care , namely , the design and distribution of different organizational solutions regarding the responsibility for and provision of home care for elderly in swedish municipalities . most of the questions were of organizational character , including , for example , the provision of home care services . in order to map the organization of home care services , the respondents were asked to specify the providing organizations ( county council , municipality , and contractors ) and performing occupations ( assistant nurse and district nurse ) . the questionnaire was tested on four municipalities before being sent out , and was found to work well . the questionnaire and a letter of information about the study were sent to directors of the social welfare services in all swedish municipalities ( n=289 ) . they were informed that participation was voluntary and that answers would be treated confidentially . moreover , if they wanted to remain anonymous , they were asked not to state their names . fifty - six per cent of the respondent municipalities were contacted by phone to clarify their answers , particularly concerning the organization of home care services . data were collected during the summer of 2002 and were analyzed with spss version 11.5 . one - way anova tests were performed to investigate differences in municipal size and proportion of elderly regarding the responsibility and organization of provision of home care . moreover , chi - square tests were performed in order to investigate whether there were any differences , in reluctance to reorganize regarding the home care organization . a total of 211 answers were received , resulting in a 73% rate of response . some of the directors responded themselves , others delegated the task to managers and civil servants at different levels in the municipal old - age care organization . the responding municipalities were evenly distributed , geographically and according to size ; the total number of citizens ( 7,127,035 ) in the responding municipalities represented 80% of the swedish population . the proportion of elderly ( 65 years ) was 17% . besides the municipality county council boundary , another inter - organizational boundary exists regarding provision of home care services , between public and private providers of home care services . elements of privatization represented only a minor part of home care services in sweden . all in all , non - public provision of home care services , as a complement to public provision , was represented in 10.5% ( 22 ) of the municipalities . the models represent different degrees of structural integration , and are based on the allocation of responsibility and provision of home care services . the responsibility was either still in the hands of county councils , as it was before the del reform , or transferred to municipalities . in municipalities where the county council was responsible for home care services , the provision could be organized in two ways ( model 1 and model 2 ) . on the other hand , where municipalities were responsible for home care services , responsibility and provision coincided in one organizational model ( model 3 ) . in the presentation below , the different organizational models appear according to degree of structural integration in the provision of home care . this model was adopted in 23% ( 48 ) of the municipalities . organizational model 2 : county councils were responsible for home care services , and qualified health care was provided by county council personnel ( district nurses ) . however , less qualified health care , as well as home help , was provided by assistant nurses employed by municipalities . county councils did not employ assistant nurses and , thus , a separation between responsibility and provision of home care services was built directly into the organizational structure . this model was adopted by 26% ( 56 ) of the municipalities . organizational model 3 : municipalities were responsible for , and provided all home care , including both home help and home care services . this model was the most common one , adopted by 51% ( 107 ) of the municipalities . a distinct pattern was identified for the distribution of municipalities with different organizational models of home care . in 55% ( 11 ) of the 20 county councils , home care was organized the same way in all municipalities ( figure 1 ) . in 35% ( 7 ) of the county councils two models of home care were represented in its municipalities . in 10% ( 2 ) of the areas thus , in 55% ( 11 ) of the county councils one model of providing home care was universally prevailing . in eight of those areas the prevailing model was organizational model 3 : municipalities provided all home care . in 75% ( 15 ) of the county councils , three - fourths or more of the municipalities had chosen the same model of organization for home care . in 85% ( 17 ) of the county councils , two - thirds or more of the municipalities were organized according to the same model of home care . thus , the organizational structure of the county councils was strongly homogeneous ; most county council areas consisted of clusters of municipalities with the same kind of home care organization . the main aim of this paper is to describe patterns of structural integration of home care , and the causes of these patterns are of secondary interest . however , some potentially influential factors are so conspicuous that they are hard to overlook . demographical characteristics may be expected to have a general impact on the municipal organization of services . further , a general preference for reorganizations may be expected to be correlated with the adoption of organizational model 3 , the most radical organizational reform . therefore , in the following , comparisons between the three types of municipalities ( i.e. municipalities organized according to model 1 , 2 or 3 ) are made regarding demography and preferences for organizational reforms . neither number of inhabitants nor proportion of elderly differed between municipalities with different organization of home care . in total , 73% ( 154 ) of swedish municipalities had completed at least one major reorganization of some kind after the del reform . of these , 26% ( 40 ) also had reorganizations in progress during the summer of 2002 . furthermore , 6% ( 13 ) of the municipalities were undergoing reorganizations during the summer of 2002 for the first time since the implementation of the del reform . however , there was also a minority of 21% ( 43 ) of the municipalities with no major completed or ongoing reorganization at all since the del reform . the introduction of a new form of structural integration with the del reform , i.e. the transferring of responsibility for home care services ( model 3 ) may be expected to express a general inclination to reorganize municipal activities . however , contrary to expectations , municipalities who had taken over the responsibility for home care services showed no significant differences regarding inclination to reorganize . however , despite the general inclination to reorganize municipal activities , only 1% ( 3 ) of the municipalities stated that their home care organization had gone through any reorganization at all , affecting the relation with county councils , after the del reform . thus , the organization of home care in sweden has been stable since the implementation of the del reform . municipalities were all demographically equal , regardless of organizational model adopted . neither number of inhabitants nor proportion of elderly differed between municipalities with different organization of home care . in total , 73% ( 154 ) of swedish municipalities had completed at least one major reorganization of some kind after the del reform . of these , 26% ( 40 ) also had reorganizations in progress during the summer of 2002 . furthermore , 6% ( 13 ) of the municipalities were undergoing reorganizations during the summer of 2002 for the first time since the implementation of the del reform . however , there was also a minority of 21% ( 43 ) of the municipalities with no major completed or ongoing reorganization at all since the del reform . the introduction of a new form of structural integration with the del reform , i.e. the transferring of responsibility for home care services ( model 3 ) may be expected to express a general inclination to reorganize municipal activities . however , contrary to expectations , municipalities who had taken over the responsibility for home care services showed no significant differences regarding inclination to reorganize . however , despite the general inclination to reorganize municipal activities , only 1% ( 3 ) of the municipalities stated that their home care organization had gone through any reorganization at all , affecting the relation with county councils , after the del reform . thus , the organization of home care in sweden has been stable since the implementation of the del reform . home care consists of two activities : social services ( home help ) and medical services ( home care services ) . one form of integrated care is the structural integration of these aspects . in all the organizational models described in the previous section , municipalities were responsible for and we identified three different models concerning the responsibility and provision of home care services in private homes in swedish municipalities . model 1 and 2 were characterized by an organizational separation of responsibility for social and medical aspects of home care : municipalities were responsible for home help services , and county councils for home care services . the differences between the two models do not concern the responsibility but the provision of home care services . in model 1 the provider is the county council , but in model 2 home care services are provided by both county councils and municipalities . thus , the degree of integration is higher in model 2 : cooperation between county councils and municipalities regarding home care services is built into the formal organizational structure . the distribution of the different organizational models for home care resulted in a sub - national rather than a national structure ; county councils tended to form clusters of municipalities with the same kind of organization . in eight of the eleven county council areas where one single model of organization for home care was universally prevailing , this resembles an all or nothing solution ; either all municipalities in an area took over the responsibility for home care services , or none adopted this solution . this radical organizational change , compared to the situation before the del reform , seems to be strongly connected with unanimity among the municipalities within a county council area . our data show that swedish municipalities during the 1990s have been inclined to change their organization generally , a result strongly supported by earlier research [ 25 , 27 , 28 , 39 ] . however , this is not so regarding the organization of home care . in 1992 , more than half of the municipalities in the country took over the responsibility for home care services from the county councils . thus , after the radical change that occurred with the introduction of the del reform , the organization of home care in sweden has been remarkably stable . no demographical or organizational characteristics associated with different organizational models were found at the municipal level . the organization of home care services is an inter - organizational matter , and the relationship between municipalities and county councils , rather than municipal characteristics , affects the design and distribution of home care organizations . the design of organization for responsibility and provision of home care is negotiated locally between municipality and county council . yet , why should this result in a similar organizational structure for all or most municipalities within the same county council area ? one explanation is processes of imitation between municipalities in the same county council ; another is collective negotiations between all concerned municipalities and the county council ; a third explanation is asymmetric power relations between the negotiating parties , where one party ( the county council ) is dominant . however , in this case processes of imitation are hardly a plausible explanation . imitation presupposes a process where an innovation is first adopted by a forerunner and then imitated by followers . the del reform was introduced at the same time in the whole country and a new organization of home care was adopted overnight in all swedish municipalities . thus , there was no time sequence involved where forerunners and followers could adopt the reform at different points in time . two of the three above - mentioned explanations are , however , plausible and they are not mutually exclusive . collective negotiations are facilitated by the fact that municipalities are organized according to county council area affiliation . regarding the power relations between county councils and municipalities the situation is more uncertain : the negotiations regarding the organization of home care services took place at the political level and we have not found any research at all neither regarding these negotiations nor regarding the power relations more generally . ( the absence of research in this area is in itself remarkable . ) however , regarding the operative level , medical services have for several reasons ( financial resources , professional status ) a stronger position than social services , in sweden as well as elsewhere [ 21 , 40 , 41 ] . whether this asymmetry at the operative level is also reflected at the political level is not clear , but our data are certainly not inconsistent with the assumption that county councils are more powerful than municipalities . thus , the distribution of the different organizational models of home care may be explained by collective negotiations and/or asymmetric power relations , but not imitation . a possible explanation for the absence of further organizational reforms in the area of home care could be a lack of perceived problems with the present organization . however , some old problems remain and others have been created or aggravated by the reform , particularly in municipalities of types 1 and 2 , e.g. increased costs , fragmentation , lack of co - ordination , and no possibilities to influence the home care activity area as a whole [ 4244 ] . thus , in spite of a general inclination to reorganize among swedish municipalities , and despite existing problems in the home care organization few efforts have been made to solve the problems through organizational reforms . how could that be ? the same factors that explain the distribution of organizational home care models ( collective negotiations and/or asymmetric power relations ) may also explain the reluctance to reorganize the home care organization . each municipality faces a combination of a necessity to negotiate with the county council , and a strong conformity among municipalities within the same county council area . thus , if one municipality wishes to change its home care organization , it has to pass a high threshold , namely , not only negotiate with a powerful county council but also sometimes negotiate with other municipalities to make them adopt the same organizational reform . even though the del reform has been extensively evaluated , international publications about the organization of home care in sweden are rare . further research from an organizational perspective is needed , preferably by way of comparisons between the three types of municipalities , regarding , e.g. quality of care and inter - professional cooperation across organizational boundaries . despite having distributed the questionnaire at the beginning of the summer vacation the rate of response was high ( 73% ) . it would probably have been even higher if another time had been chosen . in spite of the high rate of response , the fact remains that 27% of the municipalities did not answer the questionnaire . however , the missing cases were evenly distributed all over the country in municipalities of different sizes . the internal loss is partly due to our way of presenting combinations of data , and partly to unanswered questions in some questionnaires . to test its validity , the respondents , who were asked to give constructive criticism , had no objections to the design and content of the questionnaire . therefore , we consider the validity of the study to be acceptable . the reliability of the study was confirmed through follow - up telephone calls with 118 municipalities . no demographical or organizational characteristics associated with different organizational models were found at the municipal level . the organization of home care services is an inter - organizational matter , and the relationship between municipalities and county councils , rather than municipal characteristics , affects the design and distribution of home care organizations . the design of organization for responsibility and provision of home care is negotiated locally between municipality and county council . yet , why should this result in a similar organizational structure for all or most municipalities within the same county council area ? one explanation is processes of imitation between municipalities in the same county council ; another is collective negotiations between all concerned municipalities and the county council ; a third explanation is asymmetric power relations between the negotiating parties , where one party ( the county council ) is dominant . imitation presupposes a process where an innovation is first adopted by a forerunner and then imitated by followers . the del reform was introduced at the same time in the whole country and a new organization of home care was adopted overnight in all swedish municipalities . thus , there was no time sequence involved where forerunners and followers could adopt the reform at different points in time . two of the three above - mentioned explanations are , however , plausible and they are not mutually exclusive . collective negotiations are facilitated by the fact that municipalities are organized according to county council area affiliation . regarding the power relations between county councils and municipalities the situation is more uncertain : the negotiations regarding the organization of home care services took place at the political level and we have not found any research at all neither regarding these negotiations nor regarding the power relations more generally . ( the absence of research in this area is in itself remarkable . ) however , regarding the operative level , medical services have for several reasons ( financial resources , professional status ) a stronger position than social services , in sweden as well as elsewhere [ 21 , 40 , 41 ] . whether this asymmetry at the operative level is also reflected at the political level is not clear , but our data are certainly not inconsistent with the assumption that county councils are more powerful than municipalities . thus , the distribution of the different organizational models of home care may be explained by collective negotiations and/or asymmetric power relations , but not imitation . a possible explanation for the absence of further organizational reforms in the area of home care could be a lack of perceived problems with the present organization . however , some old problems remain and others have been created or aggravated by the reform , particularly in municipalities of types 1 and 2 , e.g. increased costs , fragmentation , lack of co - ordination , and no possibilities to influence the home care activity area as a whole [ 4244 ] . thus , in spite of a general inclination to reorganize among swedish municipalities , and despite existing problems in the home care organization few efforts have been made to solve the problems through organizational reforms . how could that be ? the same factors that explain the distribution of organizational home care models ( collective negotiations and/or asymmetric power relations ) may also explain the reluctance to reorganize the home care organization . each municipality faces a combination of a necessity to negotiate with the county council , and a strong conformity among municipalities within the same county council area . thus , if one municipality wishes to change its home care organization , it has to pass a high threshold , namely , not only negotiate with a powerful county council but also sometimes negotiate with other municipalities to make them adopt the same organizational reform . even though the del reform has been extensively evaluated , international publications about the organization of home care in sweden are rare . further research from an organizational perspective is needed , preferably by way of comparisons between the three types of municipalities , regarding , e.g. quality of care and inter - professional cooperation across organizational boundaries . despite having distributed the questionnaire at the beginning of the summer vacation it would probably have been even higher if another time had been chosen . in spite of the high rate of response however , the missing cases were evenly distributed all over the country in municipalities of different sizes . the internal loss is partly due to our way of presenting combinations of data , and partly to unanswered questions in some questionnaires . to test its validity , the respondents , who were asked to give constructive criticism , had no objections to the design and content of the questionnaire . therefore , we consider the validity of the study to be acceptable . the reliability of the study was confirmed through follow - up telephone calls with 118 municipalities . the organizational structure of home care in sweden was radically changed through the del reform of 1992 , and three different organizational models arose . however , the models have not been disseminated in a national organizational field , but rather in sub - national networks at the county council level within the field . the highest degree of structural integration of home care , i.e. municipal responsibility for and provision of home care services as well as social services , presupposed the most radical organizational change , and was related to unanimity among the municipalities within each county council area ; in 8 of 11 entirely homogeneous county council areas the municipalities had all chosen the most radical change ( i.e. organizational model 3 ) , and few municipalities outside these areas had chosen this solution . however , once the del reform was implemented the organizational structure of home care has remained unchanged . the inter - organizational network structure within the county councils was designed in a way that made it difficult for individual municipalities to change their organization of home care . the necessity to reach an agreement with the county council as well as with other municipalities within the region seems to have created an almost insurmountable threshold . our conclusion is a paradox : the decentralist intention of the del reform to give actors at the municipal and county council levels freedom to integrate home care according to varying local circumstances has resulted in an inter - organizational network structure leading to conformity within county council areas , and local home care organizations which are highly inert and difficult to change . it seems entirely possible for national governments to bring about structural integration of home care through major organizational reforms , at least in countries with strong political institutions . however , the use of hierarchical power in order to decentralize , by creating network structures at different sub - national levels results in inter - organizational structures , which , once established , are highly difficult to change . thus , after one change , the inter - organizational structure is frozen . therefore , if structural integration is the political goal , decentralisation to county council and municipal levels may not be the most appropriate means , because those municipalities who initially choose not to adopt an organizational solution leading to full structural integration can not change their minds later . they are locked into a frozen structure , and it probably takes another government decision to unfreeze it . anna - karin edberg , phd , associate professor department of health sciences lund university , sweden . nick gould , senior lecturer , networks and integrated care , welsh institute for health and social care , university of glamorgan , united kingdom . gunnar ljunggren , md , phd , forum / centre for care development , stockholm county council , stockholm , sweden . kirstein rummery , professor of social policy , llb(hons ) , ma , phd , department of applied social sciences , university of stirling , united kingdom .

purposeto study the design and distribution of different organizational solutions regarding the responsibility for and provision of home care for elderly in swedish municipalities.methoddirectors of the social welfare services in all swedish municipalities received a questionnaire about old - age care organization , especially home care services and related activities . rate of response was 73% ( 211/289).resultsthree different organizational models of home care were identified . the models represented different degrees of integration of home care , i.e. health and social aspects of home care were to varying degrees integrated in the same organization . the county councils ( i.e. large sub - national political - administrative units ) tended to contain clusters of municipalities ( smaller sub - national units ) with the same organizational characteristics . thus , municipalities ' home care organization followed a county council pattern . in spite of a general tendency for swedish municipalities to reorganize their activities , only 1% of them had changed their home care services organization in relation to the county council since the reform.conclusionthe decentralist intention of the reform to give actors at the sub - national levels freedom to integrate home care according to varying local circumstances has resulted in a sub - national inter - organizational network structure at the county council , rather than municipal , level , which is highly inert and difficult to change .

central serous chorioretinopathy ( csc ) is a focal retinal detachment due to fluid accumulation underneath the sensory retina . this case report presents the development of csc after the use of dexamethasone eye drops as a nasal drop . to our knowledge one case series and a case report is documented in literature , which specifically linked csc with use of intranasal steroids . in the previous publications , fluticasone or beclomethasone were used . this is the first case described in literature , suggestive of csc due to nasal use of dexamethasone drops for rhinitis . a 35-year - old male , optometrist by profession , visited our hospital with complaint of seeing a dark circle in front of his right eye with onset three days prior to presentation . patient was a non - smoker , non - alcoholic , non - hypertensive , was not on any medication and followed a healthy life style . vision was 6/18 in the right eye with best corrected visual acuity ( bcva ) of 6/6 with + 2.0 d sphere . the patient was prescribed 0.5% carboxymethylcellulose eye drops thrice a day and asked to return for follow up every two weeks . csc completely resolved , clinically and on oct , within seven weeks [ figure 2b ] . four weeks later the patient had a recurrence that again resolved spontaneously over eight weeks . in all , patient had three additional episodes of csc in the same eye over the next year [ figure 3 ] . after detailed questioning , he admitted , that he used dexamethasone eye drops , nasally for five to seven days before each episodes . this suggests the strong correlation between steroids given by any route and pathogenesis of csc . this is also first case ever of confirmed csc due to nasal intake of steroid drops . ( a ) fundus photograph showing a fluid - filled bleb - like elevation at the posterior pole figure ( b ) , ( c ) and ( d ) : showing small hyperfluorescence spot in early phase , which gradually increased in size due to leakage taking an ink blot pattern ( a ) oct shows area of detachment as hyporeflectivity between neurosensory retina and rpe ( b ) shows flattening of retinal contour after seven weeks oct showing serous retinal detachment at first and second recurrence emotional liability , neuroticism and introversion , hysteria , type a personality , pregnancy , have been suggested to predispose to csc . symptoms include sudden blurring of vision , central and paracentral scotoma , micropsia , macropsia . csc is a serous detachment of the retina due to serum leakage from the choroidal circulation through a break in the diffusion barrier , made of tight junction around the retinal pigment epithelial ( rpe ) cells . the proposed pathogenesis is that steroid use leads to reduced absorption of subretinal fluid by altering the permeability of choriocapillaris and ion transport mechanism at the mineralocorticoid level . were the first to report a statistically significant relation between exogenous corticosteroid use and csc . multiple reports have suggested the development of csc after the use of steroids in any form . to our knowledge one case series and a case report is documented in the literature which specifically links csc with use of intranasal steroids . intranasal corticosteroids are widely used for allergic rhinitis and it is often purchased as over - the - counter drug . a position paper by joint task force for the american academy of allergy , asthma and immunology and american college of allergy , asthma and immunology stated that , data suggest that the use of inhaled corticosteroids is associated with an increased risk of adverse ocular effects and that the risk increases with dose and duration of therapy , leading them to recommend that intranasal corticosteroids not be approved for over - the - counter use . any patient using steroid in any form must be informed about the possibility of development of csc and should immediately report to a treating doctor and ophthalmologist on the appearance of symptoms , and the drug should be discontinued . this is the first case in the literature , suggestive of csc after use of dexamethasone eye drop in the form of nasal drop for recurrent allergic rhinitis .

central serous chorioretinopathy ( csc ) is characterized by serous retinal detachment at the posterior pole . several factors have been implicated in the pathogenesis , and endogenous or exogenous corticosteroids are thought to play a major role . here we present a case of a 35-year - old male with complaints of a dark circle in front of his right eye . fundus examination , optical coherence tomography and fundus fluorescein angiography were performed . the patient was diagnosed with csc . csc resolved completely within seven weeks . four weeks later the csc recurred and spontaneously resolved over eight weeks . overall , the patient had three additional recurrences of csc in the same eye over the next year . a detailed history taking revealed the patient was using 0.1% dexamethasone eye drops nasally for recurrent rhinitis for few days prior to each episode of csc . this indicates the strong correlation between steroids given by any route and the pathogenesis of csc .