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atrial fibrillation ( af ) is the most common sustained arrhythmia in western countries , with an estimated 30 million patients affected by 2050 across united states and europe alone .
atrial fibrillation has a significant impact on morbidity mainly related to symptoms , heart failure , and thromboembolic events and is the most frequent arrhythmic cause of hospital admission in the usa [ 13 ] .
in addition , af is associated with excess mortality independently of thromboembolic complications . to date , the most effective treatment for af is radiofrequency catheter ablation , and pulmonary vein antrum isolation ( pvai ) is the mainstay of such an approach .
the major drawback of catheter ablation of af consists in its potential risk of periprocedural complications , with thromboembolic and hemorrhagic complications being among the most common and insidious ones [ 7 , 8 ] . despite the introduction of novel ablation technologies , such as open irrigation catheters , and the widespread use of systemic anticoagulation with heparin
, the risk of periprocedural thromboembolism remains not negligible , reaching up to 2.8% in large series [ 6 , 9 ] . with these premises ,
the development of novel strategies able to further decrease the risk of periprocedural thromboembolism without increasing the risk of bleeding is crucial .
oral anticoagulant therapy with warfarin is the current standard of care for the treatment of af , with a strikingly favorable balance between thromboembolic protection and bleeding complications when adequate monitoring of international normalized ratio ( inr ) is achieved . for years ,
discontinuation of warfarin before ablation associated with the periprocedural use of unfractioned and low molecular weight heparin and bridging with warfarin after ablation has been the most widespread anticoagulation protocol in patients undergoing catheter ablation of af [ 5 , 11 ] .
we firstly described the feasibility of af ablation without interruption of oral anticoagulation and recently reported that this approach is able to potentially abolish thromboembolic complications without increasing the risk of bleeding .
accordingly , af ablation without interruption of anticoagulation is nowadays the standard protocol in our center . in this paper , we summarize our approach to anticoagulation in patients undergoing af ablation .
patients eligible for catheter ablation of af are started on warfarin as outpatients , at least 2 months before the scheduled procedure .
all patients receive weekly inr monitoring during the 4 to 6 weeks preceding the procedure , with a target inr of 2 to 3 .
preprocedural transesophageal echocardiography ( tee ) is performed only in patients showing sub - therapeutic inr values in the month prior to the procedure .
patients who demonstrate inr values consistently above 2 in the month before the procedure are directly sent to ablation .
four venous accesses are obtained : two right and one left femoral venous accesses , and one right internal jugular vein access .
femoral venous accesses are usually obtained with the seldinger technique or with ultrasound guidance in difficult cases . the right internal jugular vein
is accessed with ultrasound guidance or under fluoroscopic guidance with a wire or a deflectable catheter advanced into this vein via the right femoral vein sheath .
a double transseptal puncture is performed with the assistance of intracardiac echocardiography ( ice ) [ 13 , 14 ] . before transseptal punctures ,
all patients receive a bolus of unfractionated heparin ( 10,000 units ) , followed by a noncontinuous infusion to maintain an activated coagulation time ( act ) > 300 seconds .
we found important the addition of unfractionated heparin to periprocedural therapeutic warfarin , since soft thrombus can still be observed on the transeptal sheath or left atrial catheters in patients with periprocedural therapeutic inr .
radiofrequency energy is delivered with an open irrigated ablation catheter with a maximum temperature of 42c , power up to 45 w , and flow rate of 30 cm/min .
intracardiac echocardiography is continuously used to monitor the electrode surface during ablation , to assist with catheter positioning and identify coagulum formation , and to monitor for complications including pericardial effusion .
an esophageal temperature probe is always inserted to assist with power titration during posterior wall ablation . at the end of the left atrial ablation , we partially reverse heparin anticoagulation with up to 40 mg of protamine guided by the act and remove the sheaths when the act is less than 250 seconds . with this approach
the majority of the major bleedings are related to cardiac tamponade , which can be effectively managed with emergent pericardiocentesis , together with heparin interruption and reversal with protamine , and warfarin reversal with fresh - frozen plasma or prothrombin complex concentrate .
if continued significant drainage or reaccumulation of the pericardial effusion occurs despite these measures , emergent open surgical exploration is considered .
notably , there is also no difference in rates of emergent surgical exploration between patients with periprocedural therapeutic inr and those who discontinue warfarin , although patients on therapeutic anticoagulation are more likely to have a larger amount of blood removed from their pericardium for stabilization and require more blood transfusion units .
all patients receive a single dose of aspirin ( 325 mg ) before leaving the electrophysiology laboratory and continue their warfarin dosage regimens to maintain a target inr of 2 to 3 .
after ablation the thromboembolic risk may be further increased by the procedure - related endothelial damage , which may activate the coagulation cascade and increase the risk of thromboembolism .
therefore , warfarin discontinuation may be associated with an increased thromboembolic risk also in the postprocedural period , since reachieving a therapeutic inr after the procedure may take several days , and the risk of left atrial thrombosis during af is strikingly time dependent .
all patients are strictly monitored for outcome and complications during overnight hospital stay , and on the following day prior to discharge using symptom assessment , serial neurological examinations , and puncture site checks .
all patients are instructed to call in case of any symptom development and to send weekly transtelephonic electrocardiogram transmissions for the first 5 months after ablation .
progress of recovery and symptoms are assessed as well by a dedicated nurse . in case of symptoms or suspected complications patients
are asked to seek medical attention at either a local emergency department or our emergency department or to follow up with their local physician .
moreover , all patients present for followup 3 to 4 months after ablation with the electrophysiologist who performed the procedure . with regard to the out - of - hospital long - term anticoagulation management , patients are referred to dedicated anticoagulation clinics with the aim of maintaining a stable therapeutic inr level .
we follow a standard , uniform , and validated protocol of long - term postprocedural anticoagulation management . briefly ,
oral anticoagulation is discontinued , regardless of the chads2 score , if patients do not experience any recurrence of atrial tachyarrhythmias , severe pulmonary vein stenosis ( pulmonary vein narrowing > 70% ) , and severe left atrial mechanical dysfunction , as assessed by transthoracic echocardiography .
patients with a chads2 score 1 experiencing early recurrence of af are maintained on warfarin for at least 6 months . in these patients
, warfarin is discontinued if there is no af recurrence in the last 3 months without antiarrhythmic drugs , and aspirin 81 to 325 mg is started . in case of new af recurrence after warfarin discontinuation in patients with a chads2 score 1 , oral anticoagulation
our approach to periprocedural anticoagulation has been extensively validated in previous work [ 12 , 13 , 17 ] . in particular , we found that a conventional anticoagulation approach , which included warfarin discontinuation with peri- and postprocedural bridging with unfractioned and low - molecular - weight heparin actually increases the risk of bleeding and thromboembolic complications , as compared to no periprocedural interruption of oral anticoagulation [ 12 , 13 , 17 ] .
our most recent report provides strong evidence that performing af ablation under therapeutic inr is a safe and effective approach to virtually abolish the risk of thromboembolic complications .
we reported a multicenter prospective comparison of three anticoagulation protocols in 9 centers performing the same ablation procedure .
a total of 6,454 patients were included in the study , of whom 2,488 underwent ablation with an 8-mm ablation catheter and preprocedural warfarin discontinuation ( group 1 ) , 1,348 underwent ablation with an open irrigated catheter and preprocedural warfarin discontinuation ( group 2 ) , and 2,618 underwent ablation with an open irrigated catheter without preprocedural warfarin discontinuation ( group 3 ) .
overall , periprocedural thromboembolic complications occurred in 39 ( 0.6% ) patients , with a rate of 1.1% in group 1 , and of 0.9% in group 2 .
these data support also the appropriateness of our approach to tee based on the intensity of anticoagulation in the month preceding the procedure , especially considering that 1,178 ( 45% ) group 3 patients had persistent af , and 498 ( 19% ) long - standing persistent af . at multivariable analysis , which adjusted for age , gender , coronary artery disease , type of af , heart failure , diabetes , hypertension , and prior stroke , the anticoagulation strategy of ablation with
a therapeutic inr was a strong independent predictor of lower periprocedural thromboembolic events ( odds ratio 0.54 , 95% confidence interval 0.32 to 0.89 , p = .017 ) .
with regard to bleedings , the pooled rate of major bleeding complications ( i.e. , bleeding requiring interventions including transfusions , hemopericardium , hemothorax , and retroperitoneal bleeding ) and pericardial effusion in patients who discontinued warfarin before the ablation procedure ( groups 1 and 2 ) was 1.1% , whereas in group 3 was 0.8% ( figure 1 ) .
if also minor bleedings were included , patients who discontinued warfarin before ablation procedure had a pooled rate of bleeding complications of 20.7% , while patients who were maintained on warfarin had a rate of 4.8% . translating such percentages into treatment effects , the net clinical benefit associated with af ablation without interruption of oral anticoagulation is overwhelming ( figure 2 ) , with an estimated 170 thromboembolic or hemorrhagic complications avoided every 1,000 patients ablated . in the most recent survey on af catheter ablation , cappato et al . reported thromboembolic and hemorrhagic complication rates in 16,039 patients undergoing af ablation between 2003 and 2006 in 521 centers distributed worldwide .
all these patients had oral anticoagulant discontinuation before the ablation procedure . accordingly , thromboembolic and bleeding complication rates in this large survey were fairly consistent with that reported in group 1 and 2 patients of our study [ 13 , 18 ] .
based on our findings it is estimable that , if periprocedural oral anticoagulation was not discontinued in all patients included in the survey , more than 2,700 thromboembolic or hemorrhagic complications would have been avoided worldwide from 2003 to 2006 .
therefore , there are cogent data supporting the benefit of af ablation without discontinuation of oral anticoagulation .
since the acute reversal of the anticoagulant effect of warfarin is possible only through infusion of fresh coagulation factors , we routinely type- and cross - match all patients , so that packed red blood cells and fresh frozen plasma are readily available for infusion in case of hemorragic complications . if the preprocedural inr is above 3.5 , we partially reverse the anticoagulant effect with one to two units of fresh frozen plasma . of interest ,
the strategy of ablation under therapeutic inr could also be more cost - effective compared to bridging therapy with enoxaparin , which is expensive and may be inconvenient for many patients .
radiofrequency catheter ablation of af without discontinuation of oral anticoagulation significantly reduces the risk of thromboembolic and minor bleeding complications .
maintenance of periprocedural therapeutic inr should be considered the anticoagulation strategy of choice among patients submitted to catheter ablation of af . | atrial fibrillation
( af ) can be cured by pulmonary vein antrum
isolation ( pvai ) in a substantial proportion of
patients .
the high efficacy of pvai is partially
undermined by a small but concrete
periprocedural risk of complications , such as
thromboembolic events and bleeding .
a correct
management of anticoagulation is essential to
prevent such complications .
performing pvai
without interruption of oral anticoagulation has
been demonstrated feasible by our group in
previous studies .
recently , we reported that
continuation of therapeutic warfarin during
radiofrequency catheter ablation consistently
reduces the risk of periprocedural
stroke / transient ischemic attack without
increasing the risk of hemorrhagic events . of
note , interrupting warfarin anticoagulation may
actually increase the risk of stroke even when
bridged with heparin .
the latter strategy is
also associated with an increased risk of minor
bleeding . with regard to major bleeding , we
found no significant difference between patients
with a therapeutic inr and those who were
bridged with heparin .
therefore , continuation of
therapeutic warfarin during ablation of af
appears to be the best anticoagulation strategy .
in this paper
we summarize our experience with
af ablation without interruption of
anticoagulation . |
breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide , accounting for 23% ( 1.38 million ) of the total new cancer cases and 14% ( 458,400 ) of the total cancer deaths in 2008 . about half of the breast cancer cases and 60% of the deaths are estimated to occur in economically developing countries . in iran ,
also , patients with advanced stages of the disease are relatively younger ( about 10 years ) than their western counterparts [ 2 , 3 ] .
exposure to environmental pollutants such as metals including cadmium , chromium , nickel , and arsenic is classified in group 1 of the international agency for research on cancer categories of carcinogen ; it also reports lead as a suspected human carcinogen ( group 2a ) and also mercury as possibly carcinogenic to humans ( group 2b ) . in order to explain the role of metals in breast cancer incidence
, we should refer to the studies in the field of estrogenicity of metals that express estrogen - like activity in breast cancer cells and suggest several pathways to explain association of metals with human cancer [ 711 ] . on the other hand , cadmium , lead , and mercury as carcinogens belong to the group of selenium , the antagonistic elements that compete with selenium uptake as anticarcinogen .
the mechanism of se as an anticarcinogenic element is unknown , but several speculative hypotheses have been advanced .
se exerts its essential role in the formation of glutathione peroxidase , a selenoenzyme that protects body against oxidative injury and free radical damage so its suggested mechanism for cancer prevention includes effects upon programmed cell death , dna repair , carcinogen metabolism , and the immune system [ 1417 ] .
therefore , it seems that , according to the results of these researches , a considerable amount of literature has been published on the determination of metals in human breast cancer tissues which show various values in malignant and benign tissues in comparison with healthy tissues [ 1728 ] . in addition , all available studies have only focused on metal concentration totally in breast tissue , but the aim of this study is to determine cd , pb , hg , and se in various parts of breast tissue ( tegmen , tumor tissue , tumor adiposity , and tegmen adiposity ) in order to compare any significant differences which may exist between different parts of breast tissues .
also , this research may be the first report of metal concentration in iranian breast cancer tissues .
in order to determine cd , pb , hg , and se concentrations in different parts of malignant breast tissue , 14 removed samples by mastectomy surgery of women patients ( in age range 3050 ) were taken from imam hospitals located in uremia and separate tegmen , tumor , tumor adiposity , and tegmen adiposity of breast cancerous tissues were dried in freeze - dryer at 64c for 20 to 30 hours .
then , about 1 g of each part of separated breast tissue was put in the digestion tubes ( polytetrafluoro ethylene ) with 5 ml concentrated hno3 for 3 hours in 100c on hot block digester until the disappearance of brown fumes .
after cooling , 1 to 3 ml h2o2 30% for 1 hour on heater was added .
after cooling , it was filtered with whatman filter paper number 1 and then diluted with deionized water to final volume of 25 ml [ 26 , 30 ] .
pb and cd measurements were performed by using aas and with the graphite furnace technique model aa-670 and se analysis was done by icp - oes ( ultima 2ce ) .
the aas instrument was calibrated using aqueous standards of 10 , 30 , and 60 g / kg for pb and 0.5 , 2 , and 5 g / kg for cd in breast cancer tissues .
there was a good linear relation between absorbance and standard concentrations of pb and cd .
linearity was evaluated by calculating the r - square value , which was 0.998 for pb and 0.999 for cd .
the detection limit with aas was calculated as 3 times the sd of the blank sample divided by the slope of calibration curves ( 1.57 g / kg for pb and 0.18 g / kg for cd ) .
the detection limit of se with icp - oes was 0.2 g / kg .
about 0.020.04 g of dried samples was put on nickel boot for measuring hg by leco ama 254 advanced mercury analyzer ( usa ) . to evaluate the analytical potency of the proposed methodology , accuracy of total hg analysis
was checked by running three samples of standard reference materials ( srm ) , national institute of standards and technology ( nist ) , srm 1633b , srm 2709 , and srm 2711 in seven replications .
the detection limit of the used instrument was 1 g / kg of dry weight . to check for contamination ,
all of used glassware was acid - washed and one blank was analyzed after five samples .
table 1 presents mean , standard deviation , and range of pb , cd , hg , and se concentration in 4 parts of tegmen , tumor , tumor adiposity , and tegmen adiposity of 14 breast cancer tissues . as it can be seen in the table ,
the concentration data of pb in kolmogorov - smirnov test was not normal ( p < 0.05 ) , so by using the kruskal - wallis nonparametric test , no significant differences between pb values in parts of the breast tissues were shown ( p = 0.820 ) . to assay the differences between cd , se , and hg ,
one - way anova test was used and no significant differences were found between metals in the separated breast cancer tissues ( resp . ,
no significant difference between the concentrations of metals in four separate parts might be due to the very close blood relationship between tissues . a noticeable point in this research
is that the preparation of healthy tissues as control samples for comparing cancer tissues was not possible , so we compared our results with previous studies . despite the possible relation between cd and breast cancer exposure ,
its values in different parts of 14 breast cancer samples show mean concentration in tegmen ( 35.51 g / kg ) , tumor ( 45.04 g / kg ) , tumor adiposity ( 41.15 g / kg ) , and tegmen adiposity ( 32.95 g / kg ) .
table 2 compares cd , pb , and se concentration in breast tissues from results of available studies .
, so there were not any significant differences between cd in cancerous ( 20470 g / kg ) and healthy ( 31700 g / kg ) ones and the maximum concentration of cd among hitherto accessible studies has been reported .
the separation of the close parts of breast tissues in this study did not show any significant difference between them and , as it can be inferred from the table , the present results are in accordance with the results of strumylaite et al . that report the minimum cd values so far .
due to the multiple carcinogenic evidence of lead , its detection has been done in various tumors such as breast ones . according to table 2 , the considerable concentrations in tegmen ( 336.18 g / kg ) , tumor ( 327.50 g / kg ) , tegmen adiposity ( 396.52 g / kg ) , and tumor adiposity ( 365.73 g / kg ) are similar to the results of majewska et al . and kubala - kuku et al . .
also mercuric chloride has been widely considered as causative of tumors . among collected studies ,
rizk and sky - peck report the mean concentration of hg ( 770 g / kg dry weight ) in breast cancer tissues and , according to the results of this study , it is supposed that the accumulation of this metal in tegmen ( 29.10 g / kg ) , tumor ( 33.26 g / kg ) , tumor adiposity ( 28.04 g / kg ) , and tegmen adiposity ( 26.13 g / kg ) can have a role in carcinogenicity . as it can be seen in table 2 , according to the studies , the accumulation of se in breast cancer tissues has been determined .
the mean concentration of se is similar to the results of rizk and sky - peck , kue et al .
the noticeable issue is that metals are just one of the effective factors in carcinogenesis or anticarcinogenesis , so their clear mechanism would have been investigated .
this study just reports the concentration of some metals in breast cancer in women 's samples from selective hospital , as we know that different factors may affect the occurrence of cancer especially breast cancer in women all over the world and environmental pollutants such as metals from several sources could enter the human body and , by accumulation , increase , and intensification , they may cause the incidence of cancer .
this study showed that there were not any significant differences between metals concentration in different parts of breast cancer tissues .
this result might be because of close relation of separated parts of fatty breast organ .
in general , in puberty and presence of estrogen hormone , breast cells have been grown rapidly .
in normal situation , after sudden increased rate of estrogen and breast cell growth , hormone balance became in equilibrium and irregular cell proliferation was interrupted .
according to recent researches and hypotheses , it could be concluded that estrogen - like properties of metals could mostly influence hormonal responses by binding to estrogen receptors and disrupt endocrine system and finally increased proliferation of cells would be occurring .
we conclude , thus , that a high level of these heavy metals is accumulated in iranian cancerous breasts and their presence can be one of the reasons for breast cancer appearance . | breast cancer is the major cause of cancer morbidity and mortality between women in the world .
metals involved in environmental toxicology are closely related to tumor growth and cancer . on the other hand ,
some metals such as selenium have anticarcinogenic properties .
the aim of this study is to determine the concentration of cadmium , lead , mercury , and selenium in separated parts of tegmen , tumor , tumor adiposity , and tegmen adiposity of 14 breast cancer tissues which have been analyzed by graphite furnace atomic absorption ( aa-670 ) and icp - oes ( ultima 2ce ) .
our results show that se and hg have maximum and minimum concentration , respectively .
statistical analysis reveals no significant differences between metal accumulations in different parts of cancer tissues ( p > 0.05 ) and this observation might be due to the close relation of separated parts of fatty breast organ .
thus , we could conclude that a high level of these heavy metals is accumulated in iranian cancerous breasts and their presence can be one of the reasons of cancer appearance . |
in the neonatal intensive care unit ( nicu ) population , hyponatremia is the most frequent encountered water and salt abnormality . with its broad differential diagnosis ,
the most frequent causes are renal salt loosing through an immature kidney and the use of drugs such as diuretics .
hypertension , however , is a relatively rare feature in children , especially in neonates , with an incidence in nicu 's ranging from 0.7% to 3.2% , and renal arterial thrombosis following umbilical arterial catheterization as the leading cause [ 2 , 3 ] .
a rare clinical presentation of unilateral renal arterial stenosis is the hyponatremic hypertensive syndrome ( hhs ) , characterized by activation of the renin angiotensin aldosterone ( raas ) system in the ischemic kidney , causing hypertension , and a counteracting effect on the other kidney , by different mechanisms leading to volume depletion and loss of electrolytes .
this syndrome is caused by unilateral renal ischemia , due to stenosis or occlusion of a ( branch of a ) renal artery , and also occurs in a variety of other underlying disorders .
so far , only a few reports of hhs in children are available , with polydipsia , polyuria , enuresis , weight loss , volume depletion , and various neurological and behavioural symptoms as presenting symptoms .
we present a case of hhs in a preterm infant , with an extremely low sodium concentration , and discuss the difficulties encountered in treatment and the irreversible neurological sequels due to this potentially life - threatening metabolic disturbance .
a preterm boy presented with extreme hyponatremia ( plasma sodium of 101 mmol / l ) at the 20th day after birth .
he was born from a nulliparous woman at a gestational age of 31 weeks and 4 days after an uncomplicated pregnancy , followed by spontaneous rupture of membranes and antenatal corticosteroid administration .
apgar scores were 9 and 10 at 1 and 5 minutes , and the birth weight was 2080 grams .
an umbilical arterial catheter was inserted directly after birth , for the purpose of blood pressure monitoring , and removed after 3 days .
furthermore an umbilical venous catheter and subsequently a peripheral central venous catheter were inserted for the purpose of parenteral feeding .
routine cerebral ultrasonography showed an image consistent with the gestational age and mild periventricular flaring .
he gained weight ( from 1900 grams at the 3rd day to 2100 grams at two weeks after birth ) . at the age of 3 weeks rejection to feeding ( until this moment consisting of 150 ml / kg / day breast milk with breast milk fortifier ) , weight loss ( to a minimum of 1960 grams ) , irritability , hyperthermia , and
cerebrospinal fluid analysis showed 219 leukocytes / mm with 12000 erythrocytes / mm , after a traumatic lumbar puncture , thus a meningitis could no be excluded and intravenous antibiotics were started .
intravenous fluids , with a total volume of 150 ml / kg / day , containing 8 mg / kg / min glucose and 5 mmol / kg / day sodium , were administered in the regional hospital for 2 days ( before return to the nicu ) .
the plasma sodium level had declined , from 140 mmol / l nine days before , to 101 mmol / l .
there were no sodium levels examined in the interval between , but the level at the onset of symptoms was established at 112
the boy returned to the nicu under suspicion of a syndrome of inappropriate antidiuretic hormone secretion ( siadh ) associated with the assumed meningitis , with initiated fluid restriction and sodium supplementation considered to be the appropriate therapy .
we saw a pale , irritated neonate with tachypnea , arterial hypertension ( 104/60 mmhg , mean 78 mmhg ) , opisthotonus , and abnormal synchronized extensions of arms and legs .
mmol / l , laboratory analysis revealed a mild hypokalemia , hypochloremia , and hypomagnesemia , with normal calcium and phosphate levels ( for detailed information on all important laboratory results , see table 1 ) .
plasma osmolarity was 219 mosmol / kg , and urea and creatinine levels were normal .
infection parameters were low , but liver enzymes and lactate were elevated ( in the blood drawn several shortly after the seizure ) .
furthermore an elevated plasma b - type natriuretic peptide ( bnp ) of 1228 pmol / l was found ( in children , there are no validated data on normal values available ) .
urinalysis showed no leucocytes , mild hematuria , low sodium and potassium , with proteinuria , glucosuria , and a urine osmolarity of 129 mosmol / kg .
the combination of hyponatremia and hypertension ( defined as a mean blood pressure of > 2 standard deviations for age and weight , in this case > 75 mmhg ) was suggestive of renal pathology .
abdominal doppler ultrasound showed a right renal arterial thrombosis , partially calcified , and an oedematous appearance of the left kidney .
it was suggested that the symptoms of this neonate resulted from an hhs secondary to a renal arterial thrombosis .
blood pressure levels further increased in the first hours to a maximum of 108/62 ( mean 96 ) mmhg .
we chose to carefully normalise the blood pressure with intravenous dihydralazine , causing the right kidney to become completely afunctional ( as demonstrated with 99 m technetium mag3 renography in combination with the findings on doppler ultrasound as mentioned before ) .
plasma sodium levels rose to , relatively fast within the first hours , above 120 mmol / l , and more slowly within the next 24 hours to normal .
the dihydralazine and sodium supplementation could be gradually stopped after a few days , after which a normal blood pressure and electrolyte levels were maintained without any additional therapy , also urinalysis returned to normal . within a few minutes after return to the nicu the boy developed convulsions ,
cerebral ultrasound and magnetic resonance imaging ( mri ) showed extensive white matter abnormalities and the presence of a sinus thrombosis of the superior sagittal , straight , and transverse sinus .
additional genetic screening revealed a mutation in the methylenetetra - hydrofolate reductase ( mthfr ) gene .
follow - up mri at one and two months of age showed extension of the white matter abnormalities , with secondary haemorrhage , vacuolisation , and cyst formation .
the gyration and myelinisation had increased , there were no signs of new ischemia , and all sinuses were recanalized .
the child showed abnormal neurological behaviour ( agitation , uncontrolled movements , and delayed motor development ) at three months of followup .
the parents of this child gave their informed consent to publication of this case report .
in this case report , we describe a 3-week - old preterm boy , with extreme hyponatremia , hypertension and a dramatic neurological outcome , as a result of hhs following umbilical arterial catheterization .
the typical combination of symptoms in hhs was first described in 1952 in adults , and the term hhs was established by brown et al . in 1965 .
the syndrome is not encountered frequently and in neonates it is even more rare , with renal arterial stenosis following umbilical arterial catheterization being one of the described causes [ 810 ] , accompanied by renal microthrombi in sepsis and an association with dexamethasone use .
the syndrome has been described more often in preterm than in term infants [ 811 ] and sometimes showed a lethal course [ 12 , 13 ] .
the high incidence of hyponatremia ( 30% ) reported in hypertensive neonates , suggests that hhs is probably more common than we think .
hhs is thought to be due to a complex interplay of different mechanisms , with unilateral renal hypoperfusion and a counteracting effect of the contralateral normal kidney as major hallmarks ( two - kidney - one - clip hypertension ) ( figure 1 ) .
the renal arterial thrombosis causes hypoperfusion of one kidney , which activates the raas system to cause hypertension .
the contralateral nonstenotic kidney reacts to this hypertension by excreting water and sodium ( pressure diuresis and natriuresis ) [ 12 , 15 ] .
the hypertension additionally stimulates the cardiac atrial natriuretic peptide ( anp ) and bnp to excrete more sodium and protein .
the resulting hypovolemia , probably together with an increased production of angiotensin ii , stimulates antidiuretic hormone ( adh ) , further aggravating the hyponatremia .
furthermore , aldosterone causes renal potassium loss , which in turn stimulates renin secretion , causing a vicious circle .
proteinuria , glucosuria , and hypercalciuria can also be present due to glomerular hyperfiltration in hypertension , increased renin activity , and probably even more extensive tubulointerstitial involvement .
in our case , the presence of an umbilical arterial catheter , and the finding of a mutation in the mthfr gene ( suggested to play role in homocysteine metabolism and enhancing atherosclerosis ) were thought to be contributing factors in the development of the renal arterial thrombosis . at the moment of presentation at the nicu
, our patient was thought to be already beyond the natriuretic phase of hhs ( probably the severe hyponatremia finally resulted in sodium retention , explaining the low urine sodium ) and most likely adh had been turned on in reaction to the volume depletion .
the initial sepsis - like presentation was retrospectively interpreted as the result of a combination of hypovolemia and central nervous system disturbances .
the prematurity was probably a major contributing factor leading to the severe outcome in this patient .
this could have led to more extreme hyponatremia because preterms have a relatively low sodium intake , reduced tubular sodium reabsorption , and decreased glomerular filtration rate , impairing free water excretion .
furthermore , recognizing the nonspecific clinical symptoms of hhs can be very difficult in a neonate .
the symptoms of hhs disappeared after normalising the blood pressure with a vasodilator agent ( dihydralazine ) , causing the ischemic kidney to become nonvital by totally abrogating the blood flow , destroying the juxtaglomerular cells , and hence stopping renin production .
there was a gradual decline in blood pressure in this patient , different from the potentially dangerous fall which can be seen with angiotensin - converting enzyme ( ace ) inhibitors . with severe volume depletion ,
cautious repletion is needed , which can probably also reduce arterial pressure by suppression of raas .
correction of longstanding hyponatremia should be managed carefully , to minimise the risk of developing cerebral shrinking .
final therapy of the underlying renal arterial stenosis was not necessary in this case , but can be achieved by balloon angioplasty , renal artery stenting , or uninephrectomy .
the remarkable irreversible neurological features in this case are most likely to be the consecutive effect of a hypertensive and hyponatremic encephalopathy , aggravated by a diminished cerebral circulation due to hypovolemia and a sinus thrombosis .
furthermore the convulsions could also have led to irreversible damage to the vulnerable preterm brain .
previous case reports in older children mainly mention reversible neurological symptoms , and even reversible findings on computer tomography or mri associated with hhs [ 19 , 2224 ] and only one infant dying from massive cerebral haemorrhage .
first , there is a lack of ( clinical and laboratory ) information about the period before the patient represented with the severe hyponatremia .
unfortunately , no detailed information on water balance or 24-hour urine volumes during the period in which the patient developed the hyponatremia was available .
as it is especially cumbersome to collect such data in newborns , this is very rarely done .
second , no data on plasma adh , rennin , and aldosterone are available to confirm our suggested diagnosis .
this case was meant to describe the complex pathophysiology of hhs , and the possible misleading clinical features in a neonate .
furthermore we want to underline the risk of severe irreversible neurological damage when there is a diagnostic delay .
we think that in evaluating a neonate with severe hyponatremia , hhs should be considered , especially if following umbilical arterial catheterization . | objective . to report the irreversible severe neurological symptoms following the hyponatremic hypertensive syndrome ( hhs ) in an infant after umbilical arterial catheterization
. design .
case report with review of the literature . setting . neonatal intensive care unit at a tertiary care children 's hospital . patient .
a three - week - old preterm infant . conclusions . in evaluating a neonate with hyponatremia and hypertension , hhs should be considered , especially in case of umbilical arterial catheterization . in case of diagnostic delay
, there is a risk of severe irreversible neurological damage . |
the first requirements of the no child left behind act ( nclb ) , which became law in 2002 , led every state to evaluate the adequate yearly progress ( ayp ) of students in math and language arts based on specific content standards in those subjects .
assessment requirements extended ( by statute , if not yet in practice ) to science in 2007 , where they encompass testing in three grade bands , 35 , 69 , and 1012 . in science , guidance for the production of content standards and curricula has been available through the national science education standards ( national research council [ nrc ] , 1996 ) and project 2061 benchmarks for science literacy ( american association for the advancement of science [ aaas ] , 1993 , 2009 ) , and through state and district administrative offices .
the standards governing ayp in all subjects were developed on a state - by - state basis because , until recently at least , there has been little support at the state level for common national standards .
most states have adopted common core standards for reading and math , although tremendous work remains to be done to implement these standards ( paulson , 2010 ; for current list , see http://www.corestandards.org/in-the-states ) .
the rationale behind standards and testing can be summarized by a familiar saying , invoked in the context of nclb by former u.s . secretary of education margaret spellings : what gets measured , gets done ( spellings , 2005 ) . in k12 education , this can be further translated as standards drive testing and testing drives curriculum ( popham , 2004 ) .
there is some evidence that standards can also drive achievement , although here the details of structure and implementation are more critical ( swanson , 2006 ) .
we make no argument in favor of or against standards - based instruction or nclb ; however , given that this is the reality of k12 public education in the united states at the current time , we do argue that understanding the quality of standards is useful and even necessary .
science will soon follow reading and math down the path of high - stakes outcomes and the drive to achieve ayp through curriculum , instruction , and testing
and so it is reasonable to examine science standards for quality and consistency across different states .
this is especially true in light of concerns that variations in defining proficiency allow states to manipulate standards to facilitate meeting ayp and to avoid sanctions ( kingsbury et al . , 2003 ; aspen institute , 2006 ) .
in fact , evidence from the national center for educational statistics ( nces ) demonstrates an incongruity between student proficiency at the state level and student performance on the gold standard of assessment , the national assessment of educational progress ( naep ) : there is a strong negative correlation between the proportions of students meeting the states proficiency standards and the naep score equivalents to those standards , suggesting that the observed heterogeneity in states reported percents proficient can be largely attributed to differences in the stringency of their standards ( nces , 2007 ) .
such findings suggest worrisome weaknesses in math and reading standards and reason for concern apropos science standards . to date , there have been few comprehensive evaluations of specific science content in state standards , and none by a professional scientific society , and their quality remains largely unknown ( gross et al . , 2005 ; hoffman and barstow , 2007 ) . in genetics ,
lists of core principles have been developed to guide learning for audiences ranging from undergraduate nonscience majors to health professionals ( hott et al .
, 2002 ; national coalition for health professional education in genetics [ nchpeg ] , 2004 ; smith et al . , 2008 ) ,
the american society of human genetics ( ashg ) has been interested in genetics education and k12 standards since it began documenting misconceptions that frequently appeared in essays submitted to its national dna day essay contest ( shaw et al . ,
2008 ) . in this paper , we present the results of an evaluation of the life sciences standards of every state to assess their adequacy with respect to genetics coverage .
as benchmarks , we developed and used a list of core genetics concepts that all students should understand by grade 12 as preparation for life in a world of healthcare and medicine that is increasingly informed by genetics .
this analysis of extant genetics standards hopefully can inform science education researchers and policy makers during the normal revision process for existing standards , which occurs at different intervals for different states , and during the development of common science standards .
a list of core concepts in genetics appropriate for students up to grade 12 was developed using an iterative process .
initial drafts of concepts were adapted by ashg staff from several previously published documents that included important or essential genetics content : development and evaluation of a genetics literacy assessment for undergraduates
( bowling et al . , 2008 ) ; the genetics concept assessment ( smith et al . , 2008 ) ; closing the gap ( dougherty , 2009 ) ; nchpeg core principles of genetics ( 2004 ) ; project 2061 benchmarks for science literacy ( aaas , 2009 ) ; national science education standards ( nrc , 1996 ) ; ap biology course description ( college board , 2010 ) ; and undergraduate nonmajor biology courses ( hott et al . , 2002 ) . using these documents as a baseline
, concepts were developed to adequately describe five major conceptual areas of genetics deemed essential to genetic literacy : nature of the genetic material , transmission genetics / patterns of inheritance , gene expression and regulation , genetic variation , and evolution . because our interest was the quality and comprehensiveness of genetics content in the state standards
, we did not develop benchmarks ( or analyze standards ) related to noncontent understandings or abilities , such as inquiry , the nature of science , or system standards .
advanced genetics topics , such as epigenetics , the regulatory roles of small rnas , chromatin remodeling , and others , also were not included on the grounds that : 1 ) they were too complex or detailed and basic genetic literacy did not require them of all students ; 2 ) many teachers would likely be unfamiliar with them ; and 3 ) most state standards would not be expected to include them .
initial drafts were reviewed , revised through 11 iterations , and ultimately approved by ashg 's information and education committee , a standing committee that advises ashg 's board of directors on issues related to genetics education .
its members have expertise in genetics content , science education , and teaching , including substantial expertise with high school curricula and pedagogy .
a total of 15 concepts were tested in a pilot analysis , and a final list of 19 concepts served as the benchmark concepts against which state science standards were compared ( table 1 ) .
table 1.scoring results for ashg 's genetics concepts averaged across all states by concept and by concept categorycore conceptaverage score ( all states)/adequacyconcept categoryaverage by category / adequacy1 .
dna is the genetic material for all species of living organisms.1.7/adequatenature of genetic material1.6/adequate2 .
alleles are passed from parent to offspring through the processes of replication , meiosis , and fertilization.1.4/inadequate5 . for traits primarily influenced by single genes , certain combinations of alleles
lead to predictable genotypic and phenotypic patterns of inheritance , illustrating mendel 's principles of segregation of alleles and independent assortment of genes.1.5/adequate6 .
polygenic ( or complex ) traits ( e.g. , height , blood glucose ) often show continuous variation within populations and are less predictable than single - gene traits.0.3/not addressed / absent7 .
this occurs through transcription of dna into rna and translation of mrna into protein.1.3/inadequategene expression and regulation0.9/inadequate9 .
different genes are turned on and off at specific times to form different types of cells and to influence the way different cells function.0.6/inadequate11 .
the functions of genes and their products can be affected by the environment and other genes at one or many steps involved in producing a trait.0.7/inadequate12 .
they can occur spontaneously during dna replication or they can be the result of damage by environmental factors.1.3/inadequategenetic variation1.1/inadequate13 .
mutations in dna , and sorting and recombination during meiosis , result in genetic variation.1.4/inadequate14 .
mutations may help , harm , or have little or no effect on an organism.1.2/inadequate16 .
some genetic diseases are inherited ( e.g. , tay - sachs ) , and others develop during life ( e.g. , cancer).0.8/inadequate17 . genetic variation and the phenotypic variation it leads to are the basis for evolution.1.5/adequateevolution1.5/adequate18 .
evolution by natural selection is a process by which inherited traits influence how likely an organism is to survive , reproduce , and pass those traits to its offspring.1.7/adequate19 .
the process of evolution occurs at a population level ( e.g. , not at the level of individual organisms ) , and takes place over generations ( e.g. , not within an individual organism 's lifespan).1.2/inadequatenumerical scores : 0 = not addressed / absent ; 1 = inadequately addressed ; 2 = adequate .
scoring results for ashg 's genetics concepts averaged across all states by concept and by concept category numerical scores : 0 = not addressed / absent ; 1 = inadequately addressed ; 2 = adequate .
the most current state standards as of summer 2009 were identified from the websites of the departments of education for all 50 states and the district of columbia .
standards related to genetics in any way ( i.e. , in all life or biological sciences categories ) from grades 912 were assembled in state - specific pdf files to aid reviewers .
where states used more than one section to group content , for example , introductory paragraphs or rubrics to supplement content listings , we tried to be as inclusive as possible .
in general , the only content excluded from consideration related to rare cases where content was explicitly identified as beyond the level at which all students should learn ( i.e. , exceeding the state 's view of essential content and , therefore , less likely to be taught or tested ) .
a simple online scoring guide was developed in - house to allow reviewers to open an appropriate pdf of the state 's standards , review them , and then use drop - down menus to record a score for each concept .
a box for written comments was also provided . in light of ongoing efforts to revise national standards documents , we chose not to analyze standards that appear likely to soon be replaced ( e.g. , the national science education standards or project 2061 benchmarks for science literacy ) .
a pilot test was conducted to evaluate the feasibility and reliability of our rating system .
we used a three - level grading system ( 0 = not addressed / absent , 1 = inadequate , 2 = adequate ) that was similar to methods used for evaluating earth science standards ( hoffman and barstow , 2007 ) .
two project staff members serving as pilot reviewers independently evaluated the standards of 10 randomly selected states against a draft set of ashg core genetics concepts that included 15 concepts ( n = 150 pairwise comparisons ) .
interrater reliability for averages across all concepts at a state - by - state level was calculated according to the methods of ebel ( 1951 ) , using the open - access calculator developed by solomon ( 2004 ) .
adequacy grades were assigned by converting state or concept averages according to : adequate = 1.52.0 ; inadequate = 0.61.4 ; not addressed / absent < 0.6 . to assemble the requisite experts to serve as evaluators of state standards , we recruited 167 members of ashg 's genetics education outreach network , a volunteer network of geneticists across the nation who work in k12 classrooms , help grade dna day essays , and/or provide career talks .
all have content expertise in genetics and varying levels of expertise with genetics education outreach . of those participating
, 77% held doctoral degrees ( mostly phd and md ) , 20% held master 's degrees , and just over 2% held bachelor 's degrees .
( ashg 's membership consists largely of academic basic researchers , clinical researchers , and genetic counselors . ) our rationale for using a large pool of experts external to the project team as reviewers was that the intent of a state 's standards apropos a specific concept should be clear . if a specific concept in the standards is not apparent to these experts , it likely will not be apparent to many curriculum developers and teachers .
using our criteria , such standards would be graded as either inadequate ( i.e. , present but not adequate ) or not addressed / absent . as hoffman and barstow ( 2007 )
have noted apropos standards , each state has its own development processes , generally involving a wide variety of people with different backgrounds , subject area expertise , teaching experience , special interests , and political agendas .
such variation is likely the case for those interpreting standards as well , thus arguing in favor of clarity .
in addition , by using a large number of external reviewers , we hoped to avoid the effects of systematic bias that may creep into analyses conducted by small , close - knit teams .
the evaluation and grading of standards is necessarily a subjective process , but by averaging across a number of independent judges , we believe a reasonably accurate assessment was attained .
just over half ( 94 ) of the evaluators reviewed two states , and the remainder ( 73 ) reviewed one state , with sufficient overlap to yield an average of 5.11 reviewers per state .
, we dropped the high and low scores from states having five or six reviewers .
thus , the analysis for all states was based on a minimum of three scores per concept ( four for 14 states ) .
averages by state across all concepts ( i.e. , per - state averages ) and averages by concept across all states ( i.e. , per - concept averages ) were calculated .
interrater reliabilities were evaluated by the methods of ebel and solomon , as described above . to establish a common reference frame for reviewers to use in their evaluation ( acknowledging both the vagaries of the state standards and likely differing interpretations ) ,
the reviewers were instructed by email as follows : please use your best judgment to match concepts with standards and do not assume too much .
an incomplete standard that evokes for you ( as an expert in genetics ) a natural elaboration encompassing the ashg core concept may not evoke that same connection for a non - expert high school teacher .
we need to evaluate what each standard actually says . at the same time , if the intent of a standard clearly matches the intent of an ashg core concept and differs only in wording , you should credit that standard appropriately .
a list of core concepts in genetics appropriate for students up to grade 12 was developed using an iterative process .
initial drafts of concepts were adapted by ashg staff from several previously published documents that included important or essential genetics content : development and evaluation of a genetics literacy assessment for undergraduates
( bowling et al . , 2008 ) ; the genetics concept assessment ( smith et al . , 2008 ) ; closing the gap ( dougherty , 2009 ) ; nchpeg core principles of genetics ( 2004 ) ; project 2061 benchmarks for science literacy ( aaas , 2009 ) ; national science education standards ( nrc , 1996 ) ; ap biology course description ( college board , 2010 ) ; and undergraduate nonmajor biology courses ( hott et al . , 2002 ) . using these documents as a baseline
, concepts were developed to adequately describe five major conceptual areas of genetics deemed essential to genetic literacy : nature of the genetic material , transmission genetics / patterns of inheritance , gene expression and regulation , genetic variation , and evolution . because our interest was the quality and comprehensiveness of genetics content in the state standards
, we did not develop benchmarks ( or analyze standards ) related to noncontent understandings or abilities , such as inquiry , the nature of science , or system standards .
advanced genetics topics , such as epigenetics , the regulatory roles of small rnas , chromatin remodeling , and others , also were not included on the grounds that : 1 ) they were too complex or detailed and basic genetic literacy did not require them of all students ; 2 ) many teachers would likely be unfamiliar with them ; and 3 ) most state standards would not be expected to include them .
initial drafts were reviewed , revised through 11 iterations , and ultimately approved by ashg 's information and education committee , a standing committee that advises ashg 's board of directors on issues related to genetics education .
its members have expertise in genetics content , science education , and teaching , including substantial expertise with high school curricula and pedagogy .
a total of 15 concepts were tested in a pilot analysis , and a final list of 19 concepts served as the benchmark concepts against which state science standards were compared ( table 1 ) .
table 1.scoring results for ashg 's genetics concepts averaged across all states by concept and by concept categorycore conceptaverage score ( all states)/adequacyconcept categoryaverage by category / adequacy1 .
dna is the genetic material for all species of living organisms.1.7/adequatenature of genetic material1.6/adequate2 .
alleles are passed from parent to offspring through the processes of replication , meiosis , and fertilization.1.4/inadequate5 . for traits primarily influenced by single genes , certain combinations of alleles
lead to predictable genotypic and phenotypic patterns of inheritance , illustrating mendel 's principles of segregation of alleles and independent assortment of genes.1.5/adequate6 .
polygenic ( or complex ) traits ( e.g. , height , blood glucose ) often show continuous variation within populations and are less predictable than single - gene traits.0.3/not addressed / absent7 .
this occurs through transcription of dna into rna and translation of mrna into protein.1.3/inadequategene expression and regulation0.9/inadequate9 .
different genes are turned on and off at specific times to form different types of cells and to influence the way different cells function.0.6/inadequate11 .
the functions of genes and their products can be affected by the environment and other genes at one or many steps involved in producing a trait.0.7/inadequate12 .
they can occur spontaneously during dna replication or they can be the result of damage by environmental factors.1.3/inadequategenetic variation1.1/inadequate13 .
mutations in dna , and sorting and recombination during meiosis , result in genetic variation.1.4/inadequate14 .
mutations may help , harm , or have little or no effect on an organism.1.2/inadequate16 .
some genetic diseases are inherited ( e.g. , tay - sachs ) , and others develop during life ( e.g. , cancer).0.8/inadequate17 . genetic variation and the phenotypic variation it leads to are the basis for evolution.1.5/adequateevolution1.5/adequate18 .
evolution by natural selection is a process by which inherited traits influence how likely an organism is to survive , reproduce , and pass those traits to its offspring.1.7/adequate19 .
the process of evolution occurs at a population level ( e.g. , not at the level of individual organisms ) , and takes place over generations ( e.g. , not within an individual organism 's lifespan).1.2/inadequatenumerical scores : 0 = not addressed / absent ; 1 = inadequately addressed ; 2 = adequate .
scoring results for ashg 's genetics concepts averaged across all states by concept and by concept category numerical scores : 0 = not addressed / absent ; 1 = inadequately addressed ; 2 = adequate .
the most current state standards as of summer 2009 were identified from the websites of the departments of education for all 50 states and the district of columbia .
standards related to genetics in any way ( i.e. , in all life or biological sciences categories ) from grades 912 were assembled in state - specific pdf files to aid reviewers .
where states used more than one section to group content , for example , introductory paragraphs or rubrics to supplement content listings , we tried to be as inclusive as possible .
in general , the only content excluded from consideration related to rare cases where content was explicitly identified as beyond the level at which all students should learn ( i.e. , exceeding the state 's view of essential content and , therefore , less likely to be taught or tested ) . a simple online scoring guide was developed in - house to allow reviewers to open an appropriate pdf of the state 's standards , review them , and then use drop - down menus to record a score for each concept .
a box for written comments was also provided . in light of ongoing efforts to revise national standards documents
, we chose not to analyze standards that appear likely to soon be replaced ( e.g. , the national science education standards or project 2061 benchmarks for science literacy ) .
a pilot test was conducted to evaluate the feasibility and reliability of our rating system .
we used a three - level grading system ( 0 = not addressed / absent , 1 = inadequate , 2 = adequate ) that was similar to methods used for evaluating earth science standards ( hoffman and barstow , 2007 ) .
two project staff members serving as pilot reviewers independently evaluated the standards of 10 randomly selected states against a draft set of ashg core genetics concepts that included 15 concepts ( n = 150 pairwise comparisons ) .
interrater reliability for averages across all concepts at a state - by - state level was calculated according to the methods of ebel ( 1951 ) , using the open - access calculator developed by solomon ( 2004 ) .
adequacy grades were assigned by converting state or concept averages according to : adequate = 1.52.0 ; inadequate = 0.61.4 ; not addressed / absent < 0.6 .
to assemble the requisite experts to serve as evaluators of state standards , we recruited 167 members of ashg 's genetics education outreach network , a volunteer network of geneticists across the nation who work in k12 classrooms , help grade dna day essays , and/or provide career talks .
all have content expertise in genetics and varying levels of expertise with genetics education outreach . of those participating
, 77% held doctoral degrees ( mostly phd and md ) , 20% held master 's degrees , and just over 2% held bachelor 's degrees .
( ashg 's membership consists largely of academic basic researchers , clinical researchers , and genetic counselors . ) our rationale for using a large pool of experts external to the project team as reviewers was that the intent of a state 's standards apropos a specific concept should be clear . if a specific concept in the standards is not apparent to these experts , it likely will not be apparent to many curriculum developers and teachers .
using our criteria , such standards would be graded as either inadequate ( i.e. , present but not adequate ) or not addressed / absent . as hoffman and barstow ( 2007 )
have noted apropos standards , each state has its own development processes , generally involving a wide variety of people with different backgrounds , subject area expertise , teaching experience , special interests , and political agendas .
such variation is likely the case for those interpreting standards as well , thus arguing in favor of clarity .
in addition , by using a large number of external reviewers , we hoped to avoid the effects of systematic bias that may creep into analyses conducted by small , close - knit teams .
the evaluation and grading of standards is necessarily a subjective process , but by averaging across a number of independent judges , we believe a reasonably accurate assessment was attained .
just over half ( 94 ) of the evaluators reviewed two states , and the remainder ( 73 ) reviewed one state , with sufficient overlap to yield an average of 5.11 reviewers per state .
, we dropped the high and low scores from states having five or six reviewers .
thus , the analysis for all states was based on a minimum of three scores per concept ( four for 14 states ) .
averages by state across all concepts ( i.e. , per - state averages ) and averages by concept across all states ( i.e. , per - concept averages ) were calculated .
interrater reliabilities were evaluated by the methods of ebel and solomon , as described above . to establish a common reference frame for reviewers to use in their evaluation ( acknowledging both the vagaries of the state standards and likely differing interpretations ) , the reviewers were instructed by email as follows : please use your best judgment to match concepts with standards and do not assume too much . an incomplete standard that evokes for you ( as an expert in genetics ) a natural elaboration encompassing the ashg core concept may not evoke that same connection for a non - expert high school teacher . we need to evaluate what each standard actually says . at the same time , if the intent of a standard clearly matches the intent of an ashg core concept and differs only in wording , you should credit that standard appropriately .
an important statistical measure for assessing the consistency of analysis ( or scoring ) by different reviewers is interrater reliability , which we calculated for the pilot and full evaluations .
values of 70% or higher are generally considered good to acceptable , and values of 80% or higher to be very good ( lombard et al . , 2010 ) .
interrater reliability in the pilot phase ( two reviewers ) , across 150 pairwise comparisons , was 63% at the per - concept level , and in only one instance ( 0.7% ) did the two reviewers differ by 2 grading steps ( i.e. , 0 , not addressed / absent , and 2 , adequate ) .
interrater reliability at the level of per - state averages ( i.e. , average of scores across all 15 concepts used in the pilot ) was 96% .
the difference in reliability at the two levels reflects the fact that states with overall weak ( or strong ) standards tend to have many individually weak ( or strong ) standards , and averaging smooths out differences that may be more apparent at the finer - grained level of individual concepts .
in addition , the very high degree of per - state reliability was not surprising , because the project staff who conducted the pilot analysis had spent considerable time assembling pdfs of the state standards and discussing scoring criteria and the meaning of agreement . also , the pilot reviewers noted that a number of concepts were difficult to align with state standards , because the benchmarking concepts themselves contained more than one distinct idea .
based on that finding , several benchmarking concepts containing more than one idea were divided into simpler , separate concepts , yielding a final draft with 19 concepts ( table 1 ) . at the conclusion of the full evaluation ,
the interrater reliability for the same 10 states that were used in the pilot was 79% .
the interrater reliability across all states analyzed ( i.e. , 50 states plus the district of columbia ) was 75% .
the per - concept reliability average was 71% , with four concepts having a reliability under 60% ( averaging 52% : concepts 1 , 11 , 18 , and 19 in table 1 ) .
however , there was virtually no correlation between interrater reliability ( by concept ) and concept score ( r = 0.16 ; unpublished data ) .
thus , although four concepts were more challenging for reviewers to evaluate in the standards ( i.e. , lower reliability ) , that difficulty did not translate into systematically higher or lower scores .
we first analyzed which genetics concepts were adequately addressed across the nation as a whole .
the 19 core concepts yielded average scores ( i.e. , across all states ) ranging from not addressed / absent ( 2 concepts ) to inadequate ( 12 concepts ) to adequate ( 5 concepts ) , and the five broader genetics categories encompassing those concepts had averages of adequate ( 2 ) and inadequate ( 3 ; see table 1 ) .
overall , average scores for many concepts were low ( e.g. , 7 out of 19 were < 1.0 ) , and the range of scores was broad both across and within categories , especially for transmission / patterns of inheritance , which comprised five concepts ranging from 0.31.5 .
no concept scored above 1.7 , and only five scored 1.5 or higher ( dna being common to all living organisms ; genes as units of information ; mendel 's laws of segregation and independent assortment ; genetic and phenotypic variation as the substrate for evolution ; and evolution by natural selection ) .
the five lowest - scoring concepts ranged from 0.30.8 ( continuous variation ; polygeny ; differential gene expression ; multifactorial causation ; and the connection between mutations and inherited vs. somatic genetic disease ) . the two lowest - scoring concepts ( 6 and 7 , at 0.3essentially not addressed ) are both related to complex traits .
when concepts were analyzed by category , the nature of the genetic material and evolution fared best , scoring adequate , while transmission / patterns of inheritance , gene expression and regulation , and genetic variation all were deemed inadequate .
average scores across all concepts were calculated by state and were normally distributed ( mean = 1.11 , median = 1.11 , and range = 0.511.72 ) . figure 1 shows the results of each state 's overall quality of genetics standards using a map depiction .
seven states ( i.e. , 14% ) had overall genetics standards deemed adequate by the reviewers ( delaware , illinois , kansas , michigan , north carolina , tennessee , and washington ) . among those states , michigan fared best , with an average of 1.72 , followed by delaware at 1.67 .
states judged to have inadequate standards and at the bottom of the ranking included alaska , colorado , idaho , louisiana , north dakota , nebraska , new jersey , oregon ( the only state to have an overall average of not addressed / absent with respect to genetics coverage ) , and wyoming , which all had average scores below 0.8 .
( for individual concept scores for each state , please see supplementary data at : www.ashg.org/education/pdf/stateconceptscores.pdf . )
figure 1.map of the united states summarizing the average quality of genetics standards on a state - by - state basis .
map of the united states summarizing the average quality of genetics standards on a state - by - state basis .
we also examined states for their comprehensiveness with respect to the benchmark genetics concepts that is , the number of individual genetics concepts ( out of the total 19 ) for which a state received scores ranking it as adequate ( independent of overall average ) .
figure 2 shows a map of the specific results by state and table 2 summarizes those results .
delaware and michigan also fared well by this measure , each having 15 concepts that scored as adequate . interestingly , both states scored inadequately on the same four concepts ( 6 , 7 , 10 , and 11 ) , which are the four lowest - scoring concepts overall ( see table 1 ) .
not surprisingly , states with high overall averages tended to have the largest number of concepts that adequately addressed the genetics benchmarks ( r = 0.80 ; unpublished data ) .
for example , kansas had a very high average of 1.6 , but addressed only 11 of 19 concepts adequately .
this is not far from the modal value of 10 adequate concepts across all states .
similarly , texas had a relatively high average score of 1.4 ( just below adequate ) , but had adequate treatment of only seven concepts . in both cases ,
the concepts that were addressed adequately tended to have consistently high ratings from all reviewers . across all states ,
the average number of benchmark concepts covered adequately was 7.4 , with an sd of 4.0 .
figure 2.map of the united states summarizing the comprehensiveness of genetics standards on a state - by - state basis .
the ashg benchmarks list included 19 concepts ( see table 1 ) ; colors indicate the total number of concepts rated by reviewers as adequate .
table 2.summary of comprehensiveness of genetics standards across all states and comparison with an independent evaluation of the overall quality of science standards.number of adequate genetics concepts ( out of 19)number of states ( including district of columbia)fordham scoring , science overallnumber of states15192a7111410b1271016c93616d7<37f15total5150columns 1 and 2 : see table 1 . columns 3 and 4 : data from the thomas b. fordham institute ( gross et al . , 2005 ) .
the fordham analysis used a letter grading system of a f and considered noncontent criteria , such as organization , seriousness , and inquiry . at the time of this study , iowa was not included because it did not have its own state standards .
map of the united states summarizing the comprehensiveness of genetics standards on a state - by - state basis .
the ashg benchmarks list included 19 concepts ( see table 1 ) ; colors indicate the total number of concepts rated by reviewers as adequate .
summary of comprehensiveness of genetics standards across all states and comparison with an independent evaluation of the overall quality of science standards .
columns 3 and 4 : data from the thomas b. fordham institute ( gross et al . , 2005 ) .
the fordham analysis used a letter grading system of a f and considered noncontent criteria , such as organization , seriousness , and inquiry . at the time of this study , iowa was not included because it did not have its own state standards .
three of the four lowest - scoring concepts deal with complex traits ( continuous variation , polygenic inheritance , and multifactorial causation [ concepts 6 , 7 , and 11 , respectively , in table 1 ] ) .
only kansas , north carolina , and tennessee were judged as having adequate coverage of complex trait genetics concepts ( unpublished data ) .
there was a modest but significant positive correlation between the overall quality of a state 's genetics standards and its treatment of complex traits ( r = 0.38 , p < 0.001 ; figure 3 ) .
figure 3.the relationship between the overall average quality of genetics standards to the average of the three concepts directly related to complex trait genetics ( concepts 6 , 7 , and 11 ; table 1 ) .
the relationship between the overall average quality of genetics standards to the average of the three concepts directly related to complex trait genetics ( concepts 6 , 7 , and 11 ; table 1 ) .
our analysis is based on the premise that the discipline of genetics / genomics ( and scientific disciplines , in general ) is structured around an intellectual core of fundamental concepts .
the dynamic nature of science has accustomed us to expect that our understanding of scientific details will change even as further research serves to strengthen the concepts at the core .
such is the case with genetics , where concepts such as the allelic nature of genes , basic processes of gene expression , mutations , genetic variation , and patterns of inheritance have been expanded and enhanced across more than a century .
other concepts , while less familiar , such as differential gene expression and the genetic basis of complex traits , have been recognized for decades as crucial , but only in the past 1020 yr have advances in technology enabled researchers to elucidate the genetic mechanisms by which they occur .
other groups ( several of which are referenced in methods ) have developed their own lists for their own purposes .
the ashg list drew heavily from those previously published lists , but our choices were guided by the specific goal of capturing the minimum number of concepts , at the appropriate level of specificity , that the authors and ashg 's information and education committee believe to be essential for genetic literacy and thus should be understood by all high school graduates . as with all such lists , our process necessitated choosing among concepts that should be included and concepts that should be omitted , and some readers may disagree with these choices .
we believe that ashg , a large and influential professional society in genetics , can make a contribution to the discussion of standards with its views on what constitutes a reasonably specific , comprehensive , and age - appropriate set of concepts in one of the major subdisciplines of biology .
consequently , our choices reflect a moderate level of specificity , and our benchmark list includes 19 concepts rather than , for example , 6 or 60 .
this acknowledges dramatic changes in genetics over the past two decades , such as the discipline 's increasing impact on medicine and direct - to - consumer genetic testing , and the fact that an understanding of additional concepts is needed to raise the standard of scientific literacy among the nation 's population .
the new concepts we propose can be taught using many different subconcepts and examples , which we do not prescribe .
overall , our analysis identifies substantial deficiencies in the treatment of genetics in state standards across the united states .
of ashg 's 19 benchmark concepts , 14 were treated inadequately ( averaged across all states , including the district of columbia ) , and 39 states had adequate coverage of fewer than 11 concepts ( table 1 and figure 2 ) .
two concepts ( continuous variation of complex traits and polygenic inheritance ) were virtually absent from state standards .
only evolution and the nature of the genetic material were adequately covered , while the broad categories of gene expression and regulation and genetic variation were not .
it is particularly troubling that the inadequate and not addressed / absent concepts represent ideas that are increasingly important as genetics / genomics matures and assumes practical importance in people 's lives . as dna sequencing and genotyping technologies
have become more powerful and less expensive ( at rates that vastly exceed moore 's law in computing ) , they have been applied more often in medical research .
it is now possible to investigate the genetic contributions to virtually any disease or normal trait , and our ability to apply knowledge of individual genetic variation to clinical treatment and outcomes is advancing .
for example , roughly 10% of drugs approved by the food and drug administration have labels with pharmacogenomics information , and genetic testing has already become common for certain conditions , such as breast cancer ( hamburg and collins , 2010 ) . as lanie et al .
have written , even if people grasp and understand these basic concepts [ mendelian genetic concepts ] , the impact of this knowledge will be limited . . .
it will not go far in helping the layperson understand the barrage of genetic information to which they are exposed through the media the vast majority of which deals with complex diseases and traits ( 2004 ) . without adequate standards dealing with mutations , gene regulation , and non - mendelian patterns of inheritance , such concepts may never make it into curricula and assessments .
the most - neglected concepts also demand higher orders of thinking than concepts that tended to be covered adequately .
for example , unlike the nature of the genetic material ( i.e. , dna ) , which deals largely with descriptive biology , gene expression ( concept 8) deals with the mechanisms of how genes operate and the functional consequences of those operations .
the latter involve understanding positive and negative feedback ( general ideas that also extend to engineering , computing , and economics ) and require thinking at levels of application , interpretation , and analysis . in parallel with being
poorly represented in state standards ( table 1 ) , assessment questions related to the concepts of gene expression ( concept 8) and differential gene expression ( concepts 9 and 10 ) elicited very few responses indicative of complete understan ding by grade 12 students on the 2000 naep ( 2006 ; table 3 ) .
table 3.students responses to genetics questions on the 2000 naep ( grade 12 ) indicate poor understanding of essential genetics conceptsnaep question ( related concept , this work , from table 1)% complete or essential answerswhat is a gene ?
( 2)21briefly explain how the information that a gene provides to a cell results in the production of a molecule such as hemoglobin .
( 8)1[e]xplain why kidney cells do not make hemoglobin , even though they contain the hemoglobin gene .
( 10)10give an example of an inherited disease that people might be able to treat by use of [ recombinant dna ] technology , and explain how you think the technology might be used to treat this disease .
( 16)5 students responses to genetics questions on the 2000 naep ( grade 12 ) indicate poor understanding of essential genetics concepts another specific area of weakness is the state standards treatment of mutations .
frequently , state standards do not distinguish between germline and somatic mutations and their connections to hereditary versus nonhereditary genetic disease ( concepts 14 and 16 , respectively ) .
failure to make this distinction may contribute to students conflating inherited disease with any disease influenced by genetics , a frequent student misconception ( shaw et al . , 2008 ) .
students poor understanding of these concepts is also reflected in their responses to a related question on the naep ( table 3 ) .
perhaps not surprisingly , dna as the genetic material ( concept 1 ) is well covered by most states , as it should be nearly 60 yr after the elucidation of dna 's structure ( average score of 1.7 , table 1 ) .
the organization of genetic information in the form of genes carried by chromosomes also fared well ( 1.5 ) .
evolution by natural selection ( concept 18 ) tied for best average score ( 1.7 ) , and was supported by strong coverage of genetic and phenotypic variation as the substrate for evolution ( concept 17 ; 1.5 ) .
these findings support earlier research indicating improvements in state standards coverage of evolution ( mead and mates , 2009 ) .
unfortunately , state standards are not doing as well framing the population - level and generational timescale at which evolution operates ( concept 19 ) .
learning goals related to single - gene inheritance patterns ( i.e. , mendelian inheritance , concept 5 ) are also adequately addressed nationwide in the state standards ( average score 1.5 ) , an encouraging but not unexpected finding .
instruction about genes , alleles , and mendelian segregation , including meiosis , is virtually ubiquitous in high school general biology , and these concepts continue to dominate instruction in equivalent undergraduate courses ( hott et al . , 2002 ) .
these concepts also lend themselves easily to problem - based learning , which engages higher - level critical thinking . unfortunately , there may be a downside to all this attention . in the context of the modern discipline 's broader view of genetics
, we may be skewing students understanding of the genotype phenotype connection ( dougherty , 2009 ) .
in essence , if we spend too much time on single - gene inheritance at the expense of polygenic inheritance and complex traits then we not only fail to convey modern genetics accurately , but we also risk giving students a false impression that most traits are inherited in the simple manner conveyed by the rare single - gene traits that are so often used as examples , such as cystic fibrosis and hemophilia .
in fact , patterns - of - inheritance misconceptions represent the second most problematic conceptual category in genetics identified by shaw et al .
not surprisingly , low scores for concepts related to complex traits and multifactorial causation ( concepts 6 , 7 , and 11 ) contributed substantially to low average scores for most states .
to get some sense of how our specific results compared with the quality of state science standards as judged more generally , we compared them with the results of an evaluation conducted by the thomas b. fordham institute ( table 2 , columns three and four ) .
differences in methodology prevent direct comparison ; however , weaknesses in state standards are apparent in both analyses .
first , states were not scored blindly , which may have allowed some unintentional reviewer bias , although our method of using multiple reviewers and dropping the high and low scores for states with five or six reviewers should have helped minimize such bias .
second , for a small number of states , it is possible that some concepts may have been addressed only at lower grade levels , in which case this analysis would have missed them .
the decision to focus exclusively on high school standards was based on a preliminary analysis that indicated that middle school standards were generally repeated in the high school standards for the states that included genetics concepts at both levels .
the repetition of genetics concepts in middle and high school standards , in the cases where we observed it , bolsters the argument by schmidt et al .
different states have different revision schedules ( and processes ) for their standards , and the performance of any given state with respect to genetics ( or any other content ) would be expected to change over time .
finally , our analysis considers only one variable ( i.e. , standards ) in the complex system that constitutes genetics education .
of course , the absence of concepts in a state 's standards does not mean that those concepts will not be taught .
knowledgeable teachers can address such concepts even in situations where high - stakes exams do not require them .
however , given the pressures of performing well on state exams and limited classroom time , teachers often prioritize the content specified by their states .
conversely , the fact that concepts are represented in a state 's standards does not guarantee that students will learn them ( schmidt et al . ,
for example , dna as the genetic material and the nature of genes were the best - covered concepts for any category on our benchmark list ( concepts 1 and 2 , table 1 ) ; however , only a minority of students gave complete or essential answers to a question dealing with these ideas on the 2000 naep .
those concepts have been taught since long before standards achieved their current prominence in education , and yet student understanding lags .
the fact that 44 states ( 86% ) had genetics standards judged to be inadequate and only 12 states ( 24% ) had 11 or more individual concepts judged to be adequate convincingly demonstrates ( in our view ) a need for improvement when states revise their life sciences standards .
ashg will encourage members of its genetics education outreach network to volunteer to assist with revisions in those states that allow such participation .
similar networks exist at other scientific professional societies and may offer a generalizable mechanism for involving scientists who are content experts , as well as knowledgeable about k12 education , in both the evaluation and improvement of content coverage in state standards .
standards are just one element of what should be an integrated and coherent teaching system , and changes to standards should be made only with full recognition of the effects those changes may have on other parts of the system .
crowded curricula , teacher professional development , and available instruction time , must all be taken into consideration . within genetics , new conceptual frameworks
for example , it may be possible to refocus instruction around complex traits , which have a greater capacity for carrying modern understandings of genetics ( as well as traditional ones ) than do the simpler , single - gene traits that are used so widely in high school classrooms now ( dougherty , 2009 ) . the nrc 's move to generate common science standards
a recent report supports the establishment of clear and common standards across states , noting that roughly one - half the teachers in all states agreed that the standards of their own states were not clear enough , and that 85% think having tougher academic standards would make at least a moderate impact on improving academic achievement
sound science instruction requires expertise in teaching ( content knowledge and pedagogy ) , exemplary curricula , strong assessment , and a supportive system ( e.g. , administration , professional development ) . in the current u.s .
system , standards are the foundation upon which curricula and instruction are built , and our work shows that the foundation , at least in genetics , is in need of repair .
we propose that the findings detailed in this paper are well positioned to guide the development of common core standards in science . | science education in the united states will increasingly be driven by testing and accountability requirements , such as those mandated by the no child left behind act , which rely heavily on learning outcomes , or
standards , that are currently developed on a state - by - state basis . those standards , in turn , drive curriculum and instruction .
given the importance of standards to teaching and learning , we investigated the quality of life sciences / biology standards with respect to genetics for all 50 states and the district of columbia , using core concepts developed by the american society of human genetics as normative benchmarks .
our results indicate that the states genetics standards , in general , are poor , with more than 85% of the states receiving overall scores of inadequate . in particular , the standards in virtually every state have failed to keep pace with changes in the discipline as it has become genomic in scope , omitting concepts related to genetic complexity , the importance of environment to phenotypic variation , differential gene expression , and the differences between inherited and somatic genetic disease .
clearer , more comprehensive genetics standards are likely to benefit genetics instruction and learning , help prepare future genetics researchers , and contribute to the genetic literacy of the u.s . citizenry . |
traction exerted by the posterior hyaloid has been implicated in the pathogenesis of several conditions such as proliferative vitreoretinopathy ( pvr ) , proliferative diabetic retinopathy ( pdr ) , penetrating eye trauma and macular holes .
therefore the surgical goal of any vitrectomy should be posterior hyaloid separation and removal of the vitreous as much as possible . despite the development of surgical techniques , at times
the surgeon may not be confident that the posterior hyaloid has actually been removed . in conditions characterized by breakdown of the blood retinal barrier such as pvr , uveitis , retinal vein occlusions and diabetic retinopathy , an intravenous injection of fluorescein sodium 1 to 2 days prior to the scheduled vitrectomy stains the vitreous green facilitating its identification.5 blood in the vitreous cavity coats the vitreous by adhering to its collagen fibrils making the normally transparent vitreous opaque and easier to visualize for removal . in eyes with no pre - existing vitreous hemorrhage , a small amount of autologous blood may be injected into the vitreous cavity to coat the vitreous.6 triamcinolone acetonide ( ta ) is a well - tolerated corticosteroid used for the treatment of diseases such as uveitis , diabetic macular edema ( dme ) and retinal vein occlusions .
once ta is injected into the vitreous cavity , its particles adhere to the vitreous gel facilitating visualization and identification ( figures 1 and 2).7 in addition , using ta may improve vitrectomy outcomes by reducing blood retinal barrier breakdown and preretinal fibrosis .
currently this is the most widely used technique to visualize the posterior hyaloid.4,6 a comparative study of fluorescein , icg , ta and tb concluded that the vitreous was best highlighted by ta.8 a recent study demonstrated that a 20% solution containing the natural dyes lutein and zeaxanthin , precipitates on the vitreous surface staining it orange.9
over the past two decades owing to refinements in instrumentation and surgical techniques , erm removal has been the typical indication for macular surgery .
the transparent erms pose a challenge even to experienced surgeons since incomplete removal of the erm is implicated in post - surgical recurrences10 which have been reported in up to 21% of cases.11 - 13 staining of the transparent erm allows identification of its entire extent and facilitates its visualization and complete removal .
staining of these erms facilitates surgery by improving visualization , characterization of the erm as diffuse or focal , and confirmation that the tissue requiring to be peeled is indeed an erm and not swollen nerve fiber layer.14 among the commercially available agents , tb is the dye of choice for erm peeling.4,15 this dye binds to degenerated cell elements and does not stain live cells or tissues with intact cell membranes since there is no uptake of the dye . as erms are mostly composed of dead glial cells ,
cataract surgeons have long used tb to stain the anterior capsule during phacoemulsification.16 erms stain prominently with 0.15% tb which is a relatively safe concentration.15 histopathological analysis of excised erm showed no retinal cells on the retinal side of the erm and no signs of apoptosis .
no rpe defects or signs of retinal toxicity have been reported in most studies as well.15,17,18 nevertheless , animal and in vitro studies show that dose dependent toxicity may appear with concentrations above 0.3%.15
the ilm is made of the basement membrane of mller cells representing an interface between the retina and the vitreous .
the significance of ilm peeling is in achieving closure of large and chronic idiopathic mhs.19 tangential traction from the ilm plays a key role in the pathogenesis of mh . in a post - mortem examination , the eye with successful mh closure
was characterized by an area of absent ilm surrounding the sealed mh.20 in contrast in another patient that had a re - opened mh , histopathological examination revealed an erm with ilm surrounding the open mh suggesting that traction from the ilm was partly responsible for recurrence.21 in other conditions such as dme and erm , ilm peeling has remained controversial .
surgical specimens often show fragments of the ilm interspersed among the erm.22 - 24 breaks in the ilm may provide access to the macular surface for cellular elements , thus ilm peeling may reduce the risk of recurrence following erm removal . even in cases with complete erm removal , recurrences may occur .
therfore by removing the ilm as well , the surgeon can be assured that the erm has been entirely removed .
thus ilm peeling has been proposed as a means to ensure complete removal of the erm and to deter recurrence.13,25 visual improvement in 65% to 90% of eyes following erm removal and a low recurrence rate of 5% with even fewer re - operations were reported and these results were achieved without any ilm peeling.26 - 28 sivalingam et al23 reported that long segments of ilm removal led to a worse visual outcome compared to less ilm removal .
in contrast , more recently bovey et al29 reported that the visual outcome of eyes in which the erm had ilm remnants were significantly more satisfactory than eyes without ilm remnants in erm .
since the ilm consists of footplates of mller cells , it is reasonable to assume that its removal may result in some type of injury .
electron microscopic evaluation of surgically removed ilm specimens revealed degenerated and necrotic mller cell processes.30 mild damage to the vitreoretinal interface following icg stained ilm removal has also been documented with electron microscopy.31 given that icg has been reported to cause retinal toxicity , it remains unclear whether this mild damage to the vitreoretinal interface is due to icg or mechanical peeling of the ilm.4 ilm peeling causes a selective delay in the recovery of focal macular erg b wave even 6 months after operation32 suggesting that some type of injury to the macular mller cells occurs after ilm peeling , since these cells are responsible for generating the b wave in the electroretinogram .
tadayoni et al 33 coined the term " dissociated optic nerve fiber layer " to describe a peculiar fundus appearance following erm stripping . in their series out of 100 eyes ,
43% manifested numerous arcuate striae slightly darker than the surrounding retina in the direction of optic nerve fibers appearing 1 to 3 months postoperatively .
mitamura et al34,35 used optical coherence tomography ( oct ) to further characterize these findings as dimples limited to the retinal nerve fiber layer .
more recently clark and colleagues36 described 3 to 5 dark striae radiating from the papilla to the macula in an arcuate fashion as early as 1 week to 1 month postoperatively on blue light autofluorescence and infrared reflectance imaging .
simultaneous oct through the striae revealed focal areas of swelling in the arcuate retinal nerve fiber layer .
these changes resolved spontaneously after a mean period of 2 months not resulting in worse post - operative visual acuity as compared to eyes without such changes.36 in some eyes , swelling of the arcuate retinal nerve fiber layer evolved into characteristic dimples of dissociated optic nerve fiber layer suggesting that both of these findings represent different time points on the same spectrum.36 it appears that both dissociated optic nerve fiber layer and the arcuate swelling of the nerve fiber layer do not affect visual function .
a number of factors including direct trauma by the surgical forceps as they grasp the ilm , subclinical trauma to the inner retina from mller cell damage , and mller cell degeneration have been speculated.36,37 more longitudinal studies are required to assess the clinical implications of these changes .
park et al13 showed that ilm peeling did not have a detrimental effect on visual acuity .
they compared the outcomes between 24 eyes that underwent pars plana vitrectomy with erm peeling but not ilm peeling , with 20 eyes that had undergone pars plana vitrectomy with both erm and ilm peeling .
they reported that visual outcomes were similar between the two groups but that recurrence rate was much higher in eyes without ilm peeling .
more recent studies have shown that ilm removal does not improve or worsen postoperative visual acuity.12,38 however , recurrence was reduced by ilm peeling.12 another purported advantage of ilm peeling is a higher likelihood of resolution of retinal striae.39 selective stains such as icg and bbg have a high afnity for basal membranes such as the ilm and a low afnity for collagen tissues such as erms .
when either dye is used to stain both layers before removing any tissue during surgery , the unstained erm is clearly depicted against the ilm , which is stained blue or green ( figure 4 ) .
some surgeons re - stain the macula to visualize the remaining ilm but others do not .
even with a single bbg staining , up to two thirds of eyes will have residual ilm remnants after erm.12,40,41 in a recent prospective study conducted by the pan american collaborative retina study group , there was little correlation between the surgeon s un - aided observation and the bbg stained observation of the ilm .
thus , if the surgeon believes in the significance of ilm peeling in erm operation , staining should be strongly encouraged.42 the first vital dye used for ilm staining was icg.43 since icg binds to the ilm , biomechanical stiffness of the ilm increases thus facilitating its peeling ( figure 5).44 however the initial enthusiasm for the use of icg has tempered following numerous reports of toxicity.4,45 a meta - analysis of chromovitrectomy with icg compared to peeling of the ilm without staining in mh surgery showed similar anatomic outcomes in both groups .
however the functional results in eyes in which the ilm was stained by icg were much worse.46 several strategies can be used to minimize potential icg toxicity .
the lowest concentration of icg which provides adequate visualization of the ilm should be used ; concentrations of 0.5% or less are relatively safe . to avoid leaving excess
icg in the eye is of high importance to limit the area of icg staining to the ilm that will be peeled . to protect the bare rpe of the mh floor , a small bubble of heavy perfluorocarbon liquid or viscoelastic
is placed over the mh . following partial fluid - air exchange , 1 to 2 drops of the icg solution
are placed over the macula for 30 to 60 seconds . then the icg is washed out and ilm peeling can be performed . an alternative to icg
is infracyanine green which , unlike icg , does not contain sodium iodide allowing it to be dissolved in 5% glucose and to generate an iso - osmotic solution . in vitro assays have shown that infracyanine green induced significantly less toxicity on rpe and retinal ganglion cell lines in comparison to icg.47 this toxicity was more time dependent rather than concentration dependent .
when the duration of exposure approached 30 minutes , early apoptotic changes were observed in both cell lines , with no significant difference in apoptotic rates at 2 different concentrations ( 0.025% and 0.05%).47 several clinical investigations have revealed that infracyanine green assisted ilm peeling during mh repair results in high anatomic and functional success rates.48 - 50 bbg also has high affinity for the ilm . in animal and in vitro studies
, bbg appears to be relatively safe at doses up to 0.25 mg / ml.4 bbg is not a fluorescent dye ( figure 6 ) .
contact with the rpe should be avoided since rpe atrophy has been documented following subretinal migration of bbg . in general , bbg appears to be a safer alternative to icg for ilm peeling .
furthermore , bbg can be beneficial as a neuroprotective agent.51 in vitro studies have shown that photoreceptors undergo apoptotic cell death when exposed to extracellular atp through the activation of p2rx7 purinergic receptors .
identification of all retinal breaks is of paramount importance for successful repair of a retinal detachment . on occasion
transretinal injection of tb into the subretinal space with a 41 gauge needle followed by heavy perfluorocarbon liquid injection into the vitreous cavity assists the identification of previously unclear retinal breaks.52
the research is persevering and numerous dyes including methyl violet , crystal violet , eosin y , sudan black b , methylene blue , toluidine blue , indigo carmine , fast green , light green , congo red , bromophenol blue , patent blue and evans blue have been investigated.53 , 54 an ideal dye should have excellent contrast and high biocompatibility.55 german investigators have designed and tested 3,3-di-(4-sulfobutyl)-1,1,1,1 tetramethyl - di-1h - benz[e]indocarbocyanine ( dss ) with good biocompatibility which can be a proper alternative for ilm staining.55 on the other hand , brazilian investigators argue that natural dyes may be safer alternatives than synthetic dyes.56 they have investigated several naturally produced dyes and reported that acai fruit ( euterpe oleracea ) , logwood ( haematoxylum campechianum ) , cochineal ( dactylopius coccus ) and old fustic ( maclura tinctoria ) extracts facilitated posterior hyaloid visualization similar to triamcinolone acetonide in cadaver eyes .
acai fruit ( euterpe oleracea ) , chlorophyll extract from alfalfa ( medicago sativa ) and cochineal ( dactylopius coccus ) extracts stained the ilm well in cadaver eyes.56 time will tell if any of these agents will end up in routine clinical use .
with the recent introduction of ocriplasmin , pharmacological vitreolysis is a reality.57 the greatest advantage of enzymatic vitreolysis is that a cleaner cleavage plane between the retina and other tissues of interest may be achieved by enzymatic means as compared to mechanical separation by vitrectomy . in the future it is likely that enzymatic vitreolysis will play an even more significant role in the management of disorders of the vitreoretinal interface .
transparent tissues such as the posterior hyaloid , erms and the ilm play an important role in several diseases of the posterior pole . surgical removal of these tissues is a principal objective which can be facilitated by staining with a variety of vital dyes . several dyes are currently used in routine clinical procedures , however the ideal staining agent has not yet been found and any dye which is injected intravitreally has the potential to become toxic . in general ,
the lowest concentration of dye that will stain the tissue of interest should be used .
the dye should also be placed for the shortest period of time to result in minimal color change as a faint stain is usually sufficient for peeling . keeping the light pipe as far away as possible from the retinal surface
ta highlights the vitreous most ideally , and tb and bbg best stain erms and the ilm , respectively . | adequate visualization and identification of the posterior hyaloid , epiretinal membranes and the internal limiting membrane are of paramount importance in modern vitreoretinal surgery .
chromovitrectomy is a term used for describing the vital dyes use in order to stain these transparent tissues and facilitate their manipulation during vitreous surgery .
this article reviews the indications , applications and characteristics of vital dyes in vitreoretinal surgery .
various dyes are currently being used in routine clinical procedures , however the ideal staining agent has not yet been found .
any dye which is injected intravitreally has the potential to become toxic .
triamcinolone acetonide is used to highlight the vitreous and is particularly beneficial in determining the attachment of the posterior hyaloid to the underlying retina .
trypan blue stains epiretinal membranes and facilitates their complete removal . both indocyanine green and brilliant blue g stain the internal limiting membrane properly
, however concerns over indocyanine green toxicity have made surgeons switch to brillliant blue g as a safer alternative . |
atherosclerosis is a pathological process that takes place in the major arteries and represents one of the causes of myocardial infarction ( mi ) , ischemic stroke , and peripheral artery disease .
atherosclerosis , a multifactorial condition determined by environmental as well as genetic factors , has a strong inflammatory component characterised by deregulation of gene expression in macrophages within atherosclerotic plaques , together with local overproduction of cytokines . among those , chemokines ( chemotactic cytokines )
have also been implicated in atherosclerosis on the basis of clinical studies and animal disease models .
the chemokine rantes , also known as cc chemokine ligand ( ccl5 ) , is involved in the activation and proliferation of t - lymphocytes and is considered as a major chemokine implicated in both the acute and chronic phases of inflammation .
tissue rantes expression is differentially regulated : in atherosclerosis , rantes mrna has been detected in the lymphocytes , macrophages , myofibroblasts , and endothelial cells of human arteries undergoing accelerated atherosclerosis but not in normal coronary arteries .
platelets , which are also implicated in the atherosclerotic process including myocardial infarction ( mi ) , are known to store rantes in intracellular granules in the basal state and to secrete it when stimulated with thrombin .
single nucleotide polymorphisms in the rantes gene promoter and intronic region have been described : c-28 g , g-403a ( relative to transcription start site ) , polymorphism in intron 1 ( in1.1 t / c ) , and in the 3untranslated regions [ 6 , 7 ] .
the case - control study investigating rantes snp g-403a in germans associated this snp with a susceptibility to coronary atherosclerosis ; in another study , in hungarians , however , this association was not significant . that rantes polymorphisms may play a role in cardiac pathology
was also suggested by a report enrolling patients with type 2 diabetes mellitus and end stage renal disease : the carriage of either rantes -403 g / a or intronic polymorphism ( in1.1 t / c ) was associated with all - cause mortality due mainly to cardiac events . concerning the effect of these snps on rantes expression , an increased gene expression was linked with the minor allele in the rantes gene at the nucleotide position -403 and -28 [ 11 , 12 ] .
however , in vitro functional data are more important for rantes intronic polymorphism t / c : in1.1c allele or haplotypes that include this variant showed a strong dominant association with rapid progression to aids .
furthermore , rantes gene polymorphisms were associated with the outcomes of hcv and hbv infections [ 13 , 14 ] . by analogy , atherosclerotic lesions ,
the underlying cause of coronary artery disease ( cad ) and mi , represent a series of highly specific cellular and molecular events of the inflammatory response . despite convincing evidence for a role of the rantes polymorphisms in different inflammatory diseases , their role in cad
requires further investigation . given the inflammatory component of coronary atherosclerosis and the lack of case - control investigations of more than one rantes polymorphism in mi
, this study examined the relationship between two rantes polymorphisms ( promoter g-403a , intron1.1 t / c ) with their haplotypes and myocardial infarction . in agreement with genetic epidemiology principles , our replication
study comprised two population cohorts ( czech , russian ) differing in ethnicity from those populations in which the rantes gene was investigated previously ( hungarian , german ) .
a total of 804 unrelated caucasian individuals were enrolled into this case - control study , of which 467 were mi cases and 337 healthy controls subjects .
two population groups were recruited : ( 1 ) czech ( olomouc ) with 223 mi patients and 140 control subjects and ( 2 ) russian ( novosibirsk ) with 244 cases and 197 controls from the monica project register .
mi events were defined on the basis of electrocardiographic ( ecg ) changes and biomarker ( troponin , alternatively creatine - kinase ) measurements .
local research ethics committees approved the studies and the subjects provided written informed consent for use of samples in these genetic studies and anonymous publication of the summary data .
genotype analysis for the rantes snps was performed by polymerase chain reaction with sequence - specific primers ( pcr - ssp ) .
the investigated snps included rantes promoter g-403a ( rs2107538 ) and rantes intron 1.1 t / c ( rs2280789 ) .
the sequences of specific primers were as follows : allele g , forward 5 atg gat gag gga aag gag g 3 , allele a , forward : 5 cat gga tga ggg aaa gga ga 3 , constant reverse : 5 ttc tct gct gac atc ctt ag 3 ; intron 1.1 t / c ; allele t , forward : 5 cct gat cag ttt ttc tgt ctt t 3 , allele c , forward : 5 cct gat cag ttt ttc tgt ctt c 3 , constant reverse : 5 tgt gag cta ctc atg act ac 3. the internal control primers and conditions for pcr were adopted from a phototyping methodology .
calculations were performed using the spss v.13.0 ( spss inc , chicago , il ) .
categorical data of the study population was reported as frequencies and percentage , the continuous data as means with standard deviation .
the distribution of the rantes genotypes and alleles were analyzed using the pearson 's contingency table -test .
a multiple variable logistic regression model was used to estimate the association between genotype and disease .
haplotype analysis was performed using an accelerated expectation - maximization algorithm ( software arlequin , version 3.000 ) .
first , we evaluated distribution of rantes polymorphisms in patients and control subjects ( for genotype and allele frequencies see table 2 ) . out of 467 and 337
individuals in each group , the genotype data for rantes intron 1 polymorphism was available in 465 patients and 330 controls as 2 patients and 7 control samples failed to amplify ; the overall failure rate was only 1.2% ( 9/804 ) . both the rantes -403 and rantes in1.1 genotypes were distributed as expected from the hardy - weinberg equilibrium ( p > .05 ) . in both the czech and russian populations
, there were no significant differences in the frequencies of genotypes between the mi cases and controls for the two rantes polymorphisms studied .
the distribution of rantes genotypes was similar also when both populations were evaluated together ( table 2 ) , except that the frequency of cc homozygous genotype in mi patients ( 2.8% ) was higher than observed in controls ( 0.9% ) , but this difference did not achieve a statistical significance .
further , we carried out standard allelic association analysis : minor allele frequencies for the two polymorphisms were calculated , and the odds ratio and 95% confidence interval for the rantes -403a and rantes in1.1c were 0.996 ( 95% ci 0.7401.340 ) and 1.054 ( 95% ci 0.7591.464 ) , respectively , ( p > .05 in both cases ) . also , when evaluating carrier status for -403a and in1.1c alleles , there were no significant differences between mi cases and controls ( p = .97 , df = 1 for aa+ag versus gg -403 genotypes ; and p = .75 , for cc+ct versus tt in1.1 genotypes ) .
next , the patients were divided into subsets according to gender and age at the time of the 1st myocardial infarction , and univariate analyses were performed .
there was no difference in genotype distribution when male and female cases were compared with controls of appropriate gender separately ( rantes g-403a : p = .863 for males and p
= .789 for females ; rantes in1.1 t / c : p = .148 for males and p = .744 for females ) .
no differences in genotype and alleles frequencies ( -403a and in1.1c ) were observed between the patients stratified according to the age of mi onset ( > 50 yr and < 50 yr ) ( table 3 ) .
also , in the logistic regression analysis , no evidence for association was found between the polymorphisms of the rantes gene and the risk for mi in our study population ( table 4 ) .
the combined genotypes ggtt and agtc were the most frequent ( case / control group 62.3%/64% and 18%/18.4% , resp . ) .
no statistically significant difference was observed in the rantes combined genotypes between the cases and controls ( = 4.53 , df = 7 , p = .717 ) .
however , we detected strong linkage disequilibrium between the two less common alleles for two rantes snps : allele a of the rantes promoter g-403a and c allele of the rantes intron 1 t / c ( d = 0.83 for patients with mi and d = .88 for control group ; p < 10 in both cases ) .
this case - control study aimed at exploring the association between coronary atherosclerosis manifested by myocardial infarction and two functional polymorphisms in the rantes ( ccl5 ) gene , the product of which is involved in atherosclerotic inflammation . in this two - population replication study ,
no significant differences in the distribution of rantes g-403a , and in1.1 t / c single nucleotide polymorphisms were observed between patients with mi and the control groups .
further , when the subjects were stratified according to their gender and age at the first mi episode , no significant relationships were revealed .
thus , our findings do not support the previous report of association between rantes promoter g-403a polymorphism and coronary artery disease .
importantly , our report represents the first investigation of two linked rantes polymorphisms and specifically analysis of their haplotypes in the slavonic population .
previous studies associating rantes snps with coronary atherosclerosis [ 8 , 9 ] concentrated on the rantes promoter polymorphism ( g-403a ) and did not explore the relationship between cardiovascular symptomatology and further polymorphisms in the rantes gene , including its intronic variant in1.1t / c .
only bger et al . , who investigated rantes promoter and intronic polymorphisms in type 2 diabetes / end stage renal disease , reported that patients carrying the rantes-403a or in1.1c allele had higher all - cause mortality risk , mainly due to cardiac events .
we , however , can not compare their data with our results , because boger 's study did not include a control population ( healthy controls ) and also estimated the effect of genetic predictors of survival and functional polymorphisms on mortality in another disease condition . a difference in exact disease phenotypes
may be another less prominent , but still relevant factor explaining the discrepancy between our negative data and the first case - control association study on rantes , which showed a trend indicating a higher risk for coronary artery disease for hungarian patients with the -403a allele .
the population size of their cohort and our slavonic population was identical ; however , the hungarian study enrolled patients with severe coronary artery disease ( cad ) not necessarily presenting with mi .
the luric ( the ludwigshafen risk and cardiovascular health ) study was the second to report the association for -403a allele .
we speculate that the deviation of distribution of g-403a genotypes from that expected from the hardy - weinberg equilibrium in controls of the luric study , may contribute to the discrepancy between luric and our findings concerning the relationship of the rantes -403a variant and cad .
though smaller in number of enrolled subjects , our czech and russian groups were strongly compliant with the hwe .
strong linkage disequilibrium was observed throughout the rantes gene , and in particular between the alleles at the four most polymorphic sites , -403 , -28 , in1.1 , and 3222 .
the in 1.1 t occurs in combinations with -403 g in 77% of genotyped european americans ( 5% had in1.1t/-403a haplotype ) , and allele in1.1c occurred with frequencies of 0.14 in european americans , 0.20 in african americans , and 0.34 in asians . in the present study , we revealed similar allele in1.1c frequencies in our investigated slavonic populations as in european americans , and confirmed strong linkage disequilibrium between two less common variants at rantes -403 and in 1.1 loci .
the functional effect of particular snps in the rantes gene and the level of its expression and/or secretion which may provide a mechanism explaining the observed association with cad , is still controversial [ 7 , 11 , 12 , 17 , 18 ] .
the -403a allele has been associated with increased transcription of the rantes gene , and further evidence of the functional importance of this variant is provided by a significant risk of atopic dermatitis , atopy , and asthma in adults but without studying the effect of the -28 or in1.1 loci .
other studies showed an upregulatory role for the -28 g allele but did not demonstrate upregulation for -403a [ 12 , 18 ] .
another study demonstrated that rantes in1.1c alelle was associated with reduced rantes expression , -28 g allele had a moderate role for expression and -403a allele was not regulatory .
discrepant data on correlations between rantes polymorphisms and level rantes expression for other common inflammatory and chronic infection diseases support the need for further investigation of the relationship between variability in rantes gene and its expression . in conclusion , in this two - population - based case - control study , no significant difference was detected in the distribution of two rantes gene polymorphisms between healthy controls and patients with myocardial infarction , indicating the lack of association between coronary atherosclerosis and rantes single nucleotide polymorphisms .
our results , therefore , do not support a major involvement of rantes g-403a and in1.1 t / c polymorphisms in genetic predisposition to myocardial infarction . | coronary artery inflammation is a critical process in the pathogenesis of myocardial infarction ( mi ) .
the chemokine ccl5/rantes ( regulated upon activation , normal t cells expressed and secreted ) is expressed in advanced atherosclerotic lesions .
functional polymorphisms of the rantes gene can , therefore , be involved in the pathogenesis of coronary artery disease .
we examined the association of polymorphisms in the rantes gene with myocardial infarction in slavonic populations of czech and russian origin .
a total of 467 post - mi patients and 337 control subjects were genotyped for rantes promoter g-403a ( rs2107538 ) and intron 1.1 t / c ( rs2280789 ) variants by pcr - ssp .
both rantes genotypes and allele frequencies did not differ between case and control groups .
haplotype - based analysis also failed to reveal an association between mi and investigated markers .
strong linkage disequilibrium was detected between particular rantes alleles .
the data do not support an association between rantes g-403a polymorphism and mi , as reported previously . |
the advent of antenatal ultrasound has allowed the diagnosis of conditions like large neck masses affecting the fetus which can compromise the airway after delivery . fetal magnetic resonance imaging ( mri )
has helped in better delineation and planning for perinatal intervention which will improve the survival of these babies .
ex - utero intra partum treatment ( exit ) is a procedure conducted for a fetus diagnosed with compromised airway . by this procedure
the airway can be secured while the fetus is on utero - placental circulation and hypoxia can be prevented .
there are many indications for exit but the commonest indication is for a fetus with compromised airway . the diagnosis of congenital high airway obstruction syndrome ( chaos ) , lesions in the mouth and neck compressing the trachea is a definite indication for exit . detailed planning and execution is essential for this procedure to be successful .
this procedure has been extensively described in literature and this is one of first few case reports from india .
we present our experience with exit procedure and the challenges and the optimal positioning of the mother , fetus and anesthesiologist which helped us in securing the airway .
a 29-year - old multi - gravida was referred to our hospital with an antenatal diagnosis of a large cystic hygroma diagnosed at 38 weeks of gestation .
fetal mri revealed a large multiseptated cystic mass ( measuring 12 8 cm ) involving both sides of the neck with compression over the trachea [ figure 1 ] . hence a difficulty in securing the airway was anticipated and exit procedure was planned .
a multispecialty team comprising of obstetricians , anesthesiologists , pediatric surgeons , neonatologists , and radiologists were involved in planning and executing the procedure . under general anesthesia
two teams of anesthesiologists were present , one for the mother and the other for the fetus .
apart from the routine monitoring , radial artery cannulation was done for invasive blood pressure monitoring .
patient was positioned in the supine position with left lateral tilt to prevent compression on the aorta .
the legs were slightly abducted in order to allow easy access to the fetus for intubation .
general anesthesia was commenced and maintained with higher concentration of isoflurane ( for complete uterine relaxation ) , oxygen , nitrous oxide , and muscle relaxants . before skin incision
, ultrasound was done to locate the placental edge which revealed two loops of umbilical cord around the fetal neck . after skin and uterine incision ,
fetal heart rate was monitored with an ultrasound probe and manually by umbilical cord pulsation .
ultrasound - guided aspiration of the cyst was done but since most of the cysts were microcystic , adequate decompression could not be obtained .
the neck was delivered up to the sternal notch [ figure 3 ] and then intubated .
intubation could be accomplished in 3 minutes and 47 seconds and airway was secured with a 3-mm i d oral endotracheal tube and then ventilated with 100% oxygen .
there was no fetal bradycardia and cord was clamped and handed over to the neonatologist for further resuscitation .
isoflurane was stopped and oxytocin 20 units in 500 ml ringer lactate solution was administered .
the neonate was taken up for definitive surgery after 4 hours of stabilization in the nicu .
fetal mri showing large multiseptated cystic mass compressing trachea fetal head and mass delivered intubation attempts failed fetus delivered up to sternal notch successful intubation
exit was initially described to remove the tracheal clip which was utilized to plug in fetuses with cdh ( congenital diaphragmatic hernia ) . by this procedure , the fetus is maintained on utero - placental circulation without hypoxia to the neonate .
later it was expanded to other procedures such as intubation for fetuses with large neck masses .
the exit procedure maximizes the chance of survival for theses fetuses with large neck masses .
martino et al . , had described respiratory distress in their five cases with antenatal detection of cervical teratoma .
two of them were planned for exit but lost one baby due to difficulty in intubation as the baby was delivered via naturalis due to preterm delivery .
our patient had a large neck mass compressing the trachea and successful intubation could be performed because of exit .
the philadelphia group has explained the procedure of exit in great detail and the procedure was performed as advised .
on reviewing the literature , mother has been positioned either in supine with left uterine displacement , modified lithotomy , or lithotomy .
murphy dj et al . had positioned the mother in the modified lithotomy position for better access during intubation of the fetus .
considering the technical difficulties for the obstetrician to deliver the fetal head in lithotomy position on one hand and easy accessibility it would offer for the intubation of the fetus on the other hand , we decided to position the mother supine with left tilt and lower limbs abducted for the anesthesiologist to stand in between the patient 's legs for better access to the fetal airway [ table 1 ] .
the ideal positioning of the fetus before intubation has been to deliver the head and neck upto thorax . in our patient ,
the fetal head with the neck mass was not delivered upto the thorax because of the two twists of the cord around the neck . as the positioning was sub - optimal
the head and neck up to the sternal notch facilitated the proper alignment of the oro - pharyngo - laryngeal axes and successful intubation .
it would be ideal to unwind the umbilical cord while the fetus is still in the relaxed uterus , but unwinding the tight loop could lead to undue traction on the cord compromising the fetal circulation .
optimal position of the mother , anesthesiologist , and fetus for intubation during exit the uterus should be relaxed in order to maintain placental circulation .
a uterine stapler minimizes blood loss and this is essential if a longer procedure is planned . due to non - availability of staplers and
as we had planned only for intubation we could perform hysterotomy without staplers and the blood loss was approximately 400 ml .
exit has a definite role in fetuses diagnosed with large neck masses where airway compromise is suspected .
optimal positioning of the mother , obstetrician , anesthesiologists , and fetus is of paramount importance for successful and safe exit , especially in fetus with cord around the neck .
thus , exit can be safely performed in indian set up , with adequate planning and execution . | ex - utero intrapartum treatment ( exit ) is performed for fetuses diagnosed with large neck masses .
a case report of a fetus diagnosed with a large cystic hygroma and cord around the neck who was delivered by exit is presented .
the airway challenges and optimal positioning is discussed . |
supplementary material is available for this article at 10.1007/s13659 - 012 - 0035-y and is accessible for authorized users .
| solenostemon scutellarioides is a commercially important ornamental plant . in present study
, it was attempted to establish a protocol for high frequency in vitro regeneration of s. scutellarioides .
caulogenesis was found to be significant in solid ms medium supplemented with 0.5 mg dm3 bap .
combination of ga3 ( 0.5 mg dm3 ) and bap ( 0.5 mg dm3 ) induced high frequency regeneration coupled with higher plant height .
the plantlets exposed to iba ( 1 mg dm3 ) exhibited significant root development in terms of first appearance of root and number of roots per shoot .
to improve its commercial acceptability in terms of plant architecture and foliage colouration , two weeks old in vitro grown plantlets were exposed to different culture conditions namely ms strength , sucrose concentration , ph and light .
a variable plant types with diverse ornamental traits were developed under different culture conditions .
electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 012 - 0035-y and is accessible for authorized users . |
tomato ( solanum lycopersicum ) is one of the most important vegetable crops cultivated worldwide .
tomato has a diploid ( 2n = 2x = 24 ) and relatively compact genome of approximately 950 mb .
genetic linkage maps of tomato have been created by crossing cultivated tomato ( s. lycopersicum ) with several wild relatives , s. pennellii , s. pimpinellifolium , s. cheesmaniae , s. neorickii , s. chmielewskii , s. habrochaites , and s. peruvianum .
introgression lines generated from a cross between s. lycopersicum and s. pennellii have contributed to the isolation of important loci and quantitative trait loci ( qtls ) related to fruit size by utilizing dna markers on the tomato - expen 2000 genetic map [ 49 ] .
such interspecies genetic mapping is effective because the divergent genomes provide many polymorphic dna markers .
in contrast , intraspecies mapping is less popular in tomato because of the low genetic diversity within cultivated tomatoes that has resulted from the domestication process and subsequent modern breeding .
recently , we developed snp , simple sequence repeat ( ssr ) , and intronic polymorphic markers using publicly available est information and bac - end sequences ( bess ) derived from
heinz 1706 , a standard line for tomato genomics [ 11 , 12 ] , and applied these markers to create linkage maps between micro - tom and either ailsa craig , a greenhouse tomato , or m82 , a processing tomato , by mapping 1,137 markers .
micro - tom , a dwarf cultivar , is regarded as a model cultivar for functional genomics of tomato because of several characteristics , including small size ( 20 cm plant height ) , short life cycle ( 3 months ) , existence of indoor cultivation protocols under normal fluorescent conditions , and high - efficiency transformation methods that have been developed for this line [ 1315 ] .
the dwarf phenotype of micro - tom is the result of mutations in at least two major recessive loci .
dwarf ( d ) encodes a cytochrome p450 protein , which functions in the brassinosteroid biosynthesis pathway .
another locus , miniature ( mnt ) , is suggested to associate with gibberellin ( ga ) signaling without affecting ga metabolism , but the causal gene has not been identified to date . in japan , micro - tom genomics resources have been extensively accumulated , mainly in the framework of the national bioresource project ( nbrp ) ( http://tomato.nbrp.jp/indexen.html ) .
large - scale ethyl methanesulfonate ( ems ) and gamma - ray - mutagenized populations have been created , and visible phenotype data have been accumulated [ 1820 ] . the availability of micro - tom genome sequence data will accelerate the mapping of mutant alleles .
. currently , about 90,000 bess are available at the sol genomics network ( sgn , http://solgenomics.net/ ) .
bac - end sequencing has been conducted for other crop species . in the rice indica cultivar
reference genome . as a result , 12,170 paired bess were mapped that covered 80% of the rice genome . recently
, bac - end sequencing has been performed in crop plants with higher genome complexity .
bess from a commercial sugarcane variety , an interspecific hybrid with complex ploidy , were generated to analyze microsynteny between sugarcane and sorghum . in wheat , which has a complex hexaploid genome , the short arm of chromosome 3a was flow sorted to make a bac library , and chromosome arm - specific bess were generated for dna marker development . in switchgrass , more than 50,000 ssrs were identified from 330,000 bess , and this enabled detailed analysis on the evolution of this species . a low level of genetic variation has been observed for cultivated peanuts .
polymorphic ssrs were accumulated from the bess and successfully used in the construction of a genetic map .
bac - end sequencing can be useful as a resource for performing comparative genomic studies through mapping of the sequences to a reference genome and by facilitating the development of polymorphic dna markers . in the present study , we generated 93,682 single - pass end sequences from a micro - tom bac library . to compare the structures between the reference tomato
heinz 1706 genome , mapping of unique ends was performed , and possible genome rearrangements and polymorphisms were identified .
micro - tom ( tomjpf00001 ) seeds were obtained from the nbrp ( mext , japan ) and sent to the clemson university genomics institute ( cugi ) for bac library construction .
the genomic dna was partially digested , and fragments were cloned into the hind iii site of pindigobac536 .
a total of 55,296 clones in escherichia coli dh10b cells were arrayed in 144 384-well plates . to analyze bess ,
the bac dnas were amplified using a templiphi large - construction kit ( ge healthcare , uk ) , and the end sequences were analyzed according to the sanger method , using a cycle sequencing kit ( big dye - terminator kit , applied biosystems , usa ) with a type 3730xl dna sequencer ( applied biosystems ) .
the resulting sequence reads were quality checked with phred [ 27 , 28 ] , allowing the identification and removal of low - quality ( qv < 20 ) sequences .
the 93,682 reads clearing the quality criteria were submitted to ddbj / genbank with accession numbers ft227487-ft321168 .
bes reads were subjected to similarity search using the blastn program [ 29 , 30 ] . to isolate unique sequences from repetitive ones ,
93,682 bess were searched against the repeat database in itag2.3 ( http://solgenomics.net/ ) using a cutoff e - value of less than 10 .
the remaining sequences were searched against the published version of the heinz 1706 genome ( sl2.40 ) , which was accessed from the sgn database ( http://solgenomics.net/ ) . from all of the blast alignments ,
bess were extracted according to the following criteria , suggested in a previous report : ( 1 ) sequence identity > 90% and alignment coverage > 50% ; ( 2 ) mapped positions of each pair of ends < 200 kb apart in the same chromosome ; ( 3 ) direction of each paired end is correct ; ( 4 ) blastn e < 10 ; ( 5 ) a minimum of one hit for one of the paired ends ; ( 6 ) no redundant chromosomal locations
. sequence polymorphisms ( snps and indels ) between micro - tom and heinz 1706 were predicted based on the blastn alignment . since we did not allow a gap exceeding 27 bases , only indels up to 26 bases in length were counted .
mapped data and snp / indel sites were made accessible through the database tomatomics at http://bioinf.mind.meiji.ac.jp / tomatomics/. bac clones are available upon request from nbrp tomato ( http://tomato.nbrp.jp/indexen.html ) .
the bac insert size distribution was deduced based on the mapping results . according to these results , 45.4% ( 6,396 out of 14,101 ) of the bacs ranged from 100 to 120 kb , with average and median sizes of 101.3 kb and 101.8 kb , respectively ( figure 1 ) . by multiplying by the number of clones ( 55,296 ) , this bac library covers
micro - tom bes mapping to the heinz 1706 genome was processed as indicated in figure 2 . by eliminating repetitive , redundant , and unmapped sequences
, 28,804 unique pair ends and 8,263 unique ends were selected .
paired - end sequences were mapped onto the reference tomato genome sequence , and 2,248 hypothetical bac contigs were constructed ( see details at tomatomics , http://bioinf.mind.meiji.ac.jp/tomatomics/ ) . the integrity of the hypothetical contigs was confirmed by linking to the dna markers on two genetic maps , amf2 and mmf2 ( see supplementary table 1 in supplementary material available online at doi:10.1155/2012/437026 ) .
the genome coverage of the hypothetical bac contigs was assessed by applying euchromatin / heterochromatin boundary information from the genetic map expen2000 .
the results indicated that the euchromatin coverage ranged between 45.1% and 71.1% ( average , 58.9% ) among the different chromosomes , while heterochromatin coverage ranged between 57.4% and 75.3% ( average , 67.3% ) .
the total physical length of the bac contigs was 495,833,423 bp , covering 65.3% of the total chromosomes ( table 1 ) . to assess the occurrence of genome rearrangements , micro - tom and the reference tomato
possible inversions , translocations , and insertions were considered . to eliminate an artificial effect ( e.g. , chimeric bac clones ) ,
only regions covered by more than two bac clones were selected . after removing regions that had cleared the criteria for extraction ( see section 2 ) but were either shown to be multicopy by manual evaluation of the blast results or displayed similarity to transposable elements , we obtained two cases of a possible rearrangement between micro - tom and heinz 1706 ( table 2 ) . on chromosome 2 ,
the size of this inversion could be 20220 kb depending on which end of the bac clone is inversed .
translocation and inversion were observed on chromosome 3 . for each of two bac clones ( mtbac041l05 and mtbac077o14 ) , one of the ends
was mapped to 6,601 kb of chromosome 3 , while the other end was mapped to 55,665 kb , more than 49 megabases apart .
snps and indels between micro - tom and heinz 1706 were identified . among the snps and indels
found , 171,792 were mapped on 12 chromosomes , and 2,635 were mapped on pseudomolecules with no chromosomal information ( sl2.40ch00 of the tomato whole - genome shotgun chromosomes ) ( table 3 and supplementary table 2 , see details at tomatomics ) . according to these results , among the mapped snps and indels , a total of 30,930 polymorphisms were found in the euchromatin ( 1 out of 3,565 bp ) , and 140,862 were found in the heterochromatin ( 1 out of 2,737 bp ) .
transversion - type snps were observed in 83,262 cases , while 60,631 were transition - type snps . among the 30,534 indels , single - base insertions ( on the sl2.40 version of the tomato whole - genome shotgun chromosomes ) were observed in 10,740 cases , and single - base deletions were seen in 17,064 cases .
the remainder were larger indels , ranging from 2 to 26 bp ( supplementary table 2 ) .
by selecting unique end sequences from 93,682 reads , 28,804 paired ends ( 14,402 pairs ) and 8,263 unpaired ends were obtained .
the majority of the nonselected sequences ( 43,598 ) were derived from repetitive regions . for the rest , 10,943 had redundant hits to the heinz 1706 genome , possibly including repetitive sequences that were not represented in the repeat database in itag2.3 ( http://solgenomics.net/ ) , 2,015 showed weak similarity , and 59 showed no similarity ( figure 2 ) .
considering that the genome has been previously estimated to be composed of 25% gene - rich euchromatin [ 31 , 32 ] , bes selection in this study ( 39.6% , ( 28,804 + 8,263)/93,682 ) ) could have eliminated repetitive regions to a moderate degree .
micro - tom was bred by crossing the home - gardening cultivars , florida basket and ohio 4013 - 3 .
the pedigree of ohio 4013 - 3 suggested that a wild relative species was used in the breeding history [ 18 , 33 ] .
the micro - tom genome is now being sequenced ( draft sequence data available at ddbj with the accession number dra000311 ) , and mapping of orphan bess to the de novo assembly of micro - tom genome data will help to clarify this question .
the total physical length of micro - tom bac contigs was 495,833,423 bp , which covers approximately 65.3% of the dna from all 12 chromosomes . in the kasalath rice bes analysis ,
chromosomal coverage in relation to the reference nipponbare pseudomolecule was about 80% , despite the lower number ( 78,427 ) of analyzed bess .
because we used the same criteria for repetitive sequence selection ( e < 10 ) , the discrepancy between the two studies might be due to the larger genome size of tomato ( 950 mb ) compared with rice ( 430 mb ) .
our micro - tom bac coverage is reasonable , taking into account the scale of the bac library used .
micro - tom has been considered as a model cultivar to promote functional genomics studies of tomato by taking advantage of its characteristics .
currently , many tools and platforms have been developed , and some of these are already available to the research community .
the present study characterized the overall polymorphisms found between micro - tom bess and the reference tomato heinz 1706 genome .
in addition , two possible genome rearrangement events , on chromosome 2 and chromosome 3 , were observed ( table 2 ) . in the case of translocation and inversion on chromosome 3 , a gene annotated as reverse transcriptase was found in the flanking region ( solyc03g104840.1 ) .
we speculate that this region was translocated by the activity of a retrotransposon , as it was in the case of sun .
enhanced expression of sun caused by a gene duplication event mediated by the retrotransposon rider led to an elongated fruit shape . in the future
, we plan to sequence the entire bac and expect that this will help us to characterize these events in more detail . in the case of the other rearrangement possibility , on chromosome 2
since these rearrangements took place in euchromatin , which is rich in genes , these regions could represent an interesting target to investigate their possible effects on phenotypic variation between micro - tom and the reference tomato .
we mapped the polymorphisms and depicted them , alongside maps showing covered regions and gaps , in figure 3 . on chromosomes 2 , 5 , and 11
, polymorphisms seemed to be concentrated in the heterochromatic regions ; however , this tendency was not clearly observed in the other chromosomes . for the other regions , the polymorphism discovery rate seemed to be somehow correlated with the bac coverage .
although our analysis indicated little possibility of large - scale genome rearrangement between micro - tom and heinz 1706 ( table 2 ) , this uneven polymorphism distribution suggests the existence of highly divergent chromosomal regions .
the gaps in the hypothetical micro - tom bac contigs could have resulted from low coverage of the bac library , but the occurrence of chromosomal segments specific to either micro - tom or
the ongoing micro - tom genome sequencing and de novo assembly of the micro - tom genome will clarify the genome structure in detail , enabling a more solid assessment of the differences between micro - tom and heinz 1706 .
we had previously developed snp markers among several cultivated tomatoes . by selecting snps through in silico analysis using public est information and
previously developed ssr markers , 1,137 markers were obtained and successfully mapped on linkage groups between micro - tom and either ailsa craig or m82 . in the present study , we identified 171,792 snps and indels and mapped them on 12 chromosomes .
the average density was 1 snp per 3,565 bp in euchromatin and 1 snp per 2,886 bp in the genome in general ( including both euchromatin and heterochromatin ) .
previously , large - scale micro - tom full - length cdna analysis and comparison of exon regions with those on the heinz 1706 genome revealed a mean sequence mismatch of 0.061% ( 1/1,640 bp ) .
we used the published version of the heinz 1706 genome sequence , which has higher coverage , giving rise to greater accuracy , although our selection may still contain sequence errors because bess are single - pass sequences .
the information provided in this study will be useful in the development of dna markers between micro - tom and cultivated tomatoes , which will facilitate a better understanding of the physiological and metabolic differences between them .
it would also be useful in the genetic mapping of micro - tom mutants through the generation of f2 segregating populations . | a total of 93,682 bac - end sequences ( bess ) were generated from a dwarf model tomato , cv .
micro - tom . after removing repetitive sequences ,
the bess were similarity searched against the reference tomato genome of a standard cultivar ,
heinz 1706 . by referring to the heinz 1706 physical map and by eliminating redundant or nonsignificant hits , 28,804 unique pair ends and 8,263 unique ends were selected to construct hypothetical bac contigs .
the total physical length of the bac contigs was 495 , 833 , 423 bp , covering 65.3% of the entire genome .
the average coverage of euchromatin and heterochromatin was 58.9% and 67.3% , respectively . from this analysis ,
two possible genome rearrangements were identified : one in chromosome 2 ( inversion ) and the other in chromosome 3 ( inversion and translocation ) . polymorphisms ( snps and indels ) between the two cultivars were identified from the blast alignments . as a result , 171,792 polymorphisms were mapped on 12 chromosomes . among these , 30,930 polymorphisms were found in euchromatin ( 1 per 3,565 bp ) and 140,862 were found in heterochromatin ( 1 per 2,737 bp ) .
the average polymorphism density in the genome was 1 polymorphism per 2,886 bp . to facilitate the use of these data in micro - tom research , the bac contig and polymorphism information are available in the tomatomics database . |
the innate immune system or the nonspecific immune system is the first and the oldest line of defense in organisms [ 1 , 2 ] .
it was the most dominant form of immunity before the evolution of the more sophisticated adaptive immunity .
it is comprised of various mechanisms which are responsible for rapid defense of the host organism against invasion by other factors in a nonspecific manner .
the innate immune system differs from the adaptive immune system in a way that while it is able to defend the body against pathogens , it is not able to impart long - lasting immunity to the host , unlike the latter . despite the evolution of the more complex and specific adaptive immunity
, innate immunity still continues to function as the primary line of defense for most organisms .
the antimicrobial action in the innate immunity is mediated by various antimicrobial proteins and peptides , which have been evolutionary conserved .
antimicrobial peptides are small peptides which demonstrate broad - spectrum antibiotic activity against various gram - positive and gram - negative bacteria , fungi , protozoa , and viruses [ 58 ] .
while the most common mechanism of action deployed by these peptides is perturbation of microbial cell membrane [ 911 ] , there are other mechanisms which are also prevalent [ 1216 ] . due to increasing resistance to antibiotics
use of antimicrobial peptides is one of the promising approaches which may lead to potential antimicrobial drugs [ 16 , 2024 ] .
it has been observed that peptides which are predominantly cationic and hydrophobic in nature show potent antimicrobial activity [ 5 , 2529 ] .
many of these peptides including indolicidin from bovine neutrophiles , tripticin from porcine neutrophil granules , puroindoline from wheat seeds , combi-1 , a synthesized antimicrobial peptide , and lys h and lys c from lysozyme have been extensively studied .
although these peptides adopt various conformations the alpha - helical conformation with polar and nonpolar groups on opposite sides of the helix tends to be the most abundant [ 3537 ] .
the antimicrobial property of these amphipathic alpha - helical peptides increases sequentially with increase in net charge .
lactoferrin is an iron - binding glycoprotein which is found in most of the exocrine secretions such as milk , tears , nasal secretions , saliva , urine , uterine secretions , and amniotic fluids [ 3941 ] as well as in secondary granules of neutrophils .
it exerts a wide antimicrobial activity against a number of bacterial , viral , and fungal pathogens in vitro [ 4348 ] .
lactoferrin exerts its antimicrobial action not just in the form of the intact molecule but the monoferric lobes and active peptides of lactoferrin also have a role in the host defense against microbial disease [ 4952 ] .
lactoferrin is a rich source of cationic and hydrophobic antimicrobial peptides , which may be used against microbes [ 53 , 54 ] .
these antibacterial peptides which are a part of the polypeptide chain of lactoferrin and are released upon the proteolysis of this molecule by various proteolytic enzymes can be developed into clinically useful lead molecules for antimicrobial therapeutics [ 55 , 56 ] .
although it has been shown that native lactoferrin exerts its antimicrobial action through sequestration of iron [ 5759 ] , it is still unclear how the antimicrobial lactoferrin peptides act against the microbes .
though a number of studies have implicated these peptides in the binding to the outer membrane proteins of various bacteria or binding to microbial proteases [ 6062 ] , the structure - function interrelationships of these peptides have not yet been established .
a number of functional peptides are produced from lactoferrin by the action of proteolytic enzymes .
it is expected that these enzymes are present in the gastrointestinal tract as well as the site of microbial infection , and hence , they may contribute in the natural function of lactoferrin in the human body .
this paper reports the comparison of these peptides with other antimicrobial peptides found in nature as well as cross - species comparison of the sequences of these antimicrobial peptides from the native sequences of lactoferrin with the intent to draw evolutionary inferences of their function .
although many antimicrobial peptides from lactoferrin have been isolated and characterized , only three of them have been studied in detail .
these are lf1 - 11 , lactoferrampin , and lactoferricin . the sequences of these peptides indicate that these peptides belong to the n - terminal half of lactoferrin ( figure 1 ) .
hydrophobicity , cationicity , and helical conformation of these antimicrobial peptides are the important characteristics that determine their antimicrobial potency [ 9 , 63 , 64 ] .
all these peptides have high pi values ( > 9 ) and is expected to interact with negatively charged elements .
all three have different sequences , structural elements , and modes of action ( table 1 ) .
an attempt to analyze and decipher the structural and functional characteristics of three peptides is made in this paper .
lf1 - 11 , as its name suggests , is the n - terminal peptide of lactoferrin , comprised of the first eleven residues of the molecule .
this peptide has been shown to be highly effective against five multidrug - resistant acinetobacter baumannii strains and methicillin - resistant staphylococcus aureus and various candida species [ 67 , 68 ] .
the potent antimicrobial effect of lf1 - 11 was attributed to the first two arginines at the n - terminus of human lactoferrin .
this conclusion was based on the fact that when the second or third arginines were replaced by alanine , the candidacidal activity of the lf1 - 11 was observed . additionally , while lf1 - 11 , lf2 - 11 , and lf3 - 11 showed comparable candidacidal activities , the same was found compromised in the case of lf4 - 11 .
the importance of the three arginines ( r2r4 ) for the potent antimicrobial activity of this peptide was established when synthetic peptides lacking the first three n - terminal residues were found to be less effective in the killing of bacteria .
also , mutant lactoferrin lacking the first five n - terminal residues displayed decreased binding to bacterial lipopolysaccharide . in yet another study ,
stallmann et al . studied the efficacy of local prophylactic treatment with human lf1 - 11 in a rabbit model of femur infection and observed that hlf1 - 11 effectively reduced the development of osteomyelitis in a rabbit model .
it was speculated that the mechanism of antimicrobial action of lf1 - 11 is mitochondrial damage , with the extracellular atp being essential but not sufficient for lf1 - 11 to exert its candidacidal activity . in later studies
, it was found that uptake of calcium by mitochondria is vital for killing of candida albicans by the lf1 - 11 . in another study , it was found that lf1 - 11 is responsible for directing the gm - csf - driven monocyte differentiation toward macrophages that produces both pro- and anti - inflammatory cytokines . it was speculated that the peptide could be used as agent to empower the innate immune response of the host for infections .
these results demonstrated the importance of the further development of lf1 - 11 as a promising drug against microbial infections in patients who may have compromised immune systems .
the cellular target for the immunomodulatory activity of lf1 - 11 was found to be myeloperoxidase , to which lf1 - 11 binds and inhibits after entering the monocytes .
a molecular modeling study by the same group demonstrated that lf1 - 11 bound at the active site of the enzyme .
the importance of the first two arginines and the cysteine at the tenth position was further substantiated by the fact that peptides which did not possess these necessary residues were not as effective in binding with myeloperoxidase .
the sequence comparison of lf1 - 11 among the six species ( table 2 ) shows that unlike human lf1 - 11 , which contains three arginines in the positions 25 ( r2r4 ) , the peptide from other species contains only one arginine ( r3 ) .
yet , it is noteworthy that the r4 has been replaced by lysine , which is also a basic residue in all the other species , thereby maintaining the highly cationic nature of the peptide throughout the species . also , in all the cases except human , arginine occurs at the seventh position also .
the most significant change is seen in the second residue which is proline in all cases except in human and camel .
notably , the hydrophobic residues , v6 , and w8 are conserved in all the cases .
lactoferrampin , comprised of residues 268284 in the n1 domain of lactoferrin , has been identified as an antimicrobial peptide and plays a key role in membrane - mediated activities of lactoferrin [ 76 , 77 ] .
it exhibits broad antimicrobial action against several gram - positive and gram - negative bacteria , notably , bacillus subtilis , escherichia coli , pseudomonas aeruginosa , and staphylococcus aureus , as well as candidacidal activity .
the antimicrobial action of this peptide was also found to be more potent than the native lactoferrin .
the structure of lactoferrampin revealed an amphipathic alpha - helix which begins with the n - terminus and ends at the 11th residue , followed by a c - terminus tail .
it is reported that the cleavage of this peptide at both the termini resulted in considerable decrease of the candidacidal activity .
the c - terminal residues of lactoferrampin are most critical for its antimicrobial action , possibly because the c - terminus consists of several residues with positive charges which are clustered together .
but truncation of c - terminal side did not alter the ability of this peptide to adopt helical conformations .
also , substitution of the basic residues at the c - terminus led to decrease in potency of this peptide [ 77 , 79 ] .
the n - terminal residues , truncated up to the sequence 270284 , are essential for maintaining the structure of this peptide in a helical conformation .
the helical conformation of this peptide was found to be critical for the potency against gram - positive bacteria as established when the bactericidal activities of two lactoferrampin peptides , lactoferrampin 265284 and lactoferrampin 268284 , were compared .
lactoferrampin 265284 , which consists of additional three residues , asp - leu - ile , showed a broader specificity since the asp - leu - ile sequence increases the tendency of this peptide to assume an alpha - helical conformation .
both the peptides possessed bactericidal activity against certain species of gram - positive and gram - negative bacteria .
compared to lactoferrampin 268284 , higher concentrations of lactoferrampin 265284 were required to kill the gram - negative bacteria , e. coli and p. aeruginosa .
the killing activity expressed as lc50 value ( the concentration that produced 50% reduction in viable counts of the microorganisms ) was found to be about 5.8 mol / l for lactoferrampin 268284 which is about 4 times higher than lactoferrampin 265284 .
the mode of action of this peptide on bacteria is by bacterial membrane binding and membrane disruption .
it is established that lactoferrampin is internalized within few minutes with the bacterial membrane permeabilization followed by cellular damage [ 81 , 82 ] .
distinct vesicle - like structures by the lactoferrampin peptide were also observed by freeze - fracture transmission electron microscopy in the membrane of c. albicans . it is speculated that this peptide exerted detergent - like activity , disturbing the hydrophobic interphase of the lipid bilayer .
several studies have revealed that the determinants for antimicrobial action are the orientation and structure of bovine lactoferrampin in bacterial membranes [ 78 , 8385 ] .
the solution structure of bovine lactoferrampin suggests that it adopts an amphipathic alpha - helical conformation across the first 11 residues of the peptide but remains comparatively random at the c - terminus [ 78 , 85 ] .
the interaction between the n - terminal tryptophan residue and model membranes of varying composition was evaluated suggesting that w1 is inserted into the membrane at the lipid / water interface .
along with this , the orientation of the phenyl side chain of f11 found to be in same direction as the indole ring of w1 also suggested that the amphipathic n - terminal helix anchors the peptide to membrane with these two residues that facilitates peptide folding [ 78 , 86 ] .
the same group has suggested that the hydrophobic patch in between the two residues as well as leu , ile , and ala side chains are responsible for interaction between the peptide and the hydrophobic core of a phospholipid bilayer .
in addition , the helix capping residues asp - leu - ile in the n - terminus of the peptide has been found to mediate the depth of membrane insertion by enhancing the affinity for negatively charged vesicles .
bovine lactoferrampin had been shown to have greater affinity to acidic phospholipids than that to neutral phospholipids .
haney et al . have speculated a two - step model of antimicrobial action by this peptide where the c - terminus positive charge cluster helps in the primary attraction of lactoferrampin to the membrane followed by the helix formation at the n - terminus that interacts to the surface of the bacterial lipid bilayer .
the sequence comparison of lactoferrampin from six different species shows uniform preponderance of cationic amino acid residues among hydrophobic residues ( table 3 ) .
the hydrophobic domain contains w1 in all the species that is involved in membrane insertion .
bovine lactoferrampin 268284 has a net positive charge of 5 + at neutral ph with hydrophobic domain .
the hydrophobic moment ( ) of the peptide which is a measure of lipophilicity was found to be 5.42 .
in contrast , the human lactoferrampin has a net charge of 2 + resulting in reduced antimicrobial activity .
however , by increasing the net positive charge near the c - terminal end of human lactoferrampin , a significant increase in its antibacterial and candidacidal activity was obtained .
the basic amino acid residues crucial for the antimicrobial action were found to be conserved among all the six species .
lactoferricin is a multifunctional , 25-residue peptide that is generated upon cleavage of native lactoferrin by pepsin and represents amino acid residues number 1741 in lactoferrin .
the lactoferricin peptide is different from the other peptides described so far as it contains a disulfide bond between residues cys 20 and cys 37 in human lactoferrin and cys 19 and cys 36 in bovine lactoferrin .
the peptide has an abundance of basic amino acids like lysine and arginine as well as hydrophobic residues like tryptophan and phenylalanine .
the first report on lactoferricin in 1992 described this peptide to be more potent as an antibacterial agent in comparison with the intact lactoferrin and it was demonstrated to cause a rapid loss of colony - forming capacity in most of its targets .
however , some strains like pseudomonas fluorescens , enterococcus faecalis , and bifidobacterium bifidum strains were found to be resistant to lactoferricin .
the antibacterial activity of this peptide was attributed to its action of releasing lipopolysaccharide from bacterial strains and , hence , disruption of cytoplasmic membrane permeability after cell binding [ 8991 ] .
apart from having a broad antibacterial spectrum , lactoferricin was found to be highly potent against candida albicans [ 89 , 92 , 93 ] .
recently , it has also been shown to have antiviral [ 94 , 95 ] and antiprotozoal activities .
it also displayed other activities like inhibition of tumor metastasis and induction of apoptosis in human leukemic cells .
the mechanism of action of lactoferricin was attributed to 11-amino - acid amphipathic alpha - helical region which is positioned on the outer surface of the n - lobe of lactoferrin .
the proline at the 26th position ( p26 ) was found to be essential for the antibacterial activity , and it was speculated to be responsible for disruption of the helical region , and hence the helicity of the peptide was predicted to be an essential aspect of the antibacterial action of this peptide .
lactoferricin was found to be produced in the human stomach , indicating that this peptide is definitely generated in vivo for host defense .
the comparison of the antimicrobial activities of lactoferricin from human , bovine , murine , and caprine showed that bovine lactoferricin was the most potent .
the minimal inhibitory concentration ( mic ) of lactoferricin b differs according to their source .
a comparison of the mic values of lactoferricin shows that bovine lactoferricin is the most potent .
the mic of bovine lactoferricin against certain e. coli strains has been found to be around 30 g / ml while that derived from human is more than 100 g / ml . the efficiency of antibacterial activity of bovine lactoferricin is due to the presence of high amount of net positive charge ( + 8 ) and hydrophobic residues ( primarily w6 , w8 , and m10 ) [ 51 , 88 ] .
the action of this peptide is also dependent on the ph [ 89 , 103 ] .
it was shown that only six central residues ( 49 ) among the twenty five residues of the peptide are required for its antimicrobial activity .
it may be noted that the tetrapeptide krds is present only in human lactoferrin while it shows variations in the sequence of others .
it has been reported that krds inhibits platelet aggregation [ 105 , 106 ] .
the bovine lactoferricin has been demonstrated to interact with the negatively charged elements in the membrane of susceptible bacteria and disrupt the cell membrane .
a synthetic peptide derived from human lactoferricin has been found to be effective in depolarizing the bacterial cytoplasmic membrane with a loss of ph gradient .
the permeabilizing effect of bovine lactoferricin causes membrane disruption resulting in inhibition of macromolecular biosynthesis and ultimately cell death .
the mode of action is however different in gram - positive and gram - negative bacteria . in gram - negative bacteria
antimicrobial peptides act on lipopolysaccharides and in gram - positive bacteria they act on lipoteichoic and teichoic acids .
in addition to antimicrobial properties , lactoferricin derived from human and bovine origin has also been found to be effective in inhibiting the classical complement pathway .
this implicates a role of these peptides in suppression of inflammatory effects caused by bacteria .
the sequence analysis of the lactoferricin from various species indicates that unlike bovine lactoferricin there is only one tryptophan at position 6 ( table 4 ) .
a further exception to this is equine which has no tryptophan residue in either of the positions .
this shows that the two tryptophans in lactoferricin are important for its optimal activity against microbes .
the solution structures of lactoferricin have been determined from bovine and human sources .
the bovine lactoferricin adopted a distorted antiparallel beta sheet , in complete contrast with its conformation in the intact lactoferrin , as observed in the structures obtained by x - ray crystallography .
however , the solution structure of human lactoferricin was closer to its structure in native lactoferrin since the amphipathic helix was preserved from gln14 to lys29 . however , the beta - sheet character was not observed in the solution structure of human lactoferricin either .
lipopolysaccharide ( lps ) , the outer membrane component of gram - negative bacteria , is one of the major causes of endotoxin - induced production of inflammatory cytokines and septic shock .
lactoferrin has been shown to neutralize the effect of lps - induced toxicity by binding to lps [ 115 , 116 ] .
the cationic peptide derived from lactoferrin which is responsible for this interaction and release of lps is first identified to be lactoferricin .
the residues from 28 to 34 of lactoferrin corresponding to the region in human lactoferricin have been identified to have a high affinity for binding to lps .
the initial binding of the peptide with e. coli has been found to be due to interaction with bacterial lps .
further studies have shown that bovine lactoferricin can arrest the lps - induced cytokine release by suppressing the il-6 response in human monocytic cells stimulated by lps .
a synthetic peptide corresponding to the antibacterial region of human lactoferricin was also found to facilitate depolarization of the bacterial cytoplasmic membrane , loss of the ph gradient , and a bactericidal effect in e. coli .
modelling studies using synthetic peptides derived from human and bovine lactoferricin have shown that these cationic peptides with their positively charged residues first interact with lps carrying negative charges .
this is followed by hydrophobic interactions between the tryptophan residues of the peptides and the lipid a molecule of lps to promote structural disorganization .
similarly , a synthetic peptide corresponding to 11 residues of human lactoferricin near its n - terminus has been found to bind to lps and neutralize the lps - induced adverse effects in vitro and in monocytes [ 121 , 122 ] . in yet another study using nmr , it has been observed that this peptide folds into a
conformation formed by its hydrophobic core and the two clusters of hydrophilic residues of the peptide targets the two phosphate moieties of lipid a in lps .
the antimicrobial peptides found in nature are classified into four groups according to a combination of their sequence homologies , functional similarities , and common three - dimensional structures .
the four groups include group 1 , which consists of linear , cationic , and amphipathic - helical peptides , for example , cecropins , magainins , bombinins , and temporins ; group 2 , which consists of -strands connected by intramolecular disulfide bridges , for example , human -defensin-2 , tachyplesins , and protegrins ; group 3 , which consists of linear peptides with an extended structure , characterized by overrepresentation of one or more amino acids , for example , tritrpticin and indolicidin ; and group 4 , which consists of peptides containing a looped structure , for example , bactenecin , brevinins , and esculentin . in the light of the above classification , human lf1 - 11 ( grrrsvqwcav )
consists of a highly variable loop region and a short -strand and is arginine rich , and hence can be classified in group 4 .
however , the same can not be said about lf1 - 11 from other species , since their conformation may be similar to the human lf1 - 11 in the structure , but they are not rich in arginines .
the arginine - rich fragment of this peptide is similar to other cationic arginine - rich peptides found in nature which have cell - penetrating activity and hence can traverse the plasma membrane of eukaryotic cells .
a significant example of arginine - rich peptide that has cell - penetrating property is arginine - rich hiv tat peptide ( grkkrrqrrrppq ) . on the other hand
, lactoferrampin belongs to group 1 which consists of linear , cationic , and amphipathic - helical peptides .
the alpha - helical amphipathic character of lactoferrampin has been compared with other group i peptides like magainins , bombinins , cecropin a , and temporins and are depicted by the helical wheel representation of the peptides in which the charged and polar residues are found aligned along one side and most of the amino acids with nonpolar side chains occupy the opposite side of the helical cylinder ( figure 3 ) .
the spatial segregation of the hydrophobic and hydrophilic residues designates the amphipathic nature of the peptides [ 127 , 128 ] .
these peptides upon interaction with target membranes fold into an amphipathic -helix with one face of the helix predominantly containing the hydrophobic amino acids and the opposite face the charged amino acids .
the presence of a prominent hydrophobic face is observed in the helical wheel representations of magainin 2 , bombinin , and temporin ( figures 3(a ) , 3(b ) , and 3(c ) ) whereas a pronounced cationic domain is present on the hydrophilic surface of the helical wheel diagram of bovine lactoferrampin like that in cecropin a ( figures 3(d ) and 3(e ) ) .
the positively charged domain is more distinct in bovine than human lactoferrampin ( data not shown ) .
the analysis suggests that there is very little similarity in the amino acid sequence within the group ; however there is a distinct trend in the distribution of different types of residue , that is , hydrophobic and charged , polar , and so forth , within the secondary structure of the helix .
sequence similarities between lactoferricin and dermaseptin and magainins suggest that lactoferricin may act as an amphipathic alpha - helix .
the active hexapeptide fragment within bovine lactoferricin peptide showed distinct similarities with lf1 - 11 and other amphipathic tryptophan and arginine - rich antimicrobial peptides found in other sources apart from lactoferrin ( table 5 ) .
bovine lactoferricin contains two tryptophan residues at positions 6 and 8 and two arginines at positions 4 and 5 .
these two amino acids have chemical properties which are one of the critical components of antimicrobial peptides .
enzymatic digestion of lactoferrin results in the generation of antimicrobial peptides which display antimicrobial properties , in some cases , with greater potency than the native lactoferrin , possibly for the protection of neonates against the invading pathogens .
these peptides , all from the n - lobe of lactoferrin , show a remarkable similarity to cationic antimicrobial peptides found in other invertebrate and vertebrate species .
though there may be minor variations in the sequence and the conformational features among these lactoferrin peptides from various species , the basic framework tends to be similar and conserved .
this indicates that these peptides play a significant role in the antimicrobial function of this protein .
the antimicrobial effect of cationic peptides of different origins is due to cytoplasmic membrane disruption of the target cell as well as immunomodulation .
the difference in their functional properties is due to the difference in their amino acid composition inspite of sharing amphipathic and cationic characteristics .
the presence of all these antimicrobial peptides in a single domain of lactoferrin suggests that the protein acts on the membrane interface and disturbs the membrane integrity resulting in its antimicrobial activity .
since lactoferrin is found in the milk and is ingested throughout the life of all neonates and most adults , it may be an excellent agent for administration to humans . in the future , | lactoferrin is a multifunctional , iron - binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function .
it contains various antimicrobial peptides which are released upon its hydrolysis by proteases .
these peptides display a similarity with the antimicrobial cationic peptides found in nature . in the current scenario of increasing resistance to antibiotics ,
there is a need for the discovery of novel antimicrobial drugs . in this context , the structural and functional perspectives on some of the antimicrobial peptides found in n - lobe of lactoferrin have been reviewed .
this paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin . |
aspirin - exacerbated respiratory disease ( aerd ) is a syndrome characterized by chronic hyperplastic rhinosinusitis , nasal polyposis , asthma , and aspirin sensitivity , as described in 1922 by szczeklik et al .
[ 1 , 2 ] . the prevalence of aerd is variable ; stevenson and szczeklik reported in 2006 that aerd occurs in 3% of adult patients with asthma in the united states , with the onset of symptoms during the third decade of life , and that it is more common in women than in men , with approximately 70% versus 30% in europe and 57% versus 43% in the usa .
the mechanisms underlying aspirin intolerance are not fully defined , with current research focusing on cyclooxygenase 1 ( cox-1 ) inhibition by aspirin and other nsaids diverting arachidonic acid metabolism from cox pathways to the lipoxygenase ( lo ) pathway .
this leads to increased synthesis of the cysteinyl - leukotrienes ( lt ) , ltc4 , ltd4 , and lte4 , resulting in bronchoconstriction , mucus hypersecretion , and possibly the development of polyps and urticaria .
the biological plausibility of this hypothesis fact has led to the search for polymorphisms in genes responsible for lt synthesis , to explore associations between these polymorphisms and local tissue levels of the proteins .
other factors such as polymorphisms in the genes for proinflammatory cytokines including tnf , il1b , il6 , and il8 are involved in chronic inflammatory and autoimmune diseases .
interleukin 1 ( il-1 ) is a cytokine associated with inflammatory responses and found in two forms , il-1 ( produced by the il1a gene ) and il-1 ( il1b ) , with both genes located on chromosome 2 .
il-1 is expressed in nasal polyps , nasal epithelium , macrophages , activated t lymphocytes , and monocytes ; its expression is regulated by adhesion molecules , and others inflammatory cytokines .
il1b polymorphisms have been associated with inflammatory bowel disease and gastric cancer among other diseases .
recently , genetic polymorphisms in proinflammatory cytokines such as il-1 have been recognized as key players in the pathogenesis of asthma .
similarly , il-8 has been implicated in the asthmatic inflammatory process , and genetic variation in this cytokine has been associated with both the susceptibility and the severity of this disease . in the present study
, we have investigated the frequencies of polymorphisms in the genes encoding these two cytokines in aerd patients .
patients with aspirin - exacerbated respiratory disease ( aerd ) ( n = 78 ) and aspirin - tolerant asthma ( ata ) ( n = 135 ) were recruited from the allergy and otolaryngology departments at the instituto nacional de enfermedades respiratorias ( iner ) . healthy control subjects ( hcs )
all participants underwent simple spirometry , inhaled methacholine challenge , and nasal challenge with lysine - aspirin ( l - asa ) according to international guidelines to determine the degree of bronchial hyperresponsiveness and confirm aerd diagnosis .
the aerd group had positive l - asa and methacholine challenges , the asthmatic group had positive methacholine challenge , but negative l - asa challenge , and the healthy control subjects were volunteers with negative l - asa and methacholine challenges .
a positive l - asa challenge was defined as a decrease of at least 40% in total nasal airflow after l - asa application compared with baseline measures ; methacholine challenge was considered positive with a decrease of at least 20% in forced expiratory volume in one second ( fev1 ) compared with baseline fev1 after the administration of different concentrations of methacholine ( beginning with 0.03 mg / ml , increasing gradually the concentration twice every 2 minutes until the concentration of 32 mg / ml ) ; in case it does not have it , it is considered negative .
the study was approved by the science and bioethics committees of iner , and all participants gave their informed consent [ 8 , 9 ] .
patients and healthy control subjects had ancestry of at least two generations born in mexico and were thus considered to be mexican mestizo in descent [ 10 , 11 ] .
peripheral blood was drawn by venipuncture , and genomic dna was obtained using the commercial bdtract dna isolation kit ( maxim biotech , san francisco , calif , usa ) .
the dna was quantified by absorption of ultraviolet light at 260 nm wavelength using an actgene spectrophotometer ( actgene , inc .
we selected two polymorphisms in two genes related to chronic inflammation : rs16944 in il1b and rs4073 in il8 .
allelic discrimination of snps rs16944 ( il1b ) and rs4073 ( il8 ) was performed by real - time pcr ( rt - pcr ) on a 7300 real time pcr system ( applied biosystems , calif , usa ) using taqman commercial probes ( applied biosystems , usa ) for each of the polymorphisms mentioned above and followed the cycling program : preread 50c , 1 minute ; absolute quantitation : 50c , 2 minutes , 1 cycle ; 95c , 10 minutes , 1 cycle ; 95c , 15 seconds , 60c 1 min , 40 cycles ; postread 50c , 1 minute .
the results were assessed taking into account the allelic discrimination and absolute quantitation in all samples ; additionally , we included four contamination controls per plate ( nontemplate controls ) .
the fluorescence signal detectors used were vic which was assigned to the b allele and fam assigned to the a allele for both snps .
statistical analysis was performed between groups of cases ( aerd and ata ) versus the healthy control subjects by test with 3 2 tables , using spss ( v. 15.0 ) software for windows , to identify the difference between the allele and genotype frequencies of each polymorphism evaluated .
in addition , odds ratios and 95% confidence intervals were calculated with epi - info ( v. 6.04 ) software .
we performed a genetic association study of the il8 ( rs4073 ) and il1b ( rs16944 ) gene polymorphisms in the three groups .
genetic data for the snps included in this study are shown in table 1 ; minor allele frequency ( maf ) of the polymorphisms tested in healthy control subjects had a similar distribution to that reported in international databases ( table 1 ) .
gene frequencies for each genotype within the three subject groups are shown in table 3 .
the frequency of genotypes aa , at , and tt of the il8 rs4073 snp was not statistically significant and different between the three studied groups .
analysis of gg and ga genotypes of the il1b ( rs16944 ) snp for aerd and ata patients versus the healthy control subjects showed nonstatistically significant associations .
interestingly , the aa genotype showed increased frequency in the aerd patients when compared to the ata group ( gf = 0.19 versus 0.07 ) ; this association was statistically significant ( p = 0.018 , or 2.98 , and ci 1.177.82 ) ( tables 3 and 4 ) and was not found when aerd or ata groups were compared to healthy control subjects .
there is no difference in aa versus ag + gg using contrast of healthy control subjects versus aerd patients ( data not shown ) .
the airways of aspirin - sensitive patients are characterized by chronic inflammation with cell infiltration even when they are not exposed to aspirin or other nsaids .
in addition to alterations in the metabolism of arachidonic acid , several proinflammatory cytokines have been associated with aerd .
the role of il-1 , however , has not been investigated previously . here , we report that aerd patients show an increased frequency of the il1b-511 polymorphism ( rs16944 aa genotype ) compared to aspirin - tolerant asthmatics .
interleukin-1 has been reported to be involved in the genesis of both asthma [ 13 , 14 ] and chronic rhinosinusitis with nasal polyposis in a turkish population .
most studies have attempted to establish the association of polymorphisms in the il1b promoter gene , mainly at positions-511 g / a ( rs16944 ) and -31 c / t .
for example , park et al . , in 2004 , did not find any association between these polymorphisms and either asthma or atopy in a korean population . in 2007 , erbek et al .
described a susceptibility for developing nasal polyps associated with the il1b-511 polymorphism ( rs16944 ) , but mfuna endam et al .
, in 2010 , failed to reproduce this finding in canadian patients with chronic rhinosinusitis . in 2003 , allen et al .
did not find any association with il1 gene polymorphisms in asthmatic families ( n = 244 ) , but they reported an association with the dna microsatellite d2s308 in these asthma families ( p = 0.00001 ) . in parallel ,
karajalein et al . evaluated 245 patients with asthma and nasal polyposis and did not find any association between the polymorphism il1b-511 c / t and nasal polyps .
evidence for the role of il-1 in pulmonary immune responses has been gathered in murine models of allergic asthma using il-1r1-deficient [ il-1r1 ( / ) ] mice ; changes observed in these mice include significant reduction of pulmonary eosinophilic inflammation , diminished goblet cell hyperplasia , and reduction of cell recruitment to the lungs , as compared to control balb / c mice .
however , there are no studies linking gene promoter polymorphisms and levels of expression of the cytokine in the lung microenvironment . in the present study
, we have demonstrated that the frequency of the il1b-511 polymorphism ( rs16944 aa genotype ) is three - fold higher in aerd ( 19.2% ) than in ata patients ( 7.4% ) , suggesting that patients carrying this polymorphism may exhibit genetic susceptibility to develop aerd .
findings on the biological functionality of the rs16944 polymorphism have not been consistent across studies .
the aa genotype has been associated with higher gastric mucosal levels of il-1 in bacterial infections , while mononuclear cells from subjects with the gg genotype showed an increased release of il-1 after stimulation with lipopolysaccharide .
recent studies suggest that the functional role of rs16944 may depend on il1b promoter region haplotypes including rs16944 [ 2326 ] .
although the findings are inconsistent , these previous studies suggest that rs16944 could affect the expression levels of il-1. our report is the first demonstration of the involvement of il1b polymorphism in aerd .
the sample size is relatively small , particularly in the aerd group , which may limit the statistical power , so it would be desirable to replicate our findings in an independent population .
future studies must investigate whether this cytokine is released in the airways of aerd patients and whether its levels relate to genetic polymorphisms in the il1b gene .
interleukin-8 has been implicated in asthma and found in high concentrations in bronchoalveolar lavage fluid of patients with acute asthma exacerbations .
in fact , polymorphic il8 alleles ( 251 t and 781c ) have been associated with asthma in a european population [ 7 , 27 ] , but not in asthmatics of korean origin ; the differences could be explained by the different ethnic populations studied . in contrast , korean asthmatics were found to show four nonsynonymous amino acid substitutions in the il-8 receptor a ( il8ra ) and an association of one synonymous variation in il8rb . in the present study ,
we did not find a significant difference in the rs4073 a / t between the aerd group and ata patients . | aspirin exacerbated respiratory disease ( aerd ) is characterized by chronic hyperplastic rhinosinusitis , nasal polyposis , asthma , and aspirin sensitivity .
the mechanisms which produce these manifestations of intolerance are not fully defined , current research focuses on cyclooxygenase 1 ( cox-1 ) inhibition , metabolism of arachidonic acid , and the cox pathway to the lipoxygenase ( lo ) route , inducing increased synthesis of leukotrienes ( lt ) .
the biological plausibility of this model has led to the search for polymorphisms in genes responsible for proinflammatory cytokines synthesis , such as il1b and il8 .
we performed a genetic association study between il8 - 251 ( rs4073 ) and il1b-511 ( rs16944 ) polymorphisms in aerd , aspirin - tolerant asthma ( ata ) , and healthy control subjects . using allelic discrimination by real - time pcr
, we found statistically nonsignificant associations between aerd , ata , and healthy control subjects for the gg and ga genotypes of il1b ( rs16944 ) .
interestingly , the aa genotype showed an increased frequency in the aerd patients versus the ata group ( gf = 0.19 versus 0.07 , p = 0.018 , or 2.98 , and 95% ci 1.177.82 ) .
this is the first observation that il1b polymorphisms are involved in aerd .
thus , future studies must investigate whether interleukin-1 is released in the airways of aerd patients and whether it relates to genetic polymorphisms in the il1b gene . |
traditionally , students learned in the classroom through interaction with the teachers , which required his physical presence and confidence in asking questions to clear his doubts
. many students may not take part in the discussion openly due to peer pressure and fear of asking a question that may appear silly and unimportant .
if the absorbing capacity of the student differs from the normal , he may find it difficult to follow the lectures , which are usually done quite fast within the time frame allotted for that particular lecture and the teacher has to disseminate the information in a cramped style .
the use of various aids like transparencies or powerpoint slides has not really helped in learning as they do not stimulate the processing of information delivered by the student . during contemporary medical education ,
portable and enduring and might enhance the recall of information leading to improved test performance .
note - taking by students is generally seen as an integral and essential part of the process of learning from lectures .
few studies have reported that the lecture handouts could improve understanding of the topic and students test performance . to further add on , many lecturers are now routinely publishing lecture handouts on the web for students to access .
this method of dissemination of information may provide many advantages over conventional paper - based distribution as it provides links to other online materials .
the technological advancement has made all the other methods redundant in the sense that students can learn at their own pace , contact the lecturer anytime , they want to post him questions and get an answer , explanation or clarification from the lecturer . at the same time
, the learning process is made easier by providing the students all the information in presentation slides .
handouts could be a valuable aid to guide the students to study in an organized way and is easy for them to cope with their exam stress .
writing in the blank spaces in handout during a lecture allows the students to be alert and active , and they learn by writing .
the present study was undertaken to find out the efficacy of the skeleton handouts against full lecture handout during ophthalmology theory lectures .
this was a pre- and post - test based experimental study involving 6 semester medical students ( n = 108 ) . in our syllabus ,
the ophthalmology theory lectures are covered during the 6 semester of medical training while the clinical posting in ophthalmology takes place during the 7 and 8 semester .
the topics of nearly equal difficulty level were identified with lecture gap of around 1-month . during the first intervention
, the full lecture handouts were distributed to each member of the class 4 days prior to concerned lecture . on the day of lecture ,
a pretest in the form of multiple choice questions was conducted for the whole class , and the post - test with the same questions was conducted soon after the lecture .
the same type of intervention was done for skeleton handouts as well after a gap of 1-month .
one hundred and eight students participated during the intervention with full handout whereas one hundred and four students participated in the skeleton handout intervention .
closed question regarding preference of students to either type of handouts was asked after both the interventions and the percentage of preference was noted .
data were processed using microsoft excel and analyzed using spss version 12 ibm , malaysia .
dependent sample t - test was used to compare the before and after test score in intervention groups , that is , skeleton handouts and full handouts .
anova with repeated measures was used to compare the change score from pretest between skeletal handouts and full handouts .
all the 108 students of the class participated in the intervention through full lecture handout in comparison to one hundred and four students participating in the skeleton handout intervention .
table 1 reveals that the posttest score ( 4.85 0.363 in skeleton handout and 2.61 0.771 in full lecture handout ) were significantly increased in comparison to respective pretest scores ( 1.85 1.275 in skeleton handouts and 1.92 1.09 in full lecture handouts ) [ table 2 ] . paired comparison of pretest and posttest scores in skeletal handout and full handout comparison of change score from pre - test between skeletal handout and full handout ( n=104 ;
those who participated in both full handout and skeleton handout interventions ) anova with repeated measures was used for the comparison of change score from pre - test between intervention groups .
skeleton handout has a mean change score from pretest 3.0 with sd 1.246 , which is higher than full handout having mean change score from pretest 0.70 with sd 1.238 .
there was a significant difference of mean change score between a skeletal handout and full handout ( p < 0.001 ) . in this study ,
the majority of the students preferring full handouts expressed that it was easy to revise at the last minute , and it could save a lot of work in the preparation and assimilation of the details about the subject taken
. however , most of the students among those who preferred skeleton handouts expressed that it encouraged them to stay focused during the class and all of them concurred that they could add points in the skeleton handouts that were being elaborated in the class .
they also said that they could get some more points and added on to the skeleton handouts by referring to information beyond textbooks .
handouts are a useful supplement that can support further learning in noncontact hours and also give a framework on which the students can learn .
they clarify expectations during the lectures and can encourage focused note - making by leaving specific areas for students to add notes in their own words or connect to other areas of learning .
provision of full slide presentation has decreased the students attentiveness as they already have the notes on what the lecturer is going to teach in the class with the belief that they can always refer to it at their leisure . however
, the skeleton handouts will provide an opportunity for the students to ponder over the subject and increase the understanding of the subject by the scaffold given .
the students who were given skeletal handouts out - performed when compared with students who were not given . in our study , the mean post - test value was significantly higher when the students were given skeleton handouts , which were similar to the one was done by russell et al .
they have concluded that learning by medical students was improved when they recorded notes in class .
however , the majority of the students in our study , preferred to have full handout ; probably for their easy referral during the examination .
when students are given full handouts , they often substitute this for class attendance and are less likely to attend the lecture , which sometimes is disheartening to the lecturer . perhaps the compromise that best assists students is the provision of partial or guided notes .
skeleton outlines or handouts can provide a scaffold for accurate student note - taking while still requiring their attention , active engagement , and attendance . guided rather than complete notes provided prior to class time can also allow students to prepare and review material for concepts in need of further explanation with the added benefits of increasing student engagement with the content and improving their accuracy of note - taking .
it has been stated in one of the literatures that the students who generated their own outlines or study questions learned more from a lecture than did students who used materials provided by the lecturer .
the limitation of our study was that we could not conduct a randomized control study , but we plan to conduct a randomized control study with long - term outcome measure in the next batch of students .
although the majority of the students wished to have full handouts of the ophthalmology lectures , they could perform better after going through skeleton handouts .
| background : although lecture handouts are commonly given to students during theory lectures , students perception , as well as their performance , can vary depending on the type of handouts they receive for information processing.methodology:this is a quasi - experimental study involving 6th semester medical students .
the study was conducted during theory lectures on ophthalmology .
the two types of notes given to the students were comprehensive handout and a skeleton handout , which included some lecture notes but required substantial annotation by the students .
pre - test and post - test in the form of multiple choice questions were conducted before and after the lecture session , respectively.results:there was a significant difference of mean score of pre- and post - test between skeletal handout ( pre = 1.85 1.275 , post = 4.85 0.363 ) and full handout ( pre = 1.92 1.09 post = 2.61 0.771 ) with p < 0.001
. however , the students responses to questionnaires indicated a strong preference for much detailed handouts as essential to preparation for examinations.conclusion:the student can improve their performance during examination while working on skeletal handouts during theory lectures in spite of showing a preference for complete handouts . |
the cell must generally define an axis , localize fate determinants , and coordinately orient the mitotic spindle .
both plant and animal cells must accomplish these tasks , but unlike animal cells , plant cells are surrounded by a cell wall that impedes cell movement . in most plant cell types ,
the future division plane is set , not by the spindle midzone position , but by the position of another cytoskeletal array .
this array , the pre - prophase band ( ppb ) , acts prior to mitosis and spindle assembly . in the plant
male germline , asymmetric divisions involve microtubule - dependent nuclear migration and a unique microtubule array at the future germ pole .
these mechanical features make asymmetric divisions , and the orientation of those divisions , particularly important for generating the overall cell pattern during plant development and indicate that plants may have needed to develop distinct methods for generation of cell asymmetry .
asymmetric divisions are universally used to create cellular diversity and allow stem cell self - renewal .
four well - studied plant developmental contexts - the embryo , root meristem , the epidermal stomatal lineage , and the male germline - exemplify key functions of asymmetric divisions ( figure 1 ) .
the creation of cellular diversity by asymmetric division begins during arabidopsis embryogenesis when the first zygotic division generates a small apical cell ( the progenitor of most of the embryo ) and a large basal cell ( the future extra - embryonic suspensor ) .
asymmetric or oriented divisions are associated with the generation of major tissue layers ; they also create what will later become the stem cell niche of the root when an asymmetric division of the hypophyseal cell yields a large basal daughter cell that generates the columella stem cells and a small apical cell that will give rise to the quiescent center ( qc ) .
the qc consists of a group of mitotically inactive cells that maintain adjacent stem cells .
plants continue producing organs post - embryonically ; much of this growth initiates from activities of the stem cell pools in the root and shoot ; however , asymmetric divisions are also vital for de novo generation of cell lineages and patterns , as seen during stomatal development . here , precursor cells of the stomatal lineage are generated by asymmetric divisions of meristemoid mother cells ( mmcs ) that are chosen , seemingly at random , from a field of equivalent protodermal cells .
the daughters of the division include a stem cell - like meristemoid and a larger stomatal lineage ground cell ( slgc ) .
the meristemoid , after completing a limited number of self - renewing asymmetric divisions , will differentiate into a guard mother cell and then divide symmetrically to form a pair of stomatal guard cells .
although the slgcs often differentiate into default epidermal cells , they can also re - enter an asymmetric division phase , become mmcs , and produce satellite meristemoids whose positions are coordinated with neighbor cells through precise orientation of their asymmetric divisions .
plants again employ intrinsically asymmetric divisions during male germline formation ; here , an asymmetric division of the haploid microspore generates two unequally sized daughter cells : a non - germline vegetative cell that exits the cell cycle and a germ cell that divides again to form twin sperm cells for each pollen grain .
basl , breaking of asymmetry in the stomatal lineage ; epi , epidermis ; gc , ground cell ; gmc , guard mother cell ; lrc , lateral root cap ; mmc , meristemoid mother cell ; pll1 , poltergeist - like 1 ; pol , poltergeist ; qc , quiescent center ; sc , stem cell ; slgc , stomatal lineage ground cell ; ssp , short suspensor ; wox , wuschel - related homeobox ; yda , yoda . figure modified from abrash & bergmann , dev cell 2009 .
a unifying tenet of asymmetric cell division is that it generates two daughter cells with distinct fates .
there are a number of ways that this endpoint can be reached , with some mechanisms requiring the establishment of polarities before division and others acting to influence the behavior of the daughters afterward .
screens for polarity regulators in animals led to the identification of the partitioning defective ( par ) proteins , the most conserved of which act in an asymmetrically localized cortical complex that establishes cell polarity and segregates fate determinants , thereby influencing the physical and fate asymmetries of daughter cells .
do plants use different proteins for analogous par - like functions ? here , we take individual elements of the par paradigm - the ability to promote asymmetric fates , polarized localization within dividing cells , and roles in segregating fate determinants - and discuss recent results from studies of plant embryos , roots , stomata , and pollen in reference to these behaviors and functions ( figure 2 ) .
basl , breaking of asymmetry in the stomatal lineage ; duo1 , duo pollen 1 ; fbl17 , f - box like protein 17 ; mapk , mitogen - activated protein kinase ; mpk , arabidopsis gene encoding a mitogen - activated protein kinase ; pan1 , pangloss 1 ; par , partitioning defective ; pll1 , poltergeist - like 1 ; pol , poltergeist ; ssp , short suspensor ; wox , wuschel - related homeobox ; yda , yoda .
mutations in several genes lead to defects in specifying or maintaining unequal daughter cell fates . the asymmetric division that creates the embryo and suspensor requires mitogen - activated protein kinase ( mapk ) signals , including core cascade elements yoda ( yda ) , a mapk kinase kinase , mpk3 and mpk6 ( mapks ) , and a sperm - supplied upstream regulator , short suspensor ( ssp ) , which activates mapk signaling in the zygote .
mutations in these components equalize presumptive embryo and suspensor cell sizes and identities . in the later hypophyseal division , two type 2c protein phosphatases , poltergeist ( pol ) and poltergeist - like 1 ( pll1 ) ,
are redundantly required to generate asymmetry ; in pol / pll1 mutants , the hypophyseal division occurs symmetrically and neither of the resulting daughter cells adopts the appropriate fate .
there is some evidence for re - use of these putative signaling elements ; for example , pol and pll1 are used for the establishment of other stem cell populations and the mapk cascades are used again for stomatal divisions .
later stomatal decisions also require unique regulators such as breaking of asymmetry in the stomatal lineage ( basl ) ; when basl is present , only 12% of meristemoids or mmcs divide asymmetrically , with the daughter cells often adopting the same fate .
pol / pll1 , ssp , yda , and mpk3/6 are likely to be elements of pathways that lead to the appropriate timing or placement of divisions ( or both ) in response to information originating from outside of the asymmetrically dividing cell .
this is consistent with the long - held view that plant cells derive their identity primarily by position ( reinforced by cell - cell communication , for example ) .
however , it is now clear that intrinsic mechanisms like unequal inheritance of proteins are also likely to contribute to generating daughter cells with different fates .
transcripts of wuschel - related homeobox 2 ( wox2 ) and wox8 , members of the homeodomain transcription factor family , while initially expressed throughout the zygote , are apparent in only the apical and basal cell , respectively , after its asymmetric division . similarly , wox5 is expressed in the hypophyseal cell but after division is found in only the apical daughter cell . in the root meristem , the nac ( nam , ataf1/2 , and cuc2 ) domain transcription factor fez promotes the asymmetric division of columella stem cells and displays a dynamic localization pattern in both the stem cells and their daughters .
fez protein is in pre - division stem cells , but immediately after division , the stem cell daughters lack fez while their terminally differentiating sisters express fez ; fez expression is only later reestablished in the daughters with stem cell identity . in male germline formation , transcripts of the f - box protein fbl17 are expressed in the microspore but the fbl17 protein is expressed only in the germ cell following asymmetric division . while fez , members of the wox family , and fbl17 show differential expression following asymmetric division , their subcellular localization and dynamics during the entire division process have not yet been reported ; thus , we do not know whether any of these proteins are truly differentially segregated .
their molecular identities are also not easily reconciled with a direct role generating intrinsic cellular polarities .
if we take the cue from the pars that polarity - generating proteins will exhibit polarized expression at the cell cortex , then what are the plant candidates ?
the pin - formed ( pin ) family of auxin transporters is tied to cellular and organismal polarity generation and several of these proteins are localized to a single face of a cell ( reviewed in ) ; however , there is scant evidence that the pins are segregated during asymmetric divisions .
maize pangloss1 ( pan1 ) , a receptor - like protein , is highly polarized in stomatal lineage cells that will undergo asymmetric division , pointing to a possible role in generating pre - divisional asymmetry .
prior to an asymmetric cell division in meristemoid cells or mmcs , basl is both nuclear and in a polarized peripheral crescent . immediately following asymmetric division ,
basl can be found in the nucleus of the smaller cell and at the periphery as well as the nucleus of the larger daughter cell , with the main activity of basl being ascribed to the peripheral pool .
ectopic expression of basl produces a localized zone of cellular outgrowth but does not appear to alter cell fates ; thus , it too fails to behave in a manner completely analogous to the pars .
it is not known how the domain of peripheral basl is established or whether basl generates or responds to an earlier cellular polarity ; interestingly , this polarity is likely transient because the basl crescent disappears from one site and is reestablished in a new polar crescent in redividing slgcs .
unlike in animal systems where the par proteins coordinate multiple aspects of asymmetric division in many developmental contexts , in examples from the germline , embryos , meristems , and stomata , we see a great diversity of regulators .
are there specific aspects of these different plant cell divisions that necessitate different controls ? or will homologues of regulators from one context participate in others ? in each of these cases , what is the connection between developmental specification of asymmetry and the execution of oriented division ?
several proteins have recently been shown to translate the position of the ppb into the subsequent new wall position , but virtually nothing is known about how the positions of the ppb and division plane are specified .
can we identify the targets of the transcription factor families or connect the highly polarized proteins ( basl and pan1 ) in a mechanistic way to the process of ppb placement ?
after their discovery , the pars became an intellectual scaffold for considering other asymmetric cell division proteins .
future plant studies should be guided by the constant consideration of both the logical insights from the pars and by the unique constraints of plant development .
| like animals , plants use asymmetric cell divisions to create pattern and diversity .
due to a rigid cell wall and lack of cell migrations , these asymmetric divisions incur the additional constraints of being locked into their initial orientations
. how do plants specify and carry out asymmetric divisions ?
intercellular communication has been suspected for some time and recent developments identify these signals as well as point to segregated determinants and proteins with par - like functions as parts of the answer . |
the importance of cd8 + t cells in viral clearance has been established in several viral models , including the nonhuman primate model of aids , in which depletion of cd8 + t cells resulted in the inability to clear infection [ 2 , 3 ] . several other evidences demonstrated the importance of the cd8 + t cell responses for controlling hiv infection .
indeed , slow aids disease progression is associated with functional cd8 + t cell responses , specific hla class i alleles are associated with slower progression of disease in hiv - infected people , and ctl escape is a major force driving hiv evolution ( reviewed in [ 4 , 5 ] ) .
based on these findings , vaccines inducing strong and long - lived cd8 + t cells are highly desirable .
genetic immunization using plasmid dna or viral vectors is particularly suitable for inducing ctl responses , because the encoded protein enters the mhc class i processing pathway through either direct transgene expression or cross - presentation by resident antigen presenting cells ( apcs ) . among viral vectors , lentiviral vectors ( lvs )
have been shown to efficiently transduce non replicating cells in vitro as well as in vivo and to be able to elicit potent humoral and cellular responses ( reviewed in ) .
lv - transduced human dcs are able to present antigenic peptides and prime transgene - specific t cells in vitro [ 7 , 8 ] .
it has been shown that lv - encoding hiv-1 polyepitopes induce broad ctl responses in mice , and we reported that a single intramuscular administration of hiv - based lv - expressing viral antigens elicits strong cell - mediated immune responses [ 10 , 11 ] . importantly , beignon and colleagues recently provided the first evidence that an lv - expressing simian immunodeficiency virus- ( siv- ) gag protein was able to induce control of viral replication in monkeys challenged with high dose of siv .
however , safety concerns relative to the use of these vectors for delivery of therapeutic genes or for a vaccine include insertional mutagenesis and/or vector mobilization following viral infection . to minimize the risk of insertional mutagenesis , integrase defective lentiviral vectors ( idlv ) have been engineered to present viral antigens in a similar but safer manner [ 13 , 14 ] .
several reports have shown that hiv- or siv - derived integrase defective viruses as well as idlv are transcriptionally active although at lower levels than the integrated wild - type counterpart , while producing extrachromosomal forms of viral dna ( e - dna ) in the absence of noticeable amounts of integrated provirus [ 14 , 15 ] . we have recently demonstrated that a single immunization with a nonintegrating lentiviral vector carrying a codon - optimized hiv-1 gp120 envelope protein in mice results in a strong and sustained immune response characterized by the presence of anti - gp120 antibodies , effector - specific t cells and generation of a specific t cell memory , without integration of the vector into the host genome .
importantly , several groups confirmed the use of idlv as an effective vaccine delivery strategy [ 1719 ] , and we provided evidence that siv - based idlv can be constructed and used as well . in this work we compared the ability to induce long - lasting cd8 + t cell response in mice upon immunization with dna plasmid or idlv expressing the hiv-1 gp120 envelope protein .
a detailed evaluation of cd8 + t cell immune response was performed at three months after the last immunization . moreover , a comprehensive analysis of the quality of cd8 + t cell response induced by idlv is reported .
a schematic depiction of the lentiviral transfer vector plasmids used in this study is shown in figure 1 .
briefly , the hiv - based transfer vector plasmid pty2-jrmz encodes the hiv-1jr - fl gp120 envelope protein sequence ( hiv - env ) and the murine granulocyte monocyte - colony stimulatory factor ( mgm - csf ) from the internal cmv promoter ; the hiv - based transfer control vector plasmid pty2-emptymz encodes only the mgm - csf from the sequence for the poliovirus internal ribosomal entry site ( pires ) . the integrase ( in ) defective packaging plasmid pchelp / in- , obtained from dr . j. reiser ( louisiana state university health sciences center , new orleans , usa ) , is necessary for production of the vector particles and contains a d116n mutation in the in genome , preventing the functions characteristic of the in protein ; plasmid pmd g , obtained from dr . d. trono ( university of lausanne , switzerland ) , produces the vescicular stomatitis virus envelope glycoprotein g ( vsv g ) .
the human epithelium kidney 293 t cell line was maintained in dulbecco 's modified eagles medium ( euroclone , life sciences division , pero , milan , italy ) supplemented with 10% fetal calf serum ( euroclone , life sciences division ) and 100 units / ml of penicillin - streptomycin - glutamine ( gibco invitrogen , paisley , uk ) .
for production of recombinant idlv expressing hiv - env ( idlv - jrmz ) , cells were transiently transfected using the calcium phosphate - based profection mammalian transfection system ( promega corporation , madison , wi , usa ) as previously described [ 16 , 22 ] using a total of 24 g of plasmid dna with a ratio of 4 : 3 : 1 ( pty2-jrmz : pchelp / in- : pmd g ) , thus producing the idlv - jrmz vaccine preparation . for production of control recombinant idlv ( idlv - empty ) , the pty2-emptymz plasmid was used in place of pty2-jrmz , thus producing the idlv - empty vaccine preparation .
after 48 hours , culture supernatants were cleared from cellular debris and passed through a 0.45 m pore size filter ( millipore corporation , billerica , ma , usa ) .
filtered supernatants were concentrated by ultracentrifugation ( beckman coulter , inc . , fullerton , ca , usa ) for 2 hours at 27 000 rpm on a 20% sucrose gradient ( sigma chemicals , co. , st .
finally , the viral pellets were resuspended in 1 pbs and stored at 80c for further analyses .
viral titres were normalised by the rt and p24 elisa assays ( innotest , innogenetics , belgium ) .
balb / c mice were kept in accordance with the european union guidelines and italian legislation .
four mice per group were intramuscularly immunized ( i ) twice with plasmid pty2-jrmz expressing hiv - env and mgm - csf ( dna - jrmz ) , ( ii ) twice with plasmid pty2-emptymz expressing only mgm - csf ( dna - empty ) ; ( iii ) once with idlv expressing hiv - env and mgm - csf ( idlv - jrmz ) and ( iv ) once with idlv expressing only mgm - csf ( idlv - empty ) , mice from dna groups were immunized intramuscularly with dna ( 80 g / mouse ) on day 0 and after 1 month were boosted with the same dose of dna .
mice from idlv groups received a single inoculum of 1 10 rt units of idlv formulated in 0.2 ml of pbs .
three months after the immunization mice were sacrificed . at sacrifice mice were first bled orbitally under metaphane - induced anesthesia , and sera were collected and kept at 80c . single cell suspension from spleen was prepared by mechanical disruption and passage through cell streiners ( bd falcon ) .
bm - derived cells were obtained from tibiae by syringe insertion into one end of the bone and flushing with rpmi medium .
the ifn- elispot assay was performed on splenocytes and bm - derived cells by using reagents from mabtech ( mabtech ab gamla vrmdv , sweden ) as described in .
the jr-9mer peptide containing the h-2 restricted hiv-1 gp120 envelope epitope ( igpgrafyt ) ( ufpeptides s.r.l . ,
ferrara , italy ) and the h-2 restricted gfp-9mer peptide ( hylstqsal ) ( ufpeptides s.r.l . , ferrara , italy ) were used at 5 g / ml as specific and unrelated stimuli , respectively . concanavalin a ( sigma , chemical co. st .
specific samples ( specific peptide - treated wells ) were subtracted from the values obtained in the unrelated peptide - treated wells ( background ) and were scored positive when were present a minimum of 50 spots per 10 cells and a fold of 2 or higher compared to the unrelated peptide - treated samples .
splenocytes from single animals ( four mice / group ) were cultured with the specific jr-9mer ( 5 g / ml ) or the unrelated gfp-9mer ( 5 g / ml ) h-2-restricted peptides in the presence of anti - mouse cd28 mab ( clone 37.51 ) at 2 g / ml .
pma ( 50 ng / ml ) in combination with ionomicin ( 2 g / ml ) was used as positive control . after 1 hour from the stimulation ,
10 g / ml of brefeldin a was added to the cultures as a protein transport inhibitor .
after 6 hours of incubation , cells were stained with percp anti - mouse cd8a , or the isotype - matched mab
. then cells were permeabilized and stained with pe - labeled anti - mouse il2 , apc - labeled ifn , and pe - cy7-labeled tnf mabs or their isotype - matched controls in pbs-0.5% saponin and analyzed by flow cytometry .
all monoclonal antibodies were from bd pharmingen ( san diego , ca , usa ) , and all chemicals were from sigma chemicals , co. ( st . louis , mo , usa ) .
acquisition and analysis were performed using facscanto and facs diva software ( bd biosciences , usa ) .
stimulator splenocytes from nave balb / c mice were pulsed for 1 hour with the envelope - specific jr-9mer h-2-restricted peptide at 5 g / ml in serum - free medium , irradiated with 3000 rads on a co source , washed once , and then added to effector splenocytes derived from immunized mice , at an effector : stimulator ratio of 10 : 1 .
after 4 days of culture , effector cells were tested in chromium release assays for the analysis of antigen - specific t cells , as described in .
specific antibody titres against hiv - gp120 envelope protein were measured by elisa as previously described . briefly , ninety - six flat - bottom well microplates ( nunc gmbh & co. kg , wiesbaden , germany ) were coated with 100 ng / well of hiv-1sf162 gp120 ( nih repository reagents , bethesda , md , usa ) in carbonate buffer ( sigma chemical co. st .
louis , mo , usa ) . after overnight incubation at 4c , wells were washed and mice sera were serially diluted and added to the plates .
louis , mo , usa ) was added to each well and incubated for an additional 90 min at 37c .
antigen - bound antibodies were revealed by the addition of abts solution ( roche diagnostic s.p.a , mi , italy ) .
cut - off values were established for each plate and in each run and were based on the average serum samples plus 2 standard deviations ( sd ) from 4 naive mice ( value ) .
serum samples with values higher than the cut - off value were considered to be positive by elisa for hiv - env antibodies .
all p - values were two tailed and considered significant if less than .05 .
all analyses were performed using spss for windows version 13.0 ( spss inc . , chicago illinois us ) .
the presence and the persistence of the immune response were evaluated at 90 days from the last immunization in mice ( 4 mice / group ) inoculated twice with plasmid dna ( dna - jrmz ) or once with idlv ( idlv - jrmz ) , both expressing codon - optimized hiv-1jr - fl gp120 envelope and murine gm - csf .
control arms ( 4 mice / group ) included mice inoculated twice with plasmid dna ( dna - empty ) or once with idlv ( idlv - empty ) , both expressing only murine gm - csf , as well as nave mice .
as determined by ifn elispot on ex vivo splenocytes , idlv - jrmz - immunized mice showed a high level of antigen specific ifn producing t cells , with spot forming cells ( sfc)/10 ranging from 473 to 960 , with an average value of 662 ( figure 2(a ) ) .
the number of antigen - specific ifn producing t cells was lower in mice immunized twice with dna - jrmz ( sfc/10 cells ranging from 35 to 345 , with an average value of 150 ) ( figure 2(a ) ) .
recently , it has been proposed that the bone marrow ( bm ) is a crucial organ for trafficking of mature t cells and contributes greatly to long - term cytotoxic memory as well as to homeostatic regulation of peripheral cd8 t cell numbers . to evaluate
if the vaccinations induced a memory response detectable three months after the last immunization in bm , ifn elispot was performed on bm - derived cells .
idlv - jrmz - treated mice showed detectable levels of antigen - specific ifn producing cells localized in bm ( sfc/10 cells ranged from 107 to 337 , with an average value of 212 ) ( figure 2(b ) ) .
this is particularly striking in light of the fact that only 2%-3% of the cells derived from bm were cd8 + .
conversely , antigen - specific ifn producing cells were not detected in bm of dna - jrmz - immunized mice ( figure 2(b ) ) .
overall , these results indicate that a single injection of idlv - jrmz is more efficient than two immunizations with dna - jrmz in inducing antigen - specific ifn producing cd8 + t cells .
in addition , the presence of immune response in spleen and bm at three months from the single immunization indicates that idlv is able to induce a long - lasting immunity . to analyze the effective ability of antigen - specific cd8 + t cells induced by the vaccines in killing target cells ,
this was deemed necessary since production of ifn by cd8 + t cells does not directly imply their ability to kill target cells , as it has been already demonstrated for instance in the case of hiv - infected patients , where the ability to produce cytokines was not associated with the cytolytic function .
the presence of ctl was investigated on splenocytes derived from immunized mice , stimulated in vitro with irradiated nave splenocytes pulsed with the specific envelope jr-9mer peptide .
specific lysis of target cells was evident in mice immunized once with idlv - jrmz ( up to 30% of specific lysis at e : t ratio of 40 : 1 ) , whereas splenocytes from mice immunized twice with dna - jrmz did not show ctl activity at any e : t ratio tested ( figure 3(a ) ) .
no specific responses were detected in the mice immunized with dna - empty , and idlv - empty and in nave mice , used as negative controls ( figure 3(a ) ) .
these data clearly indicated that only the immunization with idlv - jrmz was able to induce antigen - specific cd8 + t cells competent for both cytokine production and cytolytic effector function .
an efficient antibody response was not expected upon immunization with idlv - jrmz , due to the reported low protein expression from the non - integrated viral e - dna .
indeed , mice immunized with idlv - jrmz displayed low but detectable anti - env igg titers ( geometric median titer 400 ) whereas mice immunized with dna - jrmz did not generate any antibody response ( figure 3(b ) ) .
the presence of a specific antibody response in the case of idlv is attractive , considering a possible prime - boost vaccine protocol , using idlv as priming and the corresponding protein for boosting humoral response .
several authors have recently confirmed the efficacy of idlv vaccination in animal models [ 1720 , 26 ] . due to its safety profile and immunization efficiency ,
there is , therefore , an increasing interest in the use of this vector for vaccine purposes in different disease models , including viral infections and tumors . to further characterize the cd8 + t cell response
, we carried out a more detailed analysis of the quality of immune response induced by idlv immunization .
in particular , an intracellular staining ( ics ) for ifn , tnf , and il2 was performed in splenocytes of mice immunized with idlv - jrmz and dna - jrmz and control mice . as shown in figure 4(a ) ,
cd8 + t splenocytes from both idlv - jrmz- and dna - jrmz - immunized animals produced all three cytokines , when cultured in vitro in the presence of the envelope - specific peptide .
importantly , idlv - jrmz - immunized mice showed a statistically significant higher percentage of cd8 + t cells producing ifn , tnf , or il2 when compared to dna - jrmz - vaccinated mice ( p < .05 ) .
splenocytes from nave mice or from mice immunized with idlv - empty and dna - empty did not show any significant production of cytokines . to evaluate the presence of polyfunctional antigen - specific cd8 + t cells ,
ifn producing cd8 + t cells were gated and evaluated for production of tnf and il2 . in figure 4(b ) a representative analysis shows that the majority of the ifn-producing cd8 + t cells from the idlv - jrmz and dna - jrmz - immunized mice also produce tnf and il2 ( 86% and 75% , respectively ) .
these results indicate that while both vaccinations qualitatively induced similar cd8 + t cell responses , immunization with idlv induced a higher number of polyfunctional antigen - specific t cells .
it has been suggested that the presence of such polyfunctional cells correlates with a better prognosis in chronically infected patient with cmv and ebv and in long - term non progressor hiv-1-infected patients .
therefore , the use of idlv - based vaccines , capable of inducing high levels of polyfunctional cd8 + t cells , after a single immunization represents a very attractive and desirable vaccine strategy .
usually , the immune response induced by dna injection is weak . to circumvent this limitation in our study
we used a single intramuscular injection of idlv - delivered envelope sequence in the mouse model which performed better than two immunizations with naked dna plasmid delivering the same antigen .
the mgm - csf coding sequence was included in both immunization protocols in the effort to improve immune response .
nevertheless , two immunizations with dna were not sufficient to induce an immune response comparable to that induced by a single immunization with idlv expressing the same antigen .
the difference in the ability to induce immune response could be partly explained by considering the advantage of the vector , in terms of uptake and stability in vivo , compared with naked dna .
the delivery of dna through electroporation ( ep ) might overcome these limitations , even if safety - related issues of ep , in the case of administration of vaccines to humans , need to be better investigated . however , the aim of our study was to provide evidence of the potency of idlv as a vaccine and to characterize the cd8 + t cell response induced by this novel , safe , and promising vector for genetic immunization .
the efficiency of idlv observed in our and in other models , in terms of quantity , persistence , and quality of cd8 + t cell response , could be reasonably considered an excellent beginning for future exploitation of this vector for developing vaccine strategies aimed at inducing cd8 + t cell responses . the possibility to use idlv in heterologous prime - boost protocols as well as in mucosal vaccine , in order to improve immune response at both systemic and mucosal sites , should be further investigated .
finally , to have the proof of concept that this vector could represent a novel and effective delivery system for vaccine strategies in humans , experiments in the nonhuman primate model of aids should be performed by evaluating the strength of the immune response induced in terms of both frequency and quality of viral - specific t cells and the ability in controlling the viral challenge . | cd8 + t cells are an essential component of an effective host immune response to tumors and viral infections .
genetic immunization is particularly suitable for inducing ctl responses , because the encoded proteins enter the mhc class i processing pathway through either transgene expression or cross - presentation . in order to compare the efficiency and persistence of immune response induced by genetic vaccines , balb
/ c mice were immunized either twice intramuscularly with dna plasmid expressing a codon - optimized hiv-1 gp120 envelope sequence together with murine gm - csf sequence or with a single immunization using an integrase defective lentiviral vector ( idlv ) expressing the same proteins .
results strongly indicated that the schedule based on idlv vaccine was more efficient in inducing specific immune response , as evaluated three months after the last immunization by ifn elispot in both splenocytes and bone marrow- ( bm- ) derived cells , chromium release assay in splenocytes , and antibody detection in sera .
in addition , idlv immunization induced high frequency of polyfunctional cd8 + t cells able to simultaneously produce ifn , tnf , and il2 . |
my introduction to research occurred in the summer of 1968 , when , as a third - year undergraduate student of biochemistry , i started to work with kai simons in the department of serology and bacteriology at the university of helsinki .
this would likely not have happened had he not been the older brother of my girlfriend , majlen , who is now my wife of 44 years . having recently returned from postdoctoral study at rockefeller university in new york ,
as it turned out , kai and i were to work together for the next 13 years , and it was he who most strongly influenced my development as a scientist .
well - informed , enthusiastic , generous , thoughtful , forward - looking , and ambitious , he provided unlimited advice and support .
the sds page system to separate denatured proteins according to molecular weight , which revolutionized work on proteins , was just making its debut .
the prevailing view was that membranes provided solid , rigid , waxy walls between compartments .
there was no commonly accepted model for their structure . while few doubted that phospholipids could spontaneously organize themselves as bilayers in water ,
it was not generally accepted that bilayers represented the basic structure of cellular membranes ( stoeckenius and engelman , 1969 ) . nor was it clear where the proteins that make up half or more of the mass of most membranes were located . in one model ,
they were placed on each side of a lipid bilayer , attached to phospholipid head groups .
a competing model proposed that membranes were composed of lipoprotein complexes associated side by side , like bricks in a wall . for a while
, it was even proposed that all membranes contained the same structural protein , a hydrophobic miniprotein .
none of these models made much sense functionally , but this was of little concern in this early stage of membrane research .
that the lipid bilayer formed the basis of biological membranes and that membranes were fluid and dynamic became generally accepted in the early 1970s .
nicolson in the well - known model that still dominates our thinking about biological membranes : a fluid lipid bilayer with proteins either embedded or adsorbed to the surface ( singer and nicolson , 1972 ) .
the realization that proteins integral to the bilayer are amphiphilic ( i.e. , that they have distinct hydrophilic and hydrophobic surfaces ) and can span the bilayer was a huge step forward .
it now became possible to employ a protein - based approach to known membrane functions , such as the controlled permeability observed for ions and other polar substances .
the fluidity of membranes made it possible to approach fusion and fission and many other phenomena more rationally .
my own work focused on the effect of detergents on membranes and lipid protein interactions .
building on the existing surfactant and colloid literature and the work of charles tanford on thermodynamics of membranes and detergent binding to soluble proteins , my phd thesis presented a conceptual and practical framework for the use of detergents in the solubilization , delipidation , purification , biochemical analysis , and reconstitution of integral membrane proteins ( helenius and simons , 1975 ) .
represented a phase transition from the detergent - saturated lipid bilayer to lipid - saturated mixed micelles .
when enough detergent was added , the lipids that covered the hydrophobic surfaces of integral membrane proteins were replaced by a detergent micelle .
the membrane used in our solubilization studies was the envelope membrane of a simple animal virus , semliki forest virus ( sfv ) . because of its simplicity and homogeneity ,
we were introduced to sfv by leevi krinen , a virologist working one floor above us .
together with ossi renkonen , a lipid and carbohydrate chemist , leevi was one of my most important coaches and mentors in these early years .
the work on the virus and its membrane opened the door to international science . in 1975 , kai simons , henrik garoff , hilkka virta ( kai 's technician for many years ) , and i joined the newly founded european molecular biology laboratory ( embl ) in heidelberg . for me , it was time to leave the detergents and all the sudsy solutions with which it was so hard to work .
after reading a review about virus receptors by karl lonberg - holm and lennart philipson ( lonberg - holm and philipson , 1974 ) , i decided to take on the mystery of host - cell entry using sfv , our household virus .
this meant that i had to learn something about cells , which we had previously viewed merely as a means to replicate viruses . using electron microscopy ,
jrgen kartenbeck , a cell biologist , friend , and collaborator for years to come , saw sfv in coated indentations of the plasma membrane and in a variety of cytoplasmic vacuoles .
we found that lysosomotropic weak bases , such as ammonium chloride , inhibited infection but did nothing to prevent endocytosis and accumulation of viruses in endocytic organelles .
fortunately , young group leaders at embl , including bernhard dobberstein , daniel louvard , and graham warren , had been recruited to start a program in cell biology . from them , we learned new techniques , such as indirect immunofluorescence microscopy , and about what is now called membrane traffic .
the key to sfv entry into host cells turned out to be the drop in ph that the virus experiences in cytoplasmic vacuoles after clathrin - mediated endocytosis .
an acid - induced conformational change converts the spike glycoproteins into efficient membrane fusion machines , and the viral envelope fuses with the limiting membrane of endocytic vacuoles from the inside , releasing the capsid into the cytosol ( helenius et al . , 1980a ) .
together with jrgen kartenbeck , erik fries , kai simons , judith white , mark marsh , karl matlin , and others , i described how various viruses ( sfv , influenza virus , vesicular stomatitis virus , etc . ) took advantage of receptor - mediated endocytosis mechanisms and used low ph as the cue for penetration .
in contrast with the secretory pathway , endocytosis had not received serious attention at this time .
the uptake of pathogens by phagocytosis in macrophages and amoebae was known , and fluid uptake ( pinocytosis ) had been described .
it was found by ralph steinman and coworkers that membrane and fluid were continuously internalized , and the majority was recycled back to the plasma membrane .
furthermore , clathrin and coated vesicles were shown by keith porter , and later by richard anderson , michael brown , and joe goldstein , to serve as primary endocytic organelles for internalization of receptor - bound ligands .
this brought in the era of receptor - mediated endocytosis , and it was soon recognized that there was a pathway that not only served as a feeder to lysosomes but supported a variety of other interactions with the outside world .
perhaps one of the most important observations emerging from the virus entry work was that coated vesicles did not ferry the viruses directly to lysosomes but to prelysosomal organelles .
in other words , there existed intermediate , acidic organelles between the primary endocytic vesicles and the lysosomes .
we gave these organelles the generic name endosomes in anticipation of yet - to - be - discovered functions ( helenius et al .
cell biologists studying receptor - mediated endocytosis of physiological ligands were also finding evidence for intermediate organelles .
in particular , ann hubbard at yale and ira pastan at the national institutes of health , whose research groups studied the internalization of asialoglycoproteins and 2-macroglobulin , respectively , concluded that after uptake by clathrin - coated vesicles , ligands passed through electron - lucent vacuoles en route to lysosomes ( wall et al . , 1980 ; willingham et al . , 1980 ) .
in 1981 , i moved to yale school of medicine , having been invited by george palade , another great mentor and role model , to join the section of cell biology .
my group included judith white , mark marsh , eva bolzau , and jennifer wellsteed from embl , and we continued to work on viruses , endosomes , and endocytosis - related topics .
luckily , we were joined by ira mellman from rockefeller university , who had just joined the yale faculty .
he brought with him deep insights into the cell biology of endocytosis and membrane trafficking , and an invaluable spirit of enthusiasm and confidence .
the combined group made rapid progress on the role of early and late endosomes in molecular sorting and in virus entry .
having been overshadowed by the more popular and more populated secretory pathway , the endocytic pathway established itself in the 1980s as the important and fascinating subject of study that it still is today . since then , i have worked with many excellent students and postdocs at yale and at eth zurich .
together , we have entered new areas of research , including membrane fusion , protein folding , quality control in the endoplasmic reticulum , and glycobiology .
after several highly rewarding diversions , my current group is again focusing on virus entry and the role of endosomes , using new tools and new virus systems .
one basic premise has remained unchanged during all these years : we have relied on viruses to guide our work .
trusting that they understand cell biology better than we do , we simply follow their lead in the hope of exposing secrets buried deep inside cells . | the fluid mosaic model for biological membranes was formulated 40 years ago .
ten years later endosomes were discovered as important prelysosomal organelles . at the outset of my research career ,
i was fortunate to witness both these turning points in biochemistry and cell biology from close up , and to participate in some of the studies . in this short essay
, i will describe how this came about , and also try to provide some background as to the general starting situation in those not so distant pioneering years of membrane biology . |
over 50 million women who live in areas of high malaria transmission become pregnant every year , and thousands of these women die .
women in their first and second pregnancies are at particular risk of infection with plasmodium falciparum , which is a major risk factor for maternal and foetal mortality and is implicated in 75,000200,000 infant deaths per annum [ 2 , 3 ] .
selective accumulation of parasites in the placental space results in maternal anaemia [ 46 ] and infant low birth weight ( lbw ) [ 713 ] through preterm delivery ( ptd ) [ 12 , 13 ] and intrauterine growth restriction ( iugr ) [ 7 , 10 , 12 , 13 ] .
malaria demands up to 5% of the gross domestic product in sub - saharan africa .
pregnancy - associated malaria ( pam ) infection is one example of a severe malaria syndrome , mediated by the surface expression of variant surface antigens ( vsas ) of p. falciparum parasitised red blood cells ( prbc ) that allow adherence to vascular endothelium . in non - pregnant individuals ,
vsas adhere to the ubiquitous endothelial surface proteins intercellular adhesion molecule-1 ( icam-1 ) and cd36 or to other prbc or form rosettes around non - infected rbc . under high transmission settings with favourable breeding sites for the vector anopheles mosquito ,
adults acquire natural immunity to vsas , rendering them asymptomatic [ 15 , 16 ] .
in contrast , women who are immune to these parasites also display adverse consequences of infection when they become pregnant ; this has contributed to the previous belief that pregnancy represents an immunocompromised state .
regardless of the extent of previous exposure to p. falciparum during pregnancy , all pregnant women are at increased risk of malaria and appear to be more attractive to mosquitoes [ 17 , 18 ] .
marked differences in symptoms are apparent between varying levels of transmission ; pam in areas of low transmission can result in severe infection and lead to foetal and maternal death [ 19 , 20 ] . in these symptomatic women ,
the presence of symptoms results in prompt diagnosis and management which reduces the incidence of unfavourable pregnancy outcomes .
in contrast , women living in areas endemic for malaria and hence possessing prior immunity tend to be asymptomatic in pregnancy but harbour high , undetected parasite levels in the placenta [ 16 , 23 ] .
pam affects these women in a gravidity - dependent manner : primigravid ( pg ) women are more susceptible than multigravid ( mg ) women .
after correction for age - related susceptibility , this trend has been reported consistently and is more pronounced with increasing transmission [ 25 , 26 ] .
pam is managed during pregnancy with intermittent preventive strategies using chemotherapeutic medications or insecticide - treated nets .
the world health organization recommends that insecticide - treated nets and intermittent preventive treatment ( iptp ) should be used during pregnancy [ 1 , 22 ] .
iptp consists of two doses of sulfadoxine and pyrimethamine in the second and third trimesters .
a recent systematic review demonstrates limited protection from pam in some malaria - endemic regions . while sulfadoxine - pyrimethamine treatment remains effective in west africa , and more so in three doses than two , there is a need for novel interventions .
current efforts to control the incidence of malaria infection are being hampered by rapidly increasing numbers of insecticide - resistant mosquitoes and treatment - resistant parasites .
hence , production of a vaccine to protect women in high risk areas is an urgent public health priority .
this paper aims to address our current understanding of this subject and to determine whether enough is known about the interactions between parasite and placenta to consider this a realistically attainable feat .
placental malaria ( pm ) is a subset of pam which refers to the pathological process whereby prbc and inflammatory cells accumulate within the intervillous space ( ivs ) of the placenta . at delivery
, pm can be measured by microscopic examination of stained slides of placental blood , by histopathological evaluation of placental biopsies or by semiquantitative polymerase chain reaction ( pcr ) .
examination of blood smears is rapid , cheap , and easy but does not allow assessment of past infection , whereas this is possible with both histological visualisation of parasites and pcr - assisted grading of pigment deposition .
both the latter two methods have enabled recent determinations of how long parasites may survive in the placenta : leke et al . reported that the same parasites may be detected up to 98 days before delivery through pcr examination of parasite polymorphism .
histology does not provide an accurate diagnosis ; absence of parasites or pigment at histology does not necessarily mean that infection has not occurred .
lack of effective and reliable measures to diagnose placental pathology during pregnancy limits comparisons between studies .
it is neither practical nor ethical to investigate placental parasite densities during pregnancy due to the risk of inducing foetal loss .
however , placental parasite densities at term do not appear to correlate with densities of parasites in the peripheral blood which complicates diagnosis during gestation .
observations that densities of parasites in the placenta may be far higher than the densities in peripheral blood samples suggest that parasites accumulate selectively in the ivs [ 8 , 33 ] .
the ivs forms from the lacunae between foetal - derived syncytiotrophoblastic villi , which emerge following fertilisation and implantation of the blastocyst [ 3638 ] .
placental trophoblast invades the uterine wall , gaining blood supply from the spiral arteries that pass through the endometrium .
a recent cohort study that examined the effects of timing and frequency of p. falciparum infection on pregnancy outcomes in 2,462 subjects was unable to evaluate the effects in early pregnancy because only six women in their first trimester attended the antenatal clinic where recruitment was taking place .
this small number was excluded from the study , but this demonstrates the difficulty in gaining data during this stage of gestation .
density of peripheral parasites peaks between 13 and 16 weeks of gestation , suggesting that susceptibility to pam is increased in the first trimester [ 8 , 40 ] .
the peak in parasitaemia was first identified in a large group of women living in areas of high transmission .
although this figure is highly cited , the reliability of this finding is limited by the two methods used for determining gestational age ; calculation from last monthly period is affected by recall bias and fundal height measurement is unreliable before 24 weeks of gestation .
furthermore , malaria is known to cause iugr and therefore a reduction in uterine size , leading to inaccuracy when utilising the latter as a determinant .
the reasons for the discrepancies in foetal measurements between studies have been reviewed recently elsewhere , and these inaccuracies demonstrate a need for standardised methods for the provision of comparable data .
the use of ultrasound to determine gestational age is more accurate , and it is hoped that increasing use of this method will provide more robust results .
subsequently , a decrease in peripheral parasite density was reported after 16 weeks of gestation .
however , it was observed that this reduction in peripheral levels coincided with completion of placental development , leading to the conclusion that parasites may sequester in the placenta at this time . of note , both pg and mg women recovered at the same time , supporting this hypothesis .
following delivery , women appear to undergo rapid clearance of parasitaemia , and evidence suggests that subsequent to this they are at risk of peripheral infection . in pregnancy , the dominant receptor for adhesion is thought to be the chondroitin sulphate a ( csa ) component of the chondroitin sulphate proteoglycan ( cspg ) [ 4650 ] .
placental - parasite variants that adhere to csa show an absence of binding to cd36 and icam-1 ligands , in contrast to parasite variants taken from infected non - pregnant women [ 5153 ] .
selection of prbc that adhere to csa in vitro leads to the loss of antigens that bind to cd36 and icam-1 ; hence , it is thought that parasite variants that bind to csa are mutually exclusive to those that bind to icam-1 and cd36 [ 47 , 5355 ] . in some instances
this is an infrequent observation and may result from the presence of two antigenically distinct variant molecules on a single prbc .
however , mounting evidence supporting the mutually exclusive behaviour of antigen presentation suggests that this is unlikely [ 52 , 5860 ] .
placental - binding isolates are unable to form rosettes and do not agglutinate when exposed to immune serum from non - pregnant individuals [ 53 , 6164 ] .
csa is a sulphated glycosaminoglycan ( gag ) present on the syncytiotrophoblast in the intervillous space of the placenta , located as a side chain on the tissue anticoagulant thrombomodulin .
a plethora of functions for proteoglycans have been identified , including regulation of cell proliferation , differentiation , and adhesion , as reviewed in .
binding to placental csa may alter gene transcription or signal transduction or activate intracellular signalling mechanisms that lead to increased expression of inflammatory mediators [ 6668 ] .
csa is expressed by nearly all cells but it is unclear why parasites bind only to csa expressed by the placenta , although this may be explained by the specific patterns of sulphation of placental csa and the structure it forms on the trophoblast membrane .
previously , other placental molecules , such as hyaluronic acid ( ha ) , have been implicated in placental binding of prbc , and some parasite lines have been shown to have affinity for three receptors ; csa , ha , and cd36 .
however , the strength of binding does not match that of csa - parasite adherence alone .
it is now well established that the parasite protein able to adhere to csa in the placenta is var2csa coded for by the var2csa gene [ 58 , 72 ] .
this protein is a member of the p. falciparum erythrocyte membrane protein 1 ( pfemp1 ) family [ 16 , 73 ] .
encoded by the var multigene family , pfemp1 molecules vary greatly between variants and strains , but each protein retains its c - terminal intracellular domain , an extracellular domain , and a single transmembrane helix .
the ectodomain , responsible for discrete receptor - binding properties , consists of multiple duffy binding - like ( dbl ) domains and cysteine - rich interdomain regions ( cidrs ) [ 7476 ] .
vsas that are thought to specifically cause disease in pam , such as var2csa , are collectively referred to as vsapam .
infection with p. vivax during pregnancy is associated with maternal anaemia and foetal lbw [ 7881 ] , but is not thought to increase the risk of ptd .
pregnant women are at increased risk of infection with p. vivax , although this species does not appear to sequester in the placenta .
p. vivax is suppressed by co - infection with p. falciparum , although co - infection may lower the risk of severe anaemia [ 32 , 82 ] .
little is known about placental infection with p. malariae , p. ovale , and p. knowlesi : both p. ovale and p. malariae can infect pregnant women , but pregnancy does not appear to increase susceptibility to infection with these species [ 7 , 80 , 83 ] .
prior to the appearance of the placenta during pregnancy , women are not exposed to vsapam - expressing variants and therefore lack specific antibodies [ 47 , 62 ] . serum taken from mg women with previous exposure to these variants has been shown to inhibit adhesion of prbc isolated from pg women [ 47 , 62 , 9395 ] .
inhibition of binding was assumed to be protective because these same women proceeded to have several additional successful pregnancies .
in contrast , immune sera from pg women and adult men , with no previous exposure to vsapam - expressing variants , could not prevent binding to csa .
levels of pregnancy - specific antibodies have been correlated with the degree to which binding of prbc to csa is inhibited [ 63 , 95 , 96 ] .
levels of protective antibodies increase with gravidity [ 62 , 63 , 95 , 96 ] .
inhibition of binding by antibodies contributes to parasite clonal variation , antigenic switching and hence immune evasion , although it has been reported recently that antigenic switching may occur due to intrinsic regulatory systems .
antibodies are detected in both pg and mg women ; low levels have been detected at 14 weeks of gestation in pg women [ 91 , 98 ] , in contrast to previous reports of detection being possible only after 20 weeks [ 95 , 99 ] .
although these women appear capable of producing antibodies , they may not confer protection in pg women because the antibody repertoire is not broad enough to inhibit binding of more than one or two variants .
complex infections with numerous phenotypes may disrupt the ability to combat effectively the response to a single variant .
in addition , the presence of non - specific , non - protective immunoglobulin ( ig ) may delay or interfere with the acquisition of memory b cells .
of the five classes of ig , igg is known to be the most important in malaria immunity , and , in humans , the subclasses igg1 and igg3 have been found to correlate most with protection from disease [ 25 , 101 ] .
igg - mediated protection is thought to be achieved through anti - adhesion and opsonic activity [ 102104 ] .
anti - adhesion antibodies have been shown to correlate with reduced levels of placental parasitaemia and to act by promoting splenic removal of parasites [ 62 , 105 ] .
the anti - adhesive properties of antibodies have been studied more extensively than their cytophilic properties .
recent reports of correlations of opsonizing var2csa - specific igg and protection from pm may indicate that both means of parasite clearance need to be assessed in vitro .
furthermore , opsonic antibodies may be a more specific predictor of outcome than overall igg levels . in one study ,
strong associations between levels of antibodies and protection from anaemia were reported at delivery in the absence of correlations with protection from lbw .
anti - adhesive antibodies lower the parasite density , which is associated with an increase in birth weight and length of gestation . whether the increased birth weight is a result of decreased chronic malaria or due to increased length of gestation is not clear , but lbw is a major risk factor for infant mortality .
if a pregnant woman is unable to adequately clear parasites , chronic infection of the placenta may ensue .
, studying women in senegal , found that women could be divided into two groups .
pregnant women were either infected with parasites that persisted in the placenta until birth ( from as early as 69 days before delivery ) or were constantly being reinfected with new , antigenically distinct parasites , emerging throughout gestation . in this latter group , samples clear of parasites were taken between those that contained parasites of new genotypes , indicating that this group of women was able to achieve effective parasite clearance between each infection .
parity status , birth weight , and time of delivery did not differ between the two groups , but women in the first group described had significantly lower levels of var2csa - specific igg initially , although levels were similar by the time of delivery .
absence of antibodies in pregnancy may lead to long - lasting parasite genotypes sequestered in the placenta , which may interfere with further antibody production .
lack of vsapam - specific igg production is associated with an increased risk of developing chronic pm and anaemia .
levels of vsa - specific igg for non - placental variants do not appear to confer protection against poor pregnancy outcomes , but levels of these antibodies appear to remain constant during pregnancy [ 25 , 98 ] .
similar levels of igg that inhibit binding to icam-1 and cd36 have been found in non - pregnant and pregnant women in the same transmission settings .
mice with prior immunity to p. berghei succumb to recrudescent parasites during pregnancy and suddenly become symptomatic in the second week of gestation .
recrudescence poses a risk for women who become pregnant after emigrating from an area of malaria transmission .
compared the antibody responses of pregnant women living in two areas of cameroon with different levels of transmission ( 2.4 infective bites / day versus 0.11.1 infective bites / month ) . in the low transmission setting ,
all women , regardless of age or parity , had significantly lower levels of igg than women living in a high transmission setting .
the sample size of the latter group was smaller than the group from the low transmission setting but the antibody responses reported are in keeping with previous studies .
in the high transmission setting , pg women produced vsapam - specific igg much more rapidly than those in the area of low transmission .
these findings support earlier reports that indicate transmission intensity is important for antibody acquisition [ 94 , 105 ] , yet although these trends are well described , comparison of results from different studies is limited due to vast variations in transmission levels . where transmission intensity is greatest , even women in their second pregnancy may have been exposed to enough variants in their first pregnancy to be protected from pm .
mg women who live in areas of perennial transmission may be less able to control mixed placental infections in subsequent pregnancies than those in areas of seasonal transmission , where polyallelic infections have been correlated with lbw [ 102 , 110 , 111 ] .
this indicates that the differences in antibody responses are not simply divided by geographical variations in transmission rates , which vary greatly across areas of sub - saharan africa .
few data have been published on antibody levels in women with no previous exposure to parasites .
where pregnant women do not appear to have been exposed during pregnancy , they have been shown to be at risk of recrudescent infections that may still cause pm due to previous exposure prior to conception [ 114 , 115 ] .
also , in non - immune mice , infection with p. berghei is usually fatal and placental infection often leads to spontaneous abortion .
retrospective and prospective data have found that increasing the dose of iptp in the third trimester reduces the production of vsapam - specific igg [ 98 , 117 ] .
observations from a longitudinal study suggest that levels of antibodies in women taking iptp remain low for the duration of pregnancy , although antibody levels were seen to fluctuate throughout gestation
. this may be due to variation in the levels of parasite exposure , increased virulence of certain phenotypes , or measured differences following decay of igg antibodies .
these observations indicate that women who have received treatment in their first pregnancy will need continued therapy in subsequent pregnancies and demonstrate that results from previous cross - sectional studies may be inaccurate in representing antibody responses during gestation .
this particular study does have its limitations ; all women had favourable outcomes and there was a lack of placental histological examination at delivery .
interestingly , immunity to malaria has been seen to increase efficacy of antimalarial chemotherapy , and this effect has been reported in pam also .
this implies that production of a vaccine would have an additional benefit ; if antibody levels do not reach those required for parasite clearance , they are at least likely to increase the efficacy of pharmacological interventions . serum from mg women can recognise a number of strains of the var2csa - expressing parasite variant .
distinct strains are recognised by monoclonal antibodies ( mabs ) although maternal antibodies may cross - react with different var2csa - transcribing placental isolates from distinct geographical regions without previous exposure [ 71 , 119 ] .
approximately 42% of pregnant women in a study cohort from malawi and papua new guinea reacted to two or more globally isolated parasite lines .
high levels of cross - reactivity seen in these populations may result from serum being cross - reactive , but not necessarily cross - inhibitory , although cross - inhibition by serum has been reported by fried et al . .
cross - reactivity of serum is thought to be either due to clonally conserved epitopes on individual var2csa molecules or to polymorphic epitopes that are found in all isolates .
levels of vsapam - specific igg decline post - partum , but they have been detected at six months after delivery .
previous studies have demonstrated that in mg women after delivery , up to one in 4,000 b cells show specificity for exposed epitopes of var2csa [ 120 , 121 ] .
persistence of memory b cells has not been examined , rendering a gap in our understanding of how mg women are able to maintain immune memory once the placenta has been expelled and antigenic exposure is lost .
the cytokine balance in pregnancy is shifted towards a predominantly anti - inflammatory response by the t helper ( th)2 subset of cd4 t lymphocytes .
this balance is mediated by the maternal placental decidua and provides a specific environment to allow persistence of the semi - allograft foetus ( expressing paternal antigens ) within the mother . at the blastocyst stage of pregnancy , th1 proinflammatory cytokines , including interleukin ( il)-2 , tumour necrosis factor ( tnf- ) , and interferon gamma ( ifn- ) , are essential for implantation .
ifn- is involved in remodelling of the spiral arteries to achieve adequate placental blood flow and tnf- is necessary for induction of labour [ 123 , 124 ] . following implantation , a th2 response is favoured which permits foetal development by production of the th2 cytokines il-4 , il-5 , il-10 , and il-13 [ 125 , 126 ] and transforming growth factor ( tgf- ) [ 127 , 128 ] .
levels of progesterone and oestrogen rise in the early stages of pregnancy , and both promote a th2 response .
secreted by the corpus luteum , progesterone maintains the endometrium , providing a welcome environment for the blastocyst and for subsequent embryo development [ 126 , 130 ] .
progesterone is a potent inducer of il-4 and il-5 , which oppose ifn- while inhibiting proliferation of cd8 t cells
relaxin is thought to counterbalance th2 activity of progesterone and promotes development of t cells that produce ifn- so is likely to play a more protective role in pregnancy . in non
- pregnant individuals , both th1 and th2 cytokine profiles are associated with protection , the former more active in the acute phase , for parasite clearance , and the latter during chronic infection [ 131 , 132 ] .
, now recognised as a prevailing th2 response of second trimester pregnancy [ 125128 ] , is thought to favour parasite persistence and has been suggested to be responsible for the rise in parasite densities reported at 1316 weeks .
placental accumulation of prbc appears to stimulate th1 cytokine release by macrophages [ 133 , 134 ] to aid parasite clearance , possibly through increased phagocytic activity and by production of reactive oxygen species and nitric oxide ( no ) metabolites [ 133136 ] .
however , parasite densities may need to reach a threshold before inducing th1 responses in the placenta . in mice , strong th1 responses cause foetal loss , but
in human pregnancies induction of these cytokines has been associated with maternal anaemia , spontaneous abortions , and ptd [ 137 , 138 ] .
concentrations of tnf- appear to correspond to infiltration of monocytes and the degree of sequestration [ 133 , 135 ] .
macrophages activated by haemozoin ingestion are thought to be the main source of both tnf- and il-8 . elevated
tnf- levels can enhance cytoadherence , promote monocyte recruitment , and affect hormonal regulation and structural integrity within the placenta [ 143 , 144 ] .
higher concentrations of plasma tnf- have been found in febrile , symptomatic pregnant women and teenage mothers , perhaps indicating an association with age - related immune acquisition .
gravidity - related differences in tnf- have been reported inconsistently [ 145 , 146 ] .
the soluble tnf receptors , tnfr1 and 2 , modulate the activity of tnf- , and dysregulation of their control mechanisms may be linked to the development of severe malaria [ 147 , 148 ] .
high levels of ifn- have been found in placental and peripheral blood samples [ 133 , 134 , 149 ] , with significantly higher levels found in infected pregnant women than non - infected pregnant women .
ifn- is produced by maternal cd8 and cd4 t cells and natural killer ( nk ) cells , but it is produced also by the foetal trophoblast . typically , elevated levels of ifn- are not associated with poor pregnancy outcomes but have been correlated with gravidity - dependent protection .
protection , without pathological consequences , is likely to be related to the maternal ability to prevent ifn- levels from exceeding an unknown pathological cut - off point .
ifn- concentrations have been found to be higher in mg women in acute pm , and a subsequent drop in cytokine levels is observed , whereas in pg women this cytokine peaks at a lower level but remains there .
migration inhibitory factor ( mif ) is secreted from the foetal syncytiotrophoblast , which may be as a direct result of binding to csa [ 68 , 150 ] .
mif stimulates phagocytosis of prbc , and increased levels of mif have been seen in pm [ 150 , 151 ] .
mif encourages accumulation of macrophages , which is consistent with the increased numbers of macrophages seen on histopathological examination at delivery [ 150 , 151 ] . in one study of pg and
mg women , substantially higher levels of mif were found in placental sera than in peripheral sera , with greater concentrations of mif in placental samples from pg compared to mg individuals .
hence , it is possible that an increase in mif may provide protection in the placenta .
these differences may play a part in gravidity - specific immunity , yet equally the differences seen may be due to higher parasite densities experienced in pg women or to raised steroid levels in pg that are known to increase mif concentrations .
however , the small sample size in this study limits the reliability of the data , and future research is needed in this area .
it is currently not known how early th1 responses may be stimulated or whether these responses can be induced independently of placental binding . however , a th1 cytokine response to clear parasites also appears to occur in mg women who continue to have successful pregnancies .
this would indicate that the alteration in cytokine balance may not be as detrimental to the foetus as might be expected . similarly
, this may demonstrate the nature of the host / parasite relationship because a rapid death of both mother and foetus would be lethal for the parasite .
although numerous studies have reported elevated levels of th1 cytokines in pm , it is not a consistent finding [ 153 , 154 ] . raised levels of th2 cytokines have also been found in pm which may indicate a positive feedback response to restore th2 dominance .
local upregulation of il-10 , which acts directly on macrophages and causes a decrease in il-12 production , has been reported in multiple studies [ 133 , 134 , 146 , 155 ] . in one study cohort ,
women with pm were reported to have a predominant th2 response which did not correlate with parity , age or sex , but parasite densities were consistently low .
tgf- is thought to protect against severe pm , demonstrated by extended survival of p. berghei - infected mice when administered tgf- , although raised levels have not been consistently correlated with parasite densities of women with predominantly successful outcomes .
peripheral levels of cytokine concentrations do not correlate with cytokine levels in the placenta or with numbers of cd4 t cells measured [ 145 , 157 ] .
however , cytokine responses can be examined using p. berghei [ 108 , 114 , 158 ] .
murine models are simpler , cheaper , and raise fewer ethical issues than simian models although data from these should be used with caution due to differences in the architecture of human and murine placentas .
in addition , mice show a similar th1th2 shift during pregnancy to that seen in humans . data from cross - sectional studies which examine cytokine dynamics during pregnancy are difficult to use because of the type - one error incurred by measuring numerous levels of cytokines that do not increase or decrease independently [ 133 , 156 ] . discrepancies between studies could further be accounted for by the short - lived persistence of cytokines in the blood , indicating the need for more intense sampling or development of methods of cytokine quantification with increased sensitivity and specificity .
women may be more susceptible to malaria during pregnancy due to a reduction in t cell numbers .
reports of decreased t cell responses could be due to sequestration of t cells in the ivs [ 157 , 161 ] .
parasite - specific t cell proliferative responses may be increased in mg women who exhibit protection from pm ; fievet et al .
reported that mg women produced increased levels of th1 cytokines to csa - adhering strains of p. falciparum but not to non - csa binding strains .
cytotoxic cd8 t cells are implicated in the protective immune response to pre - erythrocytic malaria parasites .
their role in pm has not been determined , primarily since in murine models their function beyond the liver stage of the plasmodium life cycle remains controversial .
it has been a long held opinion that the mechanism of activation of cd8 t cells precludes a protective role against blood stage malaria .
this is because cd8 t cells are stimulated by antigens in association with major histocompatibility complex ( mhc ) class i molecules which are not expressed by rbc .
however , reticulocytes ( immature rbc ) express mhc class i molecules and numbers of reticulocytes increase during pregnancy , and these can be infected by mature parasites .
cd8 t cells may potentially play a protective role in pm if they are found to respond to these antigens .
recent observations of raised levels of cd8 t cells in pregnant women with absent pm following parasite clearance may indicate that they may have an anti - parasite effect via a non - ifn- pathway .
dendritic cells , macrophages , nk cells , nk t cells , and t cells interact non - specifically with foreign pathogens .
t cells are known to be important in non - specific targeting of p. falciparum although their role , in addition to that of dendritic cells and nk t cells , has not been studied in pm thus far .
nk cells rapidly produce tnf- and ifn- which can inhibit replication of p. falciparum , and their presence is implicated in the antibody - dependent nk - mediated lysis of prbc [ 170172 ] .
unique subsets of nk cells are involved in the maintenance of early pregnancy , although alterations in nk cell behaviour may predispose to infection . in an endemic area
, a higher proportion of activated nk cells were found in the placenta , and this was associated with an absence of accompanying parasitaemia .
interestingly , numbers of nk cells were not increased , but higher concentrations of ifn- were measured .
if women could stimulate immediate ifn- release , they were able to control parasitaemia more effectively . unfortunately , this study only looked at women with favourable birth outcomes , and high numbers of nk cells have previously been associated with poor outcomes .
whether or not this is related to the concentration of ifn- rather than to the number of cells remains to be seen .
in contrast , decreased activity of nk cells has been linked to severe pm episodes , which may be due to the absence of nk recognition of parasite antigens , and therefore ifn- is not produced .
this finding is from in vitro experiments , where no other co - stimulants are present , which is unlikely to be the case in vivo .
the presence of mononuclear cells in the placenta has been correlated negatively with birth weight .
stimulation of these cells promotes secretion of chemokines which contribute to the initiation of the inflammatory cascade .
both cxc/ and cc/ chemokines may increase locally in pm , namely , ifn- inducible protein ( ip-10 ) , monocyte chemoattractant protein 1 ( mcp-1 ) , macrophage - inflammatory protein 1 ( mip-1 ) , and macrophage - inflammatory protein 1 ( mip-1 ) [ 17 , 133 ] .
these substances have been associated with angiogenesis , haematopoiesis , and protection from intracellular pathogens .
local activation is supported by observation of greater densities of activated monocytes in the placenta than are found in the peripheral circulation .
phagocytosis is further mediated through complement activation . in vitro observations using plasma from non - immune donors
indicate that if complement deposition is prevented , 8095% of phagocytosis of prbc is prevented .
thus , complement may play a supplementary role to uptake of antibody - opsonised prbc in macrophage clearance observed in semi - immune women .
however , as seen with cytokine levels , excessive levels of complement activation may contribute to the pathogenesis associated with inflammation .
in healthy pregnancies , complement component 3 ( c3 ) is reduced by expression of cd55 , cd59 , and a membrane cofactor protein which are expressed by the placenta to limit local damage [ 129 , 179 ] .
pg women with pm have been shown to have over 50% more complement at delivery than non - infected pregnant women , although this particular study did not compare levels with mg women .
nevertheless , excess complement activation may overwhelm complement regulatory proteins and contribute to placental injury . raised levels of complement component 5a ( c5a ) , activated by c3a ,
excessive levels of c5a have been identified as a mediator of placental and foetal injury in mice , often resulting in spontaneous abortion and iugr .
c5a enhances macrophage and neutrophil activity , and excessive levels indicate that regulation of complement activation has been lost .
levels of activated complement need to be examined in a larger group of women with pm and correlated with gravidity , pregnancy outcomes and degree of inflammatory infiltrate .
complement and cytokine release by macrophages may occur before placental binding takes place because toll - like receptors of the innate immune response are known to respond to the p. falciparum toxin glycosylphosphatidylinositol ( pfgpi ) , promoting local inflammation .
placental - dwelling macrophages may secrete tissue factor which initiates the clotting system ; clot formation can plug the intervillous space and disrupt blood flow . raised levels of cortisol during pregnancy
have been correlated with parasite load , with significantly higher levels in pg women than in mg women .
cortisol is known to increase mif-1 , decrease cmi , suppress nk cell activity , and delay antibody production and may decrease il-10 responses [ 175 , 186 , 187 ] .
these factors could explain why pg women are more susceptible to pam than mg women .
unfortunately , this study is confounded by the young age of pg study participants compared to the mg women ( mean ages 18 and 30 , respectively ) . in an area of unstable transmission ,
women of all gravidities were found to have raised levels of cortisol compared to non - infected pregnant women .
prolactin is thought to be host protective in pam , but it may be influenced by changing cortisol levels [ 175 , 186 ] .
a study that reported a correlation between low levels of prolactin in women with high parasite densities also found that in these women levels of il-10 were also low .
human immunodeficiency virus ( hiv ) alters the typical gravidity - specific pattern of malaria risk by shifting the burden from primarily pg and secundigravid women to all pregnant women . in a retrospective analysis of 11 reports published over a15-year period ,
the proportional increase of p. falciparum infection during pregnancy attributable to hiv was estimated to be between 5.5 and 18.8% for populations with hiv prevalences ranging from 10 to 40% .
in addition , viral load has been found to increase with malaria infection in pregnant women [ 189 , 190 ] .
lower levels of vsapam - specific igg and decreased levels of opsonic igg activity have been reported in women who are hiv positive .
hiv infection lowers numbers of cd4 t cells , with lower levels correlating with more severe disease .
hiv infection further impairs cytokine responses ; il-12 may be reduced so an early th1 shift may not occur , leading to loss of the protective rise of ifn- .
table 2 summarizes the key maternal and foetal outcomes of placental malaria and the immunological basis for the pathological process leading to each outcome .
placental damage stimulates cytotrophoblast proliferation resulting in a thickened cytotrophoblast membrane which may decrease the exposure of placental and foetal tissues to inflammation .
maternal - foetal transfer of nutrients , oxygen , and waste products may be compromised by mechanical blockage and inflammatory cells .
risk of iugr is increased if infections include complex multiallelic variants [ 98 , 102 ] , are increased in frequency , or occur during the second trimester rather than the third .
a major limitation of our understanding of the pathological mechanisms involved in pm surrounds the processes that lead to iugr .
the first trimester is a critical period for foetal organogenesis ; the majority of growth takes place in the second trimester , and organ and cell maturation are completed during the third trimester . prior to placental completion , early accumulation of prbc in lacunae may induce complement activation or cytokine release which could impact negatively on embryogenesis .
the majority of lbw babies born to mothers with pm are uniformly small , implying that growth restriction has been symmetrical and most likely occurred in the first trimester .
raised levels of activated soluble inflammatory mediators are likely to cause pathology when unregulated during pm .
tnf- has been associated consistently with foetal lbw , either through iugr [ 135 , 139 ] ( associated with accumulation of high concentrations of monocytes during chronic placental infection ) or through ptd .
ifn- , although not associated convincingly with lbw [ 135 , 139 ] , can induce nk cells which may be involved in malaria - associated spontaneous foetal loss . however , it is more likely that ifn- has a protective role in pm .
ptd may be caused by a low tnf- : il-10 ratio ( due to raised levels of both tnf- and il-10 in the placenta ) , maternal anaemia secondary to an augmentation of il-10 , or a sudden acute increase in tnf- concentration .
active inflammation with monocyte infiltration may also lead to dysregulation of the insulin - like growth factor ( igf ) axis which is upregulated in the normal placenta to support growth .
vascular endothelial growth factor ( vegf ) and angiogenic factors , including angiopoietin 1 and 2 ( ang-1 , ang-2 ) , are important in the control of vascular development and remodelling during placental development .
raised levels of svegfr1 have been associated with excessive levels of c5a ; either c5a or the presence of parasite products that activate complement may be responsible for this .
dysregulation of angiopoietin levels has been reported in women with peripheral parasitaemia , and was correlated more closely in women who had lbw babies .
these levels were measured at delivery , but disturbance of the balance of these growth factors could occur earlier in gestation and impact negatively on foetal growth .
these findings support the hypothesis that the pathogenesis of pm is similar to that of pre - eclampsia ( pe ) .
a case - cohort study reported an absence of relative risk between pm and pe whereas , although this type of study design does not provide a causal link , a correlation was seen between pm and gestational hypertension [ 199 , 200 ] .
gestational hypertension may result from impaired placental blood flow , possibly from shallow trophoblast invasion and poor placental vascularisation , or from mechanical blockage of blood flow
. reduced placental perfusion , endothelial dysfunction , and placental ischaemia are thought to contribute to iugr .
women who harbour complex infections are at increased risk of anaemia , while the infants born to these women are more likely to have infected cord blood and increased risk of malaria in the first 30 months of life .
although parasite infection of cord blood is common [ 154 , 203 ] and newborns may harbour an asymptomatic parasitaemia [ 204 , 205 ] , disease is rare [ 77 , 205 ] .
protection from disease may be due to foetal haemoglobin being composed of different globin subtypes than adult haemoglobin , decreased infant exposure due to swaddling and transfer of maternal antibody [ 205207 ] .
pm may prime foetal immune t cells in utero to favour a th2 cytokine response due to exposure to maternal cytokines [ 205207 ] .
inflammation may disrupt igg transporters in the syncytiotrophoblast , thereby reducing placental transfer of igg in response to other pathogens including measles virus and streptococcus pneumoniae .
in order to produce an ideal vaccine for pam , all the protective responses that occur in natural infections need to be stimulated .
figure 2 summarizes the potential immunological interactions in pm and the protective responses that occur in mg women .
is the var2csa molecule , the structure of which differs to that of other pfemp1 molecules .
it comprises six dbl domains and a cidr , all of which display polymorphism between strains [ 69 , 116 ] . from 32 isolates from a sample of pregnant women in senegal , not one shared an identical sequence in the dbl5 domain .
the var2csa gene that codes for var2csa displays significant levels of polymorphism , demonstrated by the number of possible unique antigens that have been produced .
however , it is more conserved than other var genes , and var2csa retains 7583% of its amino acids between isolated strains .
however , the degree of antigenic variation between globally isolated parasite lines has not been determined .
vaccine developments with var2csa have been hampered by the difficulty in producing a recombinant protein of this size . to overcome this issue initially , individual domains of var2csa
have been studied to determine if a particular region of the molecule is responsible for receptor binding and induction of antibodies .
three of the six dbl regions ( dbl2-x , dbl3-x and dbl6- ) are known to bind to csa , but the remaining three ( dbl1-x , dbl4- , dbl5- ) display limited affinity for csa [ 218 , 219 ] .
it has proved difficult for study groups to adequately assess binding of different dbl regions , as interactions between recombinant proteins ( expressed by escherichia coli , baculovirus or pichia pastoris constructs [ 220222 ] ) and csa in vitro may not be representative of native binding .
furthermore , studies that have elicited adhesion inhibition have observed it in assays of csa on plastic mounts [ 103 , 220 , 224 ] , which , whilst they provide an easy laboratory method of assessing adhesive properties , do not contain the same levels of sulphation as csa on the syncytiotrophoblast . the most promising method to study
adhesion behaviour is to use bewo cells from human choriocarcinoma that provide a placental construct on which csa is naturally found [ 90 , 220 , 224 ] .
antibodies have been induced in mice and goats with recombinant dbl3 , dbl4 , and dbl5 domains [ 104 , 220222 , 225 , 226 ] .
antibodies to dbl3 and dbl5 recognised other var2csa - expressing strains from diverse geographical regions , whereas cross - reactivity was not observed following immunisation with dbl1 , 4 , or 6 .
despite displaying cross - reactivity , these antibodies did not inhibit binding , suggesting that only surface reactivity may occur with in vitro - induced antibodies .
interestingly , the most effective anti - adhesion antibodies have been induced in rats with dbl4 domains .
this domain is thought to display the lowest binding affinity for csa but is highly conserved between variants .
the dbl5 domain displays 6681% conservation between isolates , and strain - transcending adhesion inhibition has been seen under flow conditions using bewo cells lines and antibodies induced in mice and goats .
single domains from non - placental pfemp1 may also bind to csa in vitro but with much weaker affinity than binding of whole var2csa .
the full - length version induces anti - adhesion antibodies that are highly strain - specific .
whole recombinant var2csa has now been produced , and , although the ability to induce antibodies using this construct has been inconsistent , these findings demonstrate that all domains may be as important as each other in mediating binding and antibody stimulation [ 103 , 104 , 227 ]
. a combination of epitopes on each domain may form a binding complex or unique binding site by forming a quaternary structure .
if this is the case , individual polymorphisms in dbl regions may come together to alter the binding site of var2csa , which would account for the cross - reactive but not adhesion - inhibitory strain - transcending responses described .
convincing evidence is emerging that supports the formation of a binding site from the first four domains and interactions with the cidr and interdomain regions [ 228 , 229 ] .
inconsistent induction of antibodies with whole constructs could be caused by differing expression methods employed or could indicate limitations of using such a large immunogen [ 104 , 227 ] . although the biochemical interactions are beyond the scope of this review , our understanding of the true nature of parasite antigen - placenta interactions may not be far enough advanced for effective vaccine trials .
induction of cytophilic activity by igg molecules has been shown to be an effective immune mechanism , but this has been largely ignored in vaccine development studies to date . perhaps a novel way forward would involve shifting the focus towards induction of these antibodies : specifically , whether they may be induced using single domains or whole antigens ; and , if inducible , whether they display the same degree of specificity as anti - adhesive antibodies . the lack of identification of a conserved epitope thus far should not be considered disheartening as even antibodies that stimulate a protective response against one or two variant lines may be beneficial in pg women to help lower parasite load and increase efficacy of treatment .
induction of antibodies by single domains and whole var2csa has not yet been tested in humans , and it is likely that single variants will produce differing antibody responses to those found in naturally mixed allelic infections .
evidence suggests that the ability of a construct to induce antibodies depends greatly on the animal being immunised ; a recent study observed that immunisation responses varied greatly and unpredictably between mice , rabbits , and rats and the same is likely to be true with humans .
even if var2csa could be used , the highly specific antibody repertoire gained from this would only confer protection against one parasite strain .
it is not currently known if , or how , immunity develops to strains simultaneously , and this may further complicate trials of antibody stimulation in vivo . if an epitope is identified that allows women to develop inhibitory antibodies rapidly to a range of strains by focusing the immune response , it could provide better protection than that which is naturally acquired .
currently , findings indicate that a number of allelic variants will be needed in a vaccine due to the high levels of polymorphism within the parasite population .
a clear association has been shown between the presence of anti - adhesion antibodies and weight gain of foetuses , but precise pathological mechanisms by which antibodies reduce or inhibit associated inflammation are unknown .
the inhibition of binding may decrease inflammation by preventing activation of signalling pathways on the syncytiotrophoblast that lead to cytokine production .
the continued study of individual binding domains of var2csa may increase our knowledge of these mechanisms even if these studies do not lead to viable vaccine possibilities .
judging by the appearance of placental variants in the second trimester , a vaccine would be most effective given before pregnancy but could also provide protection if given during the first trimester .
this is improbable , however , because the majority of pregnancies in developing countries are not confirmed until the second trimester due to socioeconomic and cultural reasons affecting access to primary health care .
theoretically , stimulation of mabs using individual domains may be possible in prepubescent girls and repeated over successive years , but this is unlikely to be feasible .
the lack of understanding of how immunological memory is maintained hinders the validity of this option .
antibodies have been seen to persist for six months without continued antigen exposure , but it is uncertain if they would continue beyond this .
this indicates that decay of antibodies may occur by the time a woman becomes pregnant if the vaccine is given too early , although this may depend on the specific ig subclasses that are included in the vaccine .
women who lack naturally acquired immunity are not protected from peripheral binding variants and a var2csa would not inhibit binding of these parasites , resulting in peripheral infection . furthermore , selection pressure exerted by such a vaccine is likely to increase global antigenic diversity . continued efforts
to identify conserved epitopes are the aim of non - pam vaccines , and proof of principle studies have led to phase iii trials ( reviewed in ) .
the most advanced candidate , the rts , s vaccine against the circumsporozoite antigen , provided 25.659% protection in phase ii trials and became the first malaria vaccine to reach phase iii trials .
while this extensive study across seven sub - saharan countries will not be completed before 2014 , preliminary results show grounds for qualified optimism [ 232 , 233 ] .
it is unclear what protection this may provide during pregnancy because it is unlikely to achieve sterile immunity and despite women having antibodies against non - vsapam and other parasite proteins prior to pregnancy , the immunity that develops is not adequate to prevent pregnancy - associated infection .
a vaccine against p. falciparum will not protect against p. vivax which is a major problem in regions where transmission of p. falciparum is low , such as the indian subcontinent .
the protection gained from maternal anaemia due to mixed infections may be lost through a p. falciparum - specific vaccine .
further to this , the placental - binding property of p. falciparum to csa has provided a selective advantage , but p. vivax is known to accumulate in the placenta .
more recently , severe syndromes associated with p. vivax have been reported in children , which could indicate increasing virulence in this parasite .
similar receptors to the vsas have been found on the membrane of the p. vivax prbc , but sequestration is currently not thought to occur . as
yet , it is not clear if vaccination with single domains or whole var2csa is recognised by t cells . in the early stages of determining which epitopes stimulate protection , it is essential that t cell responses are considered in vaccine trials .
natural expression of antigens on prbc may stimulate specific antibodies in a different way , and natural interactions of t and b cells may also determine efficacy .
if var2csa is the key antigen in immune acquisition in pam , then it may prove possible to answer the questions below : to
which part of the molecule do antibodies react ? in models , are antibodies produced to isolated domains ?
what class of antibodies are produced using a vaccine ? how long do memory b cells persist ?
can t cell responses be induced ? if t cells are stimulated and produce a protective response that generates ifn- , then
will a tnf- response that harms the placenta also be induced ? will protective innate mechanisms be stimulated by a vaccine ? what end points would we use to measure the effectiveness of a vaccine ?
there are a number of areas that require further research to increase our understanding of both the pathogenesis and immunology of pam .
these include the following : pathological processes occurring in the first trimester ; pathological mechanisms leading to anaemia and lbw with p. vivax infections ; trials of new methods of diagnosing placental malaria infection ; analysis of peripheral cytokines as possible surrogate markers ; longitudinal studies in low transmission settings and longitudinal studies of cytokine dynamics ; whether the responses that lead to immunity and the responses that lead to pathology can be distinguished ; how hormones contribute directly to susceptibility in pam ; longitudinal studies to examine how infant cohorts are affected ; further analysis of the burden of pam outside africa .
the majority of studies described in this review are cross - sectional or retrospective analyses of antibody and parasite dynamics in pregnant women .
levels of both parasites and antibodies fluctuate during pregnancy , and true representations of these dynamics can only be obtained through longitudinal studies [ 34 , 98 , 102 ] .
in addition to the need for more robust studies , there is a risk that by focusing attention on a small part of the pathological processes occurring in pm , we will lose sight of what we already know .
for example , although binding of csa does not occur until the second trimester , the processes may not be as crucial to pathogenesis as currently thought : if binding to csa increases gene expression and secretion of inflammatory mediators , such as mif-1 that promotes macrophage aggregation , then inflammatory processes will not begin until after week 16 .
we can not be certain of the development of inflammation because there is no way of assessing when this begins in pregnant women .
the detection of peripheral levels of inflammatory markers , such as raised levels of tnfr1/2 , svegr1 , c5a , and il-10 , would be an ideal method of diagnosing active pm in asymptomatic women .
however , no marker has been found to correlate adequately with the degree of inflammation .
the pathological processes of pm are dynamic throughout gestation , but the presence of parasites in the placenta may be detrimental to embryo development before binding occurs .
this could potentially be studied using var2csa - null variants in vitro using bewo cells .
is pam vaccination possible ? at present , the creation of a vaccine looks unlikely . not enough is known about specific binding interactions between csa and var2csa , and globally conserved epitopes have not been identified .
these hurdles , combined with the task of including all necessary parasite strains in a single vaccine and the risk that a vaccine could potentially lead to an increase in the prevalence of other malaria species not covered by the vaccine , do not favour vaccine development .
if growth mechanisms are most affected in the first trimester , a var2csa vaccine would be ineffective at reducing the burden of iugr .
this paper has , however , identified some of the gaps in our knowledge , and filling these may lead to ways of overcoming these obstacles .
even if a vaccine is able only to stimulate the production of a small antibody repertoire this could boost treatment effects and decrease the financial burden of pam . in the light of growing resistance of p. falciparum to current chemotherapeutic regimens ,
even a vaccine that could result in lower doses and increased efficacy of these regimens has potential value , and research to that end should be welcomed . | pregnancy - associated malaria , a manifestation of severe malaria , is the cause of up to 200,000 infant deaths a year , through the effects of placental insufficiency leading to growth restriction and preterm delivery .
development of a vaccine is one strategy for control .
plasmodium falciparum - infected red blood cells accumulate in the placenta through specific binding of pregnancy - associated parasite variants that express the var2csa antigen to chondroitin sulphate a on the surface of syncytiotrophoblast cells .
parasite accumulation , accompanied by an inflammatory infiltrate , disrupts the cytokine balance of pregnancy with the potential to cause placental damage and compromise foetal growth .
multigravid women develop immunity towards var2csa - expressing parasites in a gravidity - dependent manner which prevents unfavourable pregnancy outcomes .
although current vaccine design , targeting var2csa antigens , has succeeded in inducing antibodies artificially , this candidate may not provide protection during the first trimester and may only protect those women living in areas endemic for malaria .
it is concluded that while insufficient information about placental - parasite interactions is presently available to produce an effective vaccine , incremental progress is being made towards achieving this goal . |
recent advances in implant dentistry have introduced a number of alternative
treatment options for the rehabilitation of partially and completely edentulous
patients . in recent years , focus has shifted to a great extent into the
restoration - driven approach for the placement of dental implants through a detailed
pre - surgical planning .
however , functional
and aesthetic replacement of multiple missing teeth with implant - supported
restorations remains a complex task and requires a comprehensive treatment
planning .
traditionally , in clinical situations where a patient presented with advanced
periodontal and/or structural problems to the teeth , the treatment of choice often
involved approaches that focused on extraction of remaining teeth , followed by hard
and soft - tissue regeneration procedures prior to the placement of the dental
implants .
these rehabilitations often required a significant amount of healing time
utilizing delayed loading protocols and removable interim restorations during the
transitional period . for many patients
this treatment approach was not attractive
due to the consequent impact of an interim removable prosthesis on psychology ,
phonetics and mastication .
for all the aforementioned reasons , as well as in cases
of patients with thin gingival biotype , in need of extensive surgeries or in
clinical cases where immediate loading is contraindicated , the clinician is forced
to suggest a smoother transition into the fixed implant restorations .
this procedure involves serial extraction of selected hopeless teeth and the
placement of implants and/or grafts through a staged approach while utilizing the
remaining teeth to support a fixed interim restoration .
an important parameter in this therapeutic approach is the
preservation of natural teeth , whether of fair or questionable prognosis until the
end of the healing process . upon successful osseointegration of the first series of
implants , the tooth - supported interim restoration is converted to an
implant - supported provisional prosthesis
this procedure allows for the extraction
of the remaining hopeless dentition and subsequent progression to the second phase
of implant rehabilitation . in preparation for the surgery ,
the duplication of the provisional prosthesis
facilitates the fabrication of a surgical template that will dictate the location
and inclination of the additional implants [ 6 - 9 ] . to achieve optimal accuracy
and effectiveness
, the surgical template should be simple in construction , have
sufficient stability and not interfere with the surgeon 's access .
a variety of surgical templates differing in design , cost , and stability have been
described in the literature .
they are usually made of thermoplastic sheets or clear
acrylic resin [ 9,11 - 14 ] . in partially
edentulous patients ,
they cover the occlusal surfaces of the adjacent teeth or rest
on any prepared teeth to gain adequate retention and support . on the contrary , when templates are derived from a
duplication of transitional removable prostheses , their precise
repositioning is
extremely difficult after tissue reflection because of a change in the architecture
of the underlying soft tissues that served as the only reference points .
serving as the communication tool for transferring to the surgeon the necessary
guidelines for implant placement , it is quite clear that a stable , well - designed
surgical template can improve the accuracy of the surgical implant installation ,
thus reducing the duration of surgery [ 8,21 - 23 ] .
even though several techniques have been reported in the literature for surgical
template stabilization in the form of surgical fixation screws , transitional implants , or sequential extraction
of the remaining teeth during implant placement , those solutions seem to be rather complicated .
therefore , whenever
a staged rehabilitation is planned , the clinician can effectively use the existing
implants as anchors for the surgical template during the second phase of implant
surgery .
the objective of this paper is to describe the utilization of previously used implant
mounts for the fabrication of stable surgical templates in such staged procedures .
the rationale and design of fixed surgical templates is illustrated through the
presentation of two clinical cases .
a 66-year old patient presented to the clinic with extensive restorations in both
arches .
the clinical and radiographic examination revealed caries under the existing
restorations combined with generalized moderate to severe bone loss due to
periodontitis .
after the fabrication of diagnostic casts , a wax - up was performed in
accordance with the aesthetic and functional requirements of the case . given the
severity of dental caries and extensive bone loss , the treatment plan advocated for
the extraction of the majority of the maxillary teeth .
furthermore , the diagnostic
wax - up confirmed a hard - tissue deficit in bucco - lingual dimension resulting in
insufficient ridge width for ideal implant positioning in the anterior maxilla .
since the patient declined the use of immediate dentures , a two - stage approach for
the extractions and implant placement was planned .
after laboratory fabrication of
the provisional restorations , all existing restorations were removed . the extraction
of several hopeless teeth was performed with emphasis on retaining as many abutments
as possible , in a satisfactory antero - posterior distribution and in strategic
locations on the maxillary arch .
the provisional restoration was relined , cemented
with provisional cement and served for the evaluation of aesthetic and functional
characteristics .
following patient 's consent , the duplication of the interim prosthesis
facilitated the construction of a tooth - supported combined radiographic - surgical
template .
the first phase of surgery included the placement of bone grafts in the
anterior region and four implants at the first premolar and molar sites in the
maxilla .
after a period of six - months and complete healing of the graft sites , the
interim restoration was removed and an implant level impression was made , followed
by the construction of a master cast ( figure
1 ) . additionally , temporary abutments were placed intraorally and the
interim restoration was converted into an implant - supported restoration . upon
delivery of the interim implant - supported restoration , a new alginate impression was
made .
master cast with implant analogs in place . in the laboratory , a vacuform matrix ( invisacryl a , great lakes orthodontics ltd , ny ,
usa )
two implant mounts ( straumann usa ,
andover , ma , usa ) were placed on the premolar analogs ( straumann usa , andover , ma ,
usa ) , and the cast was coated with a liquid separator .
two holes were drilled in the
vacuform matrix to allow it to be properly seated over the implant mounts .
the
vacuform matrix was then filled with clear , auto - polymerizing acrylic resin
( orthodontic resin , dentsply caulk , milford de , usa ) , carefully placed in the cast
and secured with rubber bands ( figure 2 ) .
the
acrylic resin was allowed to set in a pressure pot for ten minutes at which point it
was removed and the excess material was trimmed and polished ( figure 3a and 3b ) . during the second surgery the surgical
template was screw retained to the existing implants after tissue reflection ( figure 4 ) . maintaining teeth contours in the
surgical template
enabled the surgeon to visualize the proposed implant positions
based upon the ideally planned restorative positions .
vacuform template relined over implant mounts . a = intaglio view of the template after polishing ; b = cameo view of the
template after polishing . template retained on implants prior to implant positioning . during the course of treatment , the implant in site # 33 was lost due to
peri - implantitis .
a master cast was made of the remaining three implants in sites
# 36 , # 43 and # 46 ( figure 5 ) . a vacuform
matrix ( invisacryl a , great lakes orthodontics ltd , ny , usa ) fabricated from the
original wax - up
was relined over two implant mounts ( straumann usa , andover , ma ,
usa ) on implant analogs at sites # 36 and # 43 utilizing clear , auto - polymerizing
acrylic resin ( orthodontic resin , dentsply caulk , milford de , usa ) ( figure 6 ) .
the template was trimmed , polished
and sterilized prior to surgery ( figures 7a and
7b ) . at the surgical visit ,
once the tissue was reflected at site # 33 ,
the template was placed onto implants # 36 and # 43 and provided a clear visualization
of the desired implant position based upon the planned restoration ( figure 8) .
a = intaglio view of the template after polishing ; b = cameo view of the
template after polishing .
fixed provisional prostheses are preferred by most patients because of their improved
aesthetics , stability and ease of use . preserving teeth with poor or questionable
prognosis to support
a fixed provisional restoration helps to avoid excessive
loading of the grafted sites and ensures an uneventful healing process .
furthermore ,
both the patient and the clinician may avoid the additional appointments that are
necessary for subsequent relines and any other modifications needed in case of
interim removable prostheses .
this paper describes the use of a surgical template design that allows precise
implant installation in two stages .
initial implant placement includes the use of a
surgical guide that is tooth - supported , whereas second phase implant surgery
utilizes an implant - supported one .
this template may also resemble the diagnostic
wax - up or the provisional restoration , thus giving similar accuracy and helping to
achieve a satisfactory final result .
other advantages include ease of manufacture
and low cost , while using materials , techniques and equipment that are usually
available in the office or lab .
the incorporation of stable anchoring elements into the template presented herein ,
allows its use in multiple ways .
the master cast containing the first phase implant
analogs can be used to fabricate the final provisional restoration .
when additional
implants are installed , this pre - fabricated provisional restoration can be modified
to take advantage of the additional support and provide immediate loading if needed .
after the necessary time for osseointegration has elapsed and the restorative phase
commences , this surgical template can be further modified to a registration template
to help the clinician fast and accurately mount the final working cast in the
articulator .
alternative retention devices ( other than implant mounts ) can be utilized such as
positioning indicators ( straumann usa , andover , ma , usa ) ( figure 9 ) or impression components .
their stability and design
allow for unrestricted tissue reflection , adequate irrigation of the region and
enhance the predictability of the implant position and inclination and therefore
preventing the injury of adjacent anatomical structures ( teeth , sinus , mandibular
canal ) .
in addition to the corresponding
accuracy due to the existence of reference points ( restoration contours , cervical
outline ) , the surgical procedure is also accelerated , with a positive impact on the
post - operative healing . also , the parts used for the anchoring of surgical guides
provide reference points for the inclination of the previously placed implants .
finally , they assist in the uncovering procedure as they indicate the positions of
implantation , thus contributing to a less traumatic procedure . the only drawbacks
to
this technique are the additional laboratory time required for the fabrication of a
new template and the necessary construction of a new final working cast at the
completion of the healing phase .
as long as the successful implant rehabilitation relies on being restoration - driven ,
the precise design of surgical templates will continue to be a prerequisite for
predictable results .
the dentist who chooses to invest in diagnosis , planning and
construction of the surgical template will be redeemed with similar success in the
final restoration and patient satisfaction .
there is not a single template design that fulfils the requirements of
all treatment plans and clinical cases . in general ,
each surgical guide reflects a
rational combination of basic prosthetic rules , the individual prosthetic needs and
the surgeon 's preference .
it should be mentioned that the application of various
types of surgical templates that are used in routine dental practice , require a
certain learning curve in order to ensure maximum effectiveness .
the staged approach enables a smooth transition to implant restorations in advanced
clinical cases .
implant placement during sequential surgical phases can be enhanced
utilizing surgical guides anchored in pre - existing implants .
the utilization of
implant mounts presents a practical and cost - effective method for the fabrication of
an implant - retained template in a simplified manner .
the surgical guide 's stable
fixation ensures the accuracy in implant positioning based upon the planned
restoration .
| abstractbackgroundthe philosophy of a gradual transition to an implant retained prosthesis in
cases of full - mouth or extensive rehabilitation usually involves a staged
treatment concept . in this therapeutic approach
, the placement of implants
may sometimes be divided into phases . during a subsequent surgical phase of
treatment
, the pre - existing implants can serve as anchors for the surgical
template .
those modified surgical templates help in the precise transferring
of restorative information into the surgical field and guide the optimal
three - dimensional implant positioning.methodsthis article highlights the rationale of implant - retained surgical templates
and illustrates them through the presentation of two clinical cases .
the
templates are duplicates of the provisional restorations and are secured to
the existing implants through the utilization of implant mounts.resultsthis template design in such staged procedures provided stability in the
surgical field and enhanced the accuracy in implant positioning based upon
the planned restoration , thus ensuring predictable treatment outcomes.conclusionssuccessful rehabilitation lies in the correct sequence of surgical and
prosthetic procedures .
whenever a staged approach of implant placement is
planned , the clinician can effectively use the initially placed implants as
anchors for the surgical template during the second phase of implant
surgery . |
early repair is important because of risks of incarceration and strangulation of abdominal contents along with respiratory and cardiovascular compromise .
minimally invasive techniques have been useful for diagnosis and treatment of diaphragmatic hernias in both blunt and penetrating trauma .
we present the case of a 54-year - old victim of a motor vehicle crash who presented with a delayed diagnosis of a right - sided traumatic diaphragmatic hernia . by using a 4-port technique and intracorporeal suturing ,
this case highlights the difficulties associated with diagnosing diaphragmatic hernias and the role of minimally invasive techniques to repair them .
minimally invasive surgical techniques are being increasingly used to both diagnose and repair traumatic diaphragmatic injuries with excellent results .
patients are often asymptomatic or comatose , and imaging performed may not clearly depict the dh .
therefore , further studies may not be done , leading to incorrect diagnoses and treatments .
late - onset dh can present as a variety of clinical symptoms , such as cardiorespiratory or gastrointestinal complaints .
right - sided herniation may even be more difficult to detect than left - sided herniation .
thoracoscopy and laparoscopy are 2 of the approaches to minimally invasive repair of these defects .
a 54-year - old woman was brought to the emergency department by ems as a level ii trauma status after a motor vehicle crash .
she was the driver of a vehicle that was t - boned by a deer on the passenger side of the car , and she subsequently lost control of the vehicle .
she was unresponsive at the scene , and an attempt to intubate her in the field failed . she was ventilated with a bag - valve mask and later intubated in the emergency department . on arrival to the er , she was hemodynamically stable with 100% saturation .
her glasgow coma scale was 6 t . a plain chest film performed on arrival revealed elevation of the right hemidiaphragm with linear atelectasis at the right base ( figure 1 ) .
a computed tomographic ( ct ) scan of the chest and abdomen revealed right lower and middle lobe collapse and elevation of the right hemidiaphragm .
there was left lung base atelectasis , and a grade 3 liver laceration , but the great vessels were intact .
the patient also had a closed head injury with subarachnoid hemorrhage , diffuse axonal injury , and a frontal scalp hematoma .
her other injuries included fractures of her right clavicle , multiple bilateral ribs , right wrist , stable pelvic fracture , sacrum , and unstable fracture of c2 .
the patient was admitted to the icu , and her course was complicated by respiratory failure and pulmonary sepsis , which resolved with a tracheostomy and a prolonged course of antibiotics .
the patient was transferred to the surgical floor 20 days later , and after another 5 weeks of hospitalization she was discharged to a long - term rehabilitation facility .
initial chest x - ray reveals elevation of the right hemidiaphragm with linear atelectasis at the right base .
two days later , she was readmitted with shortness of breath , wheezing , and fever .
a chest x - ray showed a moderate - to - large right pleural effusion and opacity at the right lung base ( figure 2 ) . a ct scan of the chest showed right diaphragmatic hernia with herniation of bowel alongside the liver , right lower lobe atelectasis , and mediastinal shift to the left ( figure 3 ) .
bronchoscopy was performed and showed extrinsic compression and complete closure of the right lower lobe orifice and bronchi .
the patient was given a course of antibiotics , and after resolution of her pneumonia , surgery was performed .
chest x - ray on readmission shows a moderate right pleural effusion with opacity at the right lung base .
computed tomographic scan of the chest and abdomen showing right diaphragmatic hernia with stable right pleural effusion , right lower lobe atelectasis , mediastinal shift to the left , and herniation of bowel alongside the liver into the thorax . in the operating room
laparoscopy revealed a large diaphragmatic defect with herniation of the right lobe of the liver into the thoracic cavity ( figure 4 ) .
the liver was pulled back into the abdomen revealing a collapsed right lower lobe , some pleural fluid , and the edges of a large diaphragmatic defect ( figure 5 ) .
adhesions of the lower edge of the diaphragm were taken down , and the defect closed using interrupted figure - of - eight surgidac sutures with ptfe pledgets ( figure 6 ) .
herniation of the right lobe of the liver though the defect in the right diaphragm .
retraction of the liver reveals a collapsed right lung , pleural fluid , and a large defect .
a chest tube was inserted for drainage of the thorax , and the port sites were closed . the postoperative chest x - ray compared with the preoperative chest x - ray showed marked improvement in the borders of the right hemidiaphragm ( figure 7 )
. the patient 's postoperative course was complicated by a flare - up of respiratory infection , but she responded to antibiotics and was discharged to a long - term rehabilitation facility 2 weeks after the surgery .
diaphragmatic hernias ( dh ) can be categorized as occurring in the left or right diaphragm , being congenital or acquired , or having an early or late onset .
congenital diaphragmatic hernias ( cdh ) are well described and occur in 1/2000 to 1/5000 live births , with 30% to 50% diagnosed in utero .
left - sided cdhs are more common than are right - sided and bilateral ones ( 85% vs. 8% to 21% vs 2% ) , with a 1:1.25 male to female ratio .
left - sided cdh may cause severe respiratory distress , gastrointestinal symptoms , or both , in the newborn , whereas right - sided cdh may cause fewer symptoms .
traumatic dhs are mostly left - sided ( 70% to 80% ) vs. right - sided ( 15% to 24% ) vs. bilateral ( 8% ) penetrating injury results in a 5% incidence of diaphragmatic injury , which is usually diagnosed and treated by laparotomy .
laparoscopy is a useful diagnostic tool in low - velocity penetrating injuries to assess violation of peritoneum and to survey the diaphragms .
if not diagnosed promptly , even a small diaphragmatic injury may result in significant morbidity and require repair due to migration of organs aided by the combined push - pull mechanism of positive intraabdominal pressure and negative intrathoracic pressure .
in blunt injury , which includes road accidents and falls , 15% of hospitalized patients have dh .
the first one is immediate herniation of abdominal contents through a large rent in the diaphragm produced by transient but very significant elevation of intraabdominal pressure .
the second , more insidious pattern is a diaphragmatic defect without immediate herniation of intraabdominal contents .
this pattern is often initially overlooked in the acute setting and is only correctly diagnosed in 30% to 40% of cases .
the current practice of nonoperative treatment of solid organ injuries decreases the number of laparotomies in the acute trauma setting , and imaging is notoriously inaccurate in the diagnosis of diaphragmatic injuries .
the time period before onset of symptoms can be variable , sometimes measuring in years .
delayed dh of - ten presents as cardiorespiratory or gastrointestinal complaints , such as dyspnea , coughing , chest pain , abdominal pain , or vomiting .
late presentation of symptoms makes diagnosis even more difficult , and the misdiagnosis rate increases .
when a diaphragmatic hernia presents symptomatically , a 20% to 66% morbidity rate exists due to gastrointestinal obstruction , perforation , incarceration , or respiratory compromise .
other less common causes of dh can be violent coughing , obesity , heavy lifting , multiparity , and iatrogenic causes like esophagogastric surgery . our patient
presented with fever , tachycardia , and respiratory symptoms during her second admission to the hospital , due to the diaphragmatic hernia with simultaneous pneumonia . the inability to communicate with our patient added to the delay in diagnosis and treatment of her dh .
misinterpretation of radiological imaging can miss the diagnosis of dh . in cases of late - presenting cdh ,
baglaj et al report that pneumothorax and pleural effusion were the most common initial findings .
eren et al reviewed imaging of the diaphragm after trauma and suggest that plain radiography as a first - line modality , followed by ct scans , which are feasible in the trauma setting .
mri would be very helpful in visualizing the defect and should be used in patients with a high suspicion of dh .
plain chest film and ct scan of our patient on initial presentation after the motor vehicle crash , only revealed elevation of the right hemidiaphragm and right lower lung collapse ( figures 1 , 2 ) .
this reading perhaps may have been the correct diagnosis at that time , with gradual herniation later on .
interestingly , mris focusing on her spinal injuries were ordered later during hospitalization . in the literature regarding right - sided dh ,
there is controversy about whether right - sided cdh has increased mortality or any influence in the morbidity of the neonate .
the diagnosis of right - sided cdh is often delayed due to milder symptoms compared with symptoms accompanying left - sided cdh .
right - sided cdh can also be associated with a number of congenital defects and complications , and can present with other anomalies.therefore , it is important to recognize that such anomalies exist and perhaps can be present in longstanding right dh .
our patient did not have any anomalies between the lung and liver . however , takedown of adhesions within the diaphragm itself was necessary to free the crura . if this dh had been left unrepaired , it is possible that adhesions between lung , liver , bowel , and diaphragm may have formed .
close proximity of the abdominal contents to the infected lung put our patient at risk for spread of infection .
a recent review of the literature discussing cdh reports that mixed use of thoracoscopy and laparoscopy occurs .
both thoracoscopic and laparoscopic approaches to repair dh have advantages over open surgery in that they result in minimal trauma , promote early recovery , and decrease hospital stay .
thoracoscopy has been successful for repair of dh and may be easier due to good exposure in the thoracic cavity and has decreased the difficulty in reduction of herniated contents .
thoracoscopic repair of cdh in children and in select newborns could be the treatment of choice due to its safety and efficacy .
laparoscopy is a superior early diagnostic tool compared with thoracoscopy , because survey of the entire abdomen can be performed and subsequent repairs made , especially in the acute trauma setting .
laparoscopy provides better orientation and surgical exposure of both the abdominal and thoracic cavity ( through the defect ) , permitting survey for other injuries , whereas in thoracoscopy , only one hemidiaphragm can be inspected at a time .
moreover , laparoscopy allows for the repositioning of herniated contents more easily in the abdominal cavity .
another advantage of laparoscopy is that the patient is not required to undergo single - lung ventilation as needed in thoracoscopy .
a laparoscopic approach is best indicated in anteriorly or centrally located acute or chronic traumatic diaphragmatic hernia without concomitant severe abdominal or thoracic injury .
safety of laparoscopic repair of dh has been proven in many case reports but can be technically challenging .
therefore , surgical experience and meticulous technique are key to the success of laparoscopic repair .
use of a thoracoscopic or laparoscopic approach must be individualized for the patient 's clinical condition , presence or absence of other injuries , type and duration of hernia , and the surgeon 's experience with minimally invasive surgery .
given our patient 's history of blunt trauma , a laparoscopic approach to diaphragmatic repair was preferred over thoracoscopy . using laparoscopy , it was feasible to survey the rest of the abdomen , place the liver and bowel back into the abdominal cavity , and repair the diaphragmatic defect .
diagnosis of traumatic diaphragmatic hernias can be difficult in the acute setting and can present later with variable symptoms and radiological findings .
though right - sided diaphragmatic hernias are less common than left - sided ones , they occur more frequently than was once thought . plain films , ct imaging , and mri can help establish the diagnosis .
both thoracoscopy and laparoscopy are options to minimally invasive surgical approaches to repair , and the approach must be individualized by the clinical condition , presence of other injuries , type of hernia , and the skill level of the surgeon .
laparoscopy is a safe and successful approach to dh repair in the trauma patient , because further assessment and treatment of abdominal and thoracic injury can be facilitated . | background : traumatic diaphragmatic hernias are a diagnostic and therapeutic challenge due to variable presentations .
early repair is important because of risks of incarceration and strangulation of abdominal contents along with respiratory and cardiovascular compromise .
minimally invasive techniques have been useful for diagnosis and treatment of diaphragmatic hernias in both blunt and penetrating trauma.method:we present the case of a 54-year - old victim of a motor vehicle crash who presented with a delayed diagnosis of a right - sided traumatic diaphragmatic hernia . by using a 4-port technique and intracorporeal suturing ,
the hernia was repaired .
this case highlights the difficulties associated with diagnosing diaphragmatic hernias and the role of minimally invasive techniques to repair them.conclusion:minimally invasive surgical techniques are being increasingly used to both diagnose and repair traumatic diaphragmatic injuries with excellent results . |
a central database was created in august 2002 for two cohorts of participants in the maestro project : 1 ) a younger group ( aged 18 years , n = 84 ) who had the assistance of the navigator as they graduated from pediatric care and 2 ) an older group ( aged 1925 years , n = 64 ) who were transferred to adult care without this initial support .
they were later enrolled in the program , 17 years after graduation from pediatric care .
the maestro collected information from the participants by telephone every 6 months regarding medical or diabetes education visits , the presence of chronic complications , diabetes - related hospitalizations for diabetic ketoacidosis ( dka ) or severe hypoglycemia , and barriers to accessing care in the adult diabetes care system .
evaluation was completed from september to december 2004 for the year before the maestro project was created and for the year after the maestro project was in place .
sources of data included database review , audit of medical records , and a qualitative participant survey .
the number of medical and diabetes educator visits was available for the year before and year after referral to the maestro project for 101 of 164 patients ( 62% ) in the older group and 64 of 84 patients ( 76% ) in the younger group .
as seen in table 1 , the systems navigator model helped the older group reconnect with adult medical services and helped the younger group reduce their first year fall - out rate after transfer from pediatric to adult care . prior to intervention from the navigator , over 40% of
this fall - out rate was lower in the younger group , who had access to the navigator at the time of transfer from pediatric care ; only 11% of this younger group dropped out of adult medical care completely after transfer .
the older group reported pregnancy loss of 38% ; 1 case each of heart failure , legal blindness , and amputation ; 4 cases of proliferative diabetic retinopathy ; 4 deaths ( 2 from dka and 2 unrelated to diabetes ) .
both groups reported acute complications requiring visits to the emergency room or admission to a hospital .
for dka , there were 3.0 cases per 100 patient - years in the older group versus 7.9 cases per 100 patient - years in the younger group ( ns ) .
for severe hypoglycemia , there were 2.4 cases per 100 patient - years in the older group versus 4.7 cases per 100 patient - years in the younger group ( ns ) .
of the participants , 33% of the younger group and 42% of the older group experienced difficulties or frustrations with establishing regular follow - up with their adult health care team after transfer from pediatric care .
reasons for dropping out of care included difficulty scheduling appointments and knowing who to see , difficulty establishing relationships with the new team , feeling overwhelmed and lost in the system , and a lack of perceived value of adult care .
of the participants , 100% of the older group and 78% of the younger group felt that there was a need for the maestro project .
the maestro project had facilitated 374 referrals for 168 participants to endocrinologists , diabetes educators , mental health professionals , and eye specialists , and 198 individual participants had contacted the maestro project 515 times for information .
at the time of transfer from pediatric to adult care , many young adults are unable to independently navigate the adult diabetes care system .
the young adults who are enrolled in a systems navigator and support program before age 18 years are better connected to the adult health care system than those who graduated before the program existed . in the first 2 years of the maestro project , there was improved medical surveillance but no evidence of improved short - term medical outcomes .
this may be due to specific characteristics of this young adult cohort , specific characteristics of service delivery in the adult care system , or inadequate development or time for optimum evaluation of this model . | objectives to determine whether a systems navigator service , the maestro project , could increase medical surveillance for young adults with type 1 diabetes who transfer from pediatric to adult care.research design and methods there were two cohorts of participants : 1 ) a younger group ( aged 18 years , n = 82 ) who had the assistance of the navigator as they graduated from pediatric care and 2 ) an older group ( aged 1925 years ) who were transferred to adult care without this initial support but later enrolled in the program.results of the older group ( who did not have initial access to the navigator ) , 40% dropped out of adult medical care , compared with a dropout rate of 11% for the younger group , who had access to the navigator at the time of transfer from pediatric care.conclusions the systems navigator helped improve medical surveillance for both groups , although there was no evidence of improved short - term medical outcomes . |
immunization is one of the most cost - effective public health interventions and is largely responsible for the reduction of under-5 mortality rate .
vaccine preventable diseases have declined markedly worldwide since the introduction of the routine immunization program in 1978 , which was later upgraded in 1985 as the universal immunization program ( uip ) .
cutaneous side effects include local reactions like erythema , induration with or without tenderness , subcutaneous nodules , and hypertrichosis at the site of injection . rarely erythema multiforme has also been reported .
we hereby report a rare case of focal hypertrichosis in an infant following routine immunization with the pentavalent vaccine .
a 4-month - old healthy male infant presented with focal areas of increased hair growth over bilateral thighs .
the child was born out of a nonconsanguineous marriage , full - term normal vaginal delivery .
gurin ( bcg ) vaccination at birth and combination vaccine at 6 weeks and 10 weeks of age which included diphtheria , pertussis and tetanus toxoid , hepatitis b , and hemophilus influenza type b ( dtpw - hepb - hib ) . mother gave a history of localized swelling at the site of injection which was later followed by increased hair growth at the injection site bilaterally after about 2 weeks of injection .
there was no history of local trauma , topical applications , or any other oral or injectable drug administered to the child . on examination
, there were focal areas of hypertrichosis ( pigmented hair ) about 3 cm 2 cm localized over a bilateral anterolateral aspect of thighs [ figures 1 and 2 ] .
localized area of pigmented hair over anterolateral aspect of left thigh at the site of vaccination localized area of pigmented hair over anterolateral aspect of right thigh in an infant
hypertrichosis is characterized by excessive hair growth that is abnormal for the age , sex , or race of an individual , or for a particular area of the body . it can be congenital or acquired , generalized , or localized .
acquired localized hypertrichosis ( alh ) has been described as a separate entity with various underlying factors .
it has been reported to result from tattoos , sclerotherapy , and subsiding psoriasis , respectively .
it can present in an irregular pattern on the legs in chronic venous insufficiency , around the edges of a burn , at the site of insect bites , and after radical inguinal lymphadenectomy .
contact eczema , pretibial myxedema , arthritis , occupational , or self - inflicted trauma have also been associated with this phenomenon .
rarely , alh has been observed after vaccination including measles and bcg vaccination . the exact mechanism underlying this phenomenon is unknown , but it has been suggested that prolonged exposure to antigen results in the production of various cytokines by the immune system .
these cytokines can also affect nonimmune system cells such as those in the hair follicles and promote localized hair growth .
pentavalent vaccine given for routine immunization in children contains adjuvant like aluminium salts ( aluminium hydroxide and aluminium phosphate ) .
persistent tender or itchy subcutaneous nodules and granulomas have been previously reported with these agents .
the classic histopathological appearance of these injection site reactions consists of a nodular inflammatory infiltrate with lymphoid follicles within the deep dermis and subcutaneous tissue with large collections of macrophages and eosinophilic infiltrate .
finely granular refractile material can be found within macrophages , and the diagnosis can be confirmed by electron probe microanalysis .
the development of focal hypertrichosis following pentavalent vaccine has not been described previously in indian literature .
though it is a benign side effect , the authors here want to throw some light on this rare and less known side effect of pentavalent vaccine , which is routinely given to infants as per the uip .
further research might help in revealing the exact mechanism for localized hypertrichosis at the injection site following pentavalent vaccine .
| hypertrichosis is characterized by increased hair growth independent of androgen excess .
there are various causes of acquired localized hypertrichosis including chronic irritation and inflammation .
localised hypertrichosis following pentavalent vaccine has not been described previously in indian literature .
we hereby report a case of a 4-month - old healthy infant presenting with focal circumscribed areas of hypertrichosis over anterolateral aspect of bilateral thighs at the site of vaccination . |
according to the report of who , approximately 450 million people suffer from mental or behavioral disorders like stress .
this amounts to 12.3% of the global burden of disease and is predicted to rise up to 15% by 2020 .
it is estimated that 7590% of visits to primary care physicians are related to stress either acutely or because of chronic problems associated with stress .
an october 2008 american psychological association ( apa ) press release on stress in america claimed that 8 of 10 americans cite the economy as a significant source of stress , up from 66 percent six months earlier . in june 2008 , more people were reporting symptoms associated with stress compared to the previous year , with nearly half polled indicating that stress had increased in the past years .
stress is nonspecific response of the body known to alter the physiological homeostasis of the organism resulting in various neuronal , endocrine , and visceral dysfunctions .
the ability to develop and maintain resistance against a variety of stressors encountered in human life is crucial for survival .
stress is one of the basic factors which cause a number of diseases such as atherosclerosis , coronary heart disease , aging , and liver disease .
the desire to control the coping mechanisms has led to the origin of the science of adaptation .
physiologists define stress as how the body reacts to a stressor , real or imagined , a stimulus that causes stress .
acute stressors affect an organism in the short term ; chronic stressors over the longer term selye researched the effects of stress .
if exhaustion is extended , long term damage may result as the body and the immune system are exhausted and function is impaired resulting in decompensation .
the result can manifest itself in obvious illnesses such as ulcers , depression , diabetes , and trouble with the digestive system or even cardiovascular problems , along with other mental illnesses .
adaptogens are substances that help organisms to adapt to unfavorable stressful conditions , which could be physical , chemical , biological , or mental conditions .
some pioneer researchers in this field put forth specific criteria that need to be fulfilled to qualify as an adaptogens , which include ability to produce a nonspecific response ( i.e. , increases resistance against multiple physical , chemical , or biological stressors ) ; it brings any dysfunctioning body system back into balance and must be innocuous , that is , must not influence normal body functions more than required .
however , most researches on adaptogens have focused on natural substances ( specifically plants ) , and the term phytoadaptogens is now commonly used for adaptogens of plant origin .
the ayurvedic plant pandanus odoratissimus linn belonging to the family pandanaceae pandanus comprises 500600 species and is distributed mainly in subtropical and tropical regions ; out of these , 36 species have been recorded in india .
it is widely distributed in india over coastal districts of orissa ( especially in ganjam ) , andhra pradesh , tamil nadu , and to some extent in parts of uttar pradesh .
preliminary phytochemical analysis positive tests for alkaloids , steroids , carbohydrates , phenolic compounds , glycosides , proteins and amino acids , and so forth .
pandanus odoratissimus contain the active chemical constituents which serve the medicinal properties : 2-phenyl ethyl alcohol , 2-phenyl ethyl methyl ether , terpinen-4-ol , 3-hydroxy-2-isopropenyl - dihydrobenzofuran-5-carboxylic acid methyl ester , 3-methyl-3-buten-1yl acetate , 3-methyl-3 - 1 yl - cinnamate , 3-methyl-3-buten-1yl - acetate-3-methyl-2-buten-1-yl cinnamate , 3,4-bis(4 hydroxy-3-methoxy benzyl ) tetrahydrofuran , -terpineol , -carotene , -sitosterol , benzyl - benzoate , pinoresinol , germacrene b , vitamin c , vinidine , tangerine , 5,8-hydroxy-7-methoxy - flavon .
the leaves contain the pyridine alkaloids , pandamarilactone-1 ( c18h23no4 ) , pandamarilactone-31 ( c19h25no4 ) , and pandamarilactone-32 ( c18h21no3 ) .
lignans andbenzofurans have been isolated from roots of pandanus odoratissimus [ 6 , 7 ] .
the evaluation of these plants and of their active natural principles is a logic way of searching for new drugs to treat this disease .
pandanus odoratissimus is said to be restorative , indolent , promoting a feeling of well - being , and acting as a counter to tropical climates .
ayurvedic science has found the medicinal action of essential oil yielded by the screw pine 's highly scented flowers to be useful in headaches and earaches and as a liniment for rheumatic pains .
it may be chewed as a breath sweetener or used as a preservative in foods .
it is also believed to have health - related properties , including antiviral , antiallergy , antiplatelet , anti - inflammatory , antioxidant , and anticancer action .
pandanus odoratissimus is said to be restorative , indolent , promoting a feeling of well - being , and acting as a counter to tropical climates .
the leaves of the plant have been mentioned valuable in leprosy , scabies , leucoderma , cephalalgia , coxalgia and otalgia , wounds , ulcers and colic , antioxidants .
the oil of the male flowers is considered stimulant and antispasmodic and is administered for headache and rheumatism .
fresh kewda ( pandanus odoratissimus linn ) medicinal plants were collected from the surroundings of lonavala hill station , pune , maharashtra ( india ) , in april 2009 ( figure 1 ) and authenticated by the botanical survey of india , pune , maharashtra , india ( voucher number mim_ppa-18 ) .
selected plant was powdered and material was separately weighed and used for extraction by the basis of polarity of solvent [ 6 , 1114 ] .
the concurrent extraction ( maceration ) of powdered plant material ( 1 kg ) was soaked in 2.5 litres of ( 99.9% ethanol + distilled water ) ( 60 : 40 ) hydroethanol for 7 days at room temperature .
after 7 days , the hydroethanol soluble materials of both plants were filtered off separately [ 10 , 15 ] .
aqueous extract was freezed under 70c for three days and then lyophilized . 1 g of lyophilized pandanus odoratissimus linn ( lepo ) is equivalent to 2.01 g of dried pandanus odoratissimus linn hypocotyls .
lyophilized plant extract was diluted in distilled water to obtain a dose of 1 g / kg b.w .
this dose has been proved to be optimal in a dose - dependent study [ 11 , 12 ] .
albino mice ( swiss strain ) of either sex weighing between 18 and 25 g were procured from central animal house serum institute , hadapsar , pune , for experimental purpose . after procuring
, the animals were acclimatized for seven days under standard husbandry condition with 12 : 12 hours light or dark cycle .
healthy swiss albino mice the chosen experimental animals were maintained in our animal house ( 12 : 12 dark : light cycle ) , with adequate ventilation and hygienic conditions , maintained with normal pelleted diet ( nutrivate life science , sinhagad road , pune , baramati agro ltd .
a group of animals were housed in polypropylene cage , paddy husk bed covered with stainless steel wire mesh with provision for water and feed . after obtaining prior permission from institutional animal ethical committee ( iaec ) protocol approval number jscopr / iaec/02/2008 - 09
drugs and chemicals used were as follows : marketed sample of withania somnifera ; ashwagandha , 60 caplets himalaya herbal healthcare , bangalore , india;marketed sample of pandanus odoratissimus ; kewra water ( kewda water ) , top - op ; spice of india , india . marketed sample of withania somnifera ; ashwagandha , 60 caplets himalaya herbal healthcare , bangalore , india ; marketed sample of pandanus odoratissimus ; kewra water ( kewda water ) , top - op ; spice of india , india .
acute oral toxicity study for the proprietary formulation was carried out using oecd guideline-425 ( modified , adopted march 23 , 2006 ) , the sequential test that uses a maximum of five animals . a test dose of 2000 or exceptionally 5000 mg / kg may be used in situation where experiment has information indicating that the test material is likely to be nontoxic .
the test procedure minimizes the number of animals required to estimate the oral acute toxicity of a chemical and in addition estimation of ld50 , confidence intervals .
the test also allows the observation of signs of toxicity and can also be used to identify chemicals that are likely to have low toxicity [ 11 , 12 ] .
as suggested , after acclimatization of animals for 4 - 5 days , study was carried out as follows .
healthy , young adult albino swiss mice ( 1825 gm ) , nulliparous , and nonpregnant were used for this study .
food , but not water , was withheld for 3 - 4 hours and further 1 - 2 hours after administration of sample under study .
since this first test animal survived , four other animals were dosed ( orally ) at subsequent days , so that a total of five animals were tested .
animals were observed individually at least every 5 minutes once during first 30 minutes after dosing , periodically at 2 hrs during the first 24 hours ( with special attention during the first four hours ) , and daily thereafter , for a total of 14 days ( table 1 ) .
swiss albino mice ( 1825 g ) were for forced swim endurance stress test .
group ii was taken as a standard and was with treated with ws ( 100 mg / kg p.o ) .
( po ) ( 1 ml / kg ) and groups iv and v were taken as test groups and treated with ( 100 mg / kg and 200 mg / kg p.o ) of the lepo , respectively , for the all models , namely , forced swimming endurance stress test , anoxic stress tolerance test , and chronic cold resistant stress test .
swiss albino mice ( 1825 g ) were used for forced for forced swim endurance stress test .
the animals were divided in to 5 groups and all were treated as mentioned above in scheduled drug treatments .
on the 8th day the mice were subjected to swimming stress by keeping them in polypropylene tank of dimension 37 37 30 cm filled with water to a height 25 cm .
mice were allowed to swim till complete exhaustion and the end point was taken when animal starting drowning .
the blood was collected through retroorbital puncture to estimate biochemical parameters like serum glucose and corticosterone and blood cells [ 1 , 16 ] .
swiss albino mice ( 1825 g ) were used for forced for anoxic stress tolerancetest .
the animals were divided into 5 groups and all were treated as mentioned above in scheduled drug treatments .
each animal was kept in the airtight vessel and time was noted using a stopwatch .
the moment an animal showed first convulsion , it was removed immediately from the vessel and resuscitated if needed .
the time duration from the entry of the animal in the hermetic ( conical flask ) vessel to the appearance of the first convulsion was taken as the time of anoxic stress tolerance . the mean time to convulsion was recorded and animal was removed at onset of convulsion .
the blood was collected through retroorbital puncture to estimate biochemical parameters like serum glucose and corticosterone and blood cells [ 1 , 17 ] .
swiss albino mice ( 1825 g ) were used for forced for chronic cold resistant stresstest .
the animals were divided into 5 groups and all were treated as mentioned above in scheduled drug treatments .
in each animals were subjected to cold stress by exposing them to 4 10c daily for 2 hours .
blood was collected through orbital plexus under light ether anesthesia to estimate biochemical parameters like blood glucose , corticosteroid , and blood cell count ( rbc and dlc ) .
result was analyzed statistically by using one - way anova followed by dunnett 's multiple comparison test against the respective control .
in swimming test model , seven day treatment increased swimming time to 125 , 133 , 145 by test drug and 416 by withania sominifera as compare to control group 113 sec .
test groups iii , iv , and v swam for 125 , 133 , and 145 sec , respectively .
when swimming time of groups iii , iv , and v was compared with group i , it showed significant ( p < 0.01 ) rise in the swimming time .
200 mg / kg lepo treated animals showed reading very close to 100 mg / kg of ws treated animals .
consequently higher dose of lepo is more potent with respect to lower dose of extract .
both the doses were able to increase the swimming endurance when compared with animals who received normal saline water ( table 2 ) . on 8th day level of glucose in serum
was found to be in control group ( i ) 141.6 1.6 . in the standard group which is treated with ws
the serum glucose level was found to be 98.3 4.4 mg / dl , and the test control groups ( iii , iv and v ) treated with po , lepo ( 100 mg / kg ) and lepo ( 200 mg / kg ) serum glucose level were showed the 105 2.8 , 110 1.0 and 131.6 1.6 simultaneously .
the level of glucose was increased during the time of stress but it came down due to pretreatment of lepo . the level of corticosterone in serum ( on 8th day )
was recorded as 140 mg / dl for control group for control group - i , who received by saline water . and
the group - ii treated with ws ( withania somnifera ) was found to have the 107.3 6.3 mg / dl level of corticosterone in serum .
groups iii , iv , and v which were treated with marketed preparation of po ( kewda water ) , lepo 100 , and 200 mg.kg , respectively , demonstrate the corticosterone level in plasma 123.6 1.6 , 126.6 3.3 , 126.6 1.6 mg / dl , respectively ; marketed sample of po ( kewda water ) showed significant decrease in the level of corticosterone ( table 4 ) . on 8th day
all animal groups hematological parameters were checked the group - i , served as normal control treated with saline water , group - ii , served as slandered control treated with ws , in the rbc count parameter were found as 9.3 and 7.53 million / cumm simultaneously .
the rbc count of groups iii , iv , and v was found to be 8.53 , 7.56 , and 7.93 million / cumm with respect to group i in which it reduced due to pretreatment of extracts .
the neutrophils count of animals of groups i , ii , iii , and iv was as found to be 13.3 , 10.3 , 11.29 , 12.3 , and 12.3% , respectively .
the neutrophils count of groups ii , iii , and iv reduced due to pretreatment of extracts with respect to group i. the lymphocytes count of animals of groups i , ii , iii , iv , and v was found as 59.66 , 55.33 , 56.98 , 59.6 , and 58.33% , respectively .
the lymphocyte count of groups iii and iv reduced due to pretreatment of extracts with respect to group i. the eosinophil count of animals of group i , ii , iii , iv , and v was found as 3.3 , 1.4 , 2.1 , 2.6 , and 1.5 , respectively .
the eosinophil count of groups iii and iv reduced due to pretreatment of extracts with respect to group i. the monocyte count of animals of groups i , ii , iii , and iv was found as 3 , 1 , 1.6 , 1.3 , and 1.3% , respectively .
the monocyte count of groups iv and v reduced due to pretreatment of extracts with respect to group i. consequently forced swimming endurance test significantly altered the hematological parameters , that is , decreased rbc and dlc counts .
pretreatment with ws and lepo significantly ( p < 0.01 ) inhibited the stress induced changes in these parameters . on the other hand these values also express that higher dose of lepo was very effective , but both the doses of test drug had shown significant adaptogenic activity ( table 3 ) . in anorexia stress tolerance test group
i ( control ) treated with saline water ( 1 ml ) was kept in hermetic vessel to for 18.33 min .
group ii taken as a standard treated with withania somnifera survive in hermetic vessel for 69 min ; groups iii , iv , and v animals survive in hermetic vessel 41 , 33 , and 28 min , respectively .
the anoxia tolerance test was determined by taking the appearance of convulsion as end point .
the lepo at two doses ( 100 and 200 mg / kg ) and po ( kewada water ) at dose ( 1 ml ) showed significant ( p < 0.01 ) increasing tolerance stress time in 8th day as compared with the control ( table 5 ) . on 8th day level of glucose in serum control group ( i )
was found to be 120.33 ; in the standard group which is treated with ws the serum glucose level it was found to be 81.6 1.6 mg / dl . in the test drug groups ( iii , iv , and v ) , 95 2.8 , 111.6 1.6 , and 101.33 0.6 , the level of glucose was increased during the time of stress but it came down due to pretreatment of lepo . the level of corticosterone in serum ( on 8th day )
was recorded as 141 1.6 mg / dl for control group who served by saline water ( 1 ml ) .
group - ii , treated with ws , and corticosterone level were found as the 85 2.8 mg / dl level of corticosterone in serum .
groups iii , iv , and v which were treated with marketed preparation of po ( kewda water ) and lepo 100 and 200 mg / kg , respectively , demonstrate the corticosterone level in plasma 103.3 1.6 , 118.3 4.4 , and 110 5.7 mg / dl , respectively .
200 mg / kg of lepo and marketed sample of pandanus odoratissimus linn ( po ; kewda water ) showed significant decrease in the level of corticosterone ( table 7 ) . on 8th day rbc count of animals of group
i treated as control , group ii treated as a standard , group iii for marketed preparation po ( kewda water ) , and groups iv and v treated with lepo was found as 10.3 , 7.4 , 7.5 , 7.6 , and 7.6 million / cumm , respectively .
the rbc count of groups iii , iv , and v with respect to group i reduced due to pretreatment of extracts .
the neutrophils count of animals of groups i , ii , iii , and iv was found as 16 , 10.3 , 10.6 , 11.05 , and 11.3% , respectively .
the neutrophils count of groups iii and v reduced due to pretreatment of marketed sample and extracts with respect to group i. the lymphocytes count of animals of groups i , ii , iii , and iv was found as 66 , 50.6 , 53.6 , 58 , and 54.6% , respectively .
the lymphocyte count of groups iii , iv , and v reduced due to pretreatment of marketed sample and extracts with respect to group i. the eosinophil count of animals of groups i , ii , iii , iv , and v was found as 3 , 0.6 , 1 , 1.3 , and 1.3 , respectively .
the eosinophil count of groups ii , iii , and iv reduced due to pretreatment of po ( kewda water ) and extracts with respect to group i. the monocyte count of animals of groups i , ii , iii , and iv was found as 3.3 , 1.3 , 1.3 , 1.6 , and 1.6% , respectively .
the monocyte count of groups ii , iii , and iv reduced due to pretreatment of extracts with respect to group i. consequently anoxia stress tolerance test significantly altered the hematological parameters , that is , increased rbc and dlc counts .
pretreatment with marketed sample and extracts significantly ( p < 0.01 ) inhibited the stress induced changes in these parameters . on the other hand , these values also express that higher dose of lepo was very effective , but both the doses of test drug had shown significant adaptogenic activity ( table 6 ) . on 8th day level of glucose in serum control group ( i ) was found to be 116 1.6 . in the standard group which is treated with ws
the serum glucose level was found to be 70 3.1 mg / dl . in the test
drug groups iii , iv , and v , 101.6 1.6 , 112.3 1.4 , 116.6 3.3 , the level of glucose was increased during the time of stress but it came down due to pretreatment of po ( kewda water ) and lepo .
the level of corticosterone in serum ( on 8th day ) was recorded as 143.3 1.6 mg / dl for control group i saline water ( 1 ml ) .
group ii treated with ws was found to have the 104.6 2.4 mg / dl level of corticosterone in serum .
groups iii , iv , and v which were treated with marketed preparation po ( kewda water ) and lepo 100 and 200 mgkg , respectively , demonstrate the corticosterone level in plasma 110 5.7 , 118 1.6 , and 113.3 6.6 ng / dl , respectively . marketed sample and lepo showed significant decrease in the level of corticosterone ( table 9 ) . on 8th day in group
i treated as control and group ii treated as a standard with ws , the rbc count for animals was found as 9.6 and 7.4 million / cumm .
the rbc count of groups iii , iv , and iv was found to be 7.5 , 7.9 , and 7.7 million / cumm with respect to group i in which it reduced due to pretreatment of marketed sample and extracts .
the neutrophils count of animals of groups i , ii , iii , and iv was found as 13.3 , 10.3 , 10.6 , 11.3 , and 11.6% , respectively . the neutrophils count of groups ii , iii , and iv reduced due to pretreatment of marketed sample and extracts with respect to group i. the lymphocytes count of animals of groups i , ii
, iii , iv , and v was found as 61.6 , 52 , 52.3 , 56 , and 55% , respectively .
the lymphocyte count of groups iii and iv reduced due to pretreatment of extracts with respect to group i. the eosinophil count of animals of groups i , ii , iii , iv , and v was found as 3.3 , 1.3 , 1.6 , 1.6 , and 1.3 , respectively .
the eosinophil count of groups iii and iv reduced due to pretreatment of extracts with respect to group i. the monocyte count of animals of groups i , ii , iii , and iv was found as 2.3 , 1 , 1.3 , 1.3 , and 1% , respectively .
the monocyte count of groups iv and v reduced due to pr - treatment of po ( kewda water ) and lepo with respect to group i. consequently cold stress test significantly altered the hematological parameters , that is , decreased rbc and dlc counts . pretreatment with ws and lepo significantly ( p < 0.01 ) inhibited the stress induced changes in these parameters . on the other hand
these values also express that higher dose of lepo was very effective , but both the doses of test drug had shown significant adaptogenic activity ( table 8) .
stress alters the equilibrium of various hormones which have a significant impact on the immune response in general . the status of immune system immunosuppression versus immunopotentiation will depend upon the net effect of these changes .
plant adaptogens are smooth prostressor which reduces the reactivity of host defense system and decreases the damaging effect of various stressors due to increase of basal level mediators involved in the stress response .
the forced swimming is the most widely used method for assessing the antistress property of a compound .
this paradigm is based on the observation that animal when forced to swim in water eventually assumed characteristics immobile posture , devoid of any activity .
the appearance of immobility , therefore , reflects a state of tiredness , fatigue , and reduced stamina with the end point being the moment when the mice could not swim further and started drowning [ 3 , 18 ] .
the increased swimming time has been observed in mice pretreated with pandanus odoratissimus with enhanced physical performances significantly longer than untreated ( control ) group and thus confirmed selected indian medicinal plants having the adaptogenic nature . during stress , blood glucose level increases which are found to be significantly reduced in pandanus odoratissimus treated mice .
lowering of stress induced hyperglycemia is an indication of antistress , adaptogenic activity of plant . in response to stress , acth is released , which acts on the adrenal cortex to stimulate the synthesis and release of cortisol . increased plasma cortisol influences the mobilization of stored fat and carbohydrate reserves which in turn increased blood glucose level .
stressed animals spleen contracts and releases the more amounts of blood cells ( rbc 's and wbc 's ) into circulations .
( this stage in acute stress phenomenon ) [ 1 , 17 ] . the stress induced rbc and wbc count is decreased by the lepo . increased swim duration and decreased ( this stage in chronic stress phenomenon ) in rbc and wbc count in mice pretreated with pandanus odoratissimus are similar to the changes produced by reference drug withania somnifera .
increase in adaptation during this stress by any drug could be considered as its major antistress effect .
the result of the study demonstrates that the pandanus odoratissimus extract significantly prolonged the meantime to convulsion , which therefore confirms its antistress property .
prolongation of meantime to convulsion could be a result of its powerful antioxidant and free radical scavenging activities [ 3 , 18 ] , the mechanism by which stress rises which enhances the activity of hypothalamo - hypophyseal axis ( hpa ) resulting in liberation of catecholamine and corticosteroids .
adrenaline in turn stimulates 2 receptor on the pituitary gland causing greater release of acth , which can stimulate the adrenal medulla as well as cortex .
spleen constricts to release more blood cells ( rbc and wbc ) during stress , so their weights decrease in stress .
this stress induced changes were significantly reversed by the test extract at lower and higher doses .
response to stress is highly contradictory with regard to blood sugar level . a significant hyperglycemia in cold restraint stress model was observed . under stressful condition corticosterone in mice
hyper secretion of corticosterone helps the maintenance of internal homeostasis through the process of gluconeogenesis .
the treatment with the lyophilized hydroethanol extract of leaves of pandanus odoratissimus significantly reduced the cold stress related hyperglycemias .
the re - ducting the hyperactivity of hormonal secretions of adrenal cortex and it 's beneficial to maintain homeostatic mechanism .
pretreatment with lyophilized hydroethanol extract of leaves of pandanus odoratissimus ( lepo ) and reference drug withania somnifera ( ws ) reduced the hematological parameters significantly .
lepo at dose of 100 and 200 mg / kg and the marketed sample have shown significantly that they possess the ability to prevent alteration due to stress .
the above results are promising and further dose variant studies would be helpful in substantiating the claim of adaptogenic action of this drug . thus with all above results it can be claimed and concluded that the marketed and the lyophilized hydroethanol extract of leaves of pandanus odoratissimus linn ( lepo ) have adaptogenic property . | background . the leaves of pandanus odoratissimus linn have been widely used in ayurveda to treat a variety of common and stress related disorders . in the present investigation , hydroethanol extract of leaves of pandanus odoratissimus linn ( lepo ) were evaluated for antistress activity in normal and stress induced mice .
furthermore , the extract was studied for nootropic ( adaptogenic ) activity in mice and in vitro antioxidant potential to correlate with its adaptogenic and antistress activity .
lepo ( 100 and 200 mg / kg p.o ) was evaluated against forced swimming endurance stress test , anoxia stress tolerance and immobilization stress and chronic cold resistant stress tests , and biomarkers ( serum glucose , corticosterone , wbc , rbc , and dlc count ) to assess the antistress activity in mice .
withania somnifera ( ws ) ( 100 mg / kg p.o ) was selected as reference standard .
the parameters like anoxia stress tolerance time were recorded in anoxia stress and estimation of biochemical marker levels and determination of organs weight were carried out in immobilization stress models . results .
concomitant treatment with lepo 200 mg / kg significantly increased in anoxia stress tolerance time .
dose dependent significant reduction in serum glucose , corticosterone , and wbc , rbc , and dlc was observed in immobilisation stress model as compared to stressed group .
leop 200 mg / kg and ws 100 mg / kg significantly reversed / inhibited the stress induced changes in these parameters .
the results from the present study indicate that these values also express that dose dependent significant adaptogenic activity in stressed animals . conclusion .
the present study provides scientific support for the antistress ( adaptogenic ) and nootropic activities of lyophilized hydroethanol extract of pandanus odoratissimus linn and substantiate the traditional claims for the usage of pandanus in stress induced disorders . |
a shared understanding of a patient s status between physicians , the patient , and caregivers is important in shared decision - making . not only can it reduce the discrepancy in expectations regarding treatment objectives
precise awareness of disease status , i.e. , shared understanding of cancer stages between cancer patients and their caregivers , is essential for shared decision - making .
discrimination between advanced and terminal cancer is important because the treatment goals are different . in cases of advanced cancer , which is not curable but
responds to treatment , disease - directed therapy is still very important because it prolongs life .
however , for terminal cancer , therapy can not prolong survival significantly due to the progressive nature of the disease [ 2 - 5 ] .
therefore , palliative care that provides psychosocial support and controls symptoms is the main treatment option for patients with terminal cancer .
however , studies suggest that patients and their families often misperceive the patient s cancer stage and prognosis [ 9 - 11 ] , leading to unrealistic expectations regarding the treatment effect and inappropriate treatment decisions [ 9,12 - 14 ] . in addition , many end - of - life care studies are based on the assumption that there is a shared definition of language regarding the cancer stage among patients , caregivers , the general population , and physicians . for example , yun et al
. assessed patients awareness of terminal illness soon after they were considered terminal by their doctor with the following question : do you know your disease stage ? response options included ,
advanced stage , terminal stage , and other . the authors regarded
terminal stage as the correct awareness of the terminal nature of the illness . without a shared understanding regarding the definition of terminal stage cancer of physicians and respondents , the validity of such research could be compromised .
therefore , the lack of a common understanding of words used in cancer care is a critical problem in communication within the clinical practice and the research environment .
however , few studies evaluating public awareness regarding the definition of cancer stages have been reported . in a study conducted in korea , lee et al .
6-month life expectancy and treatment refractoriness were regarded as the correct answers . in that study ,
29.4% of the participants from the general population misunderstood locally advanced cancer and metastatic / recurrent disease as terminal stage disease .
however , this study was limited in that the response options were not mutually exclusive , and the authors asked for a conceptual definition rather than using an actual situation . in this study ,
a survey was conducted to evaluate the understanding of the definition of advanced versus terminal cancer in the general population and determined associated socio - demographic factors .
in 2012 , the survey examined members of the general population to explore their views on cancer and cancer care .
a nationwide home visiting survey was conducted from november 1 , 2012 to december 1 , 2012 .
after stratification by the region , samples were systematically extracted according to the population ratio .
inclusion criteria were as follows : ( 1 ) a member of the general population aged 40 years or older and younger than 70 years and ( 2 ) a member of the general population who was never diagnosed with any type of cancer .
this study was approved by the institutional review board of the national cancer center . to assess the understanding of the definition of advanced versus terminal cancer in the general population
, we developed a clinical vignette of an advanced breast cancer patient with lung metastases , who is however not considered to have a terminal stage cancer , as defined in terms of medical context .
the detailed patient history of the vignette is as follows : a hypothetical patient ( mrs .
kim ) was diagnosed with breast cancer 4 years ago and received chemotherapy due to lung metastases detected 1 year ago . while chemotherapy was effective during the first round of treatment , the tumor subsequently continued to grow .
her doctor said that complete remission was impossible , but prolonged survival could be achieved by tumor size reduction using chemotherapy combined with another regimen . after presenting the hypothetical case of mrs .
respondents could choose one of the following stages : ( 1 ) early , ( 2 ) advanced , ( 3 ) terminal stage , and ( 4 ) do nt know .
multinomial logistic regression was performed to determine related factors for respondents answers . for selection of covariates for adjustment in the multinomial logistic regression model , univariate analysis
was performed for each of the socio - demographic characteristics and answered stages . characteristics with p - values less than 0.1
all statistical analyses were performed using the stata software ver . 13.0 ( statacorp . ,
college station , tx ) ; p - values less than 0.05 were considered statistically significant .
in 2012 , the survey examined members of the general population to explore their views on cancer and cancer care .
a nationwide home visiting survey was conducted from november 1 , 2012 to december 1 , 2012 .
after stratification by the region , samples were systematically extracted according to the population ratio .
inclusion criteria were as follows : ( 1 ) a member of the general population aged 40 years or older and younger than 70 years and ( 2 ) a member of the general population who was never diagnosed with any type of cancer .
to assess the understanding of the definition of advanced versus terminal cancer in the general population , we developed a clinical vignette of an advanced breast cancer patient with lung metastases , who is however not considered to have a terminal stage cancer , as defined in terms of medical context .
the detailed patient history of the vignette is as follows : a hypothetical patient ( mrs .
kim ) was diagnosed with breast cancer 4 years ago and received chemotherapy due to lung metastases detected 1 year ago . while chemotherapy was effective during the first round of treatment , the tumor subsequently continued to grow .
her doctor said that complete remission was impossible , but prolonged survival could be achieved by tumor size reduction using chemotherapy combined with another regimen . after presenting the hypothetical case of mrs .
respondents could choose one of the following stages : ( 1 ) early , ( 2 ) advanced , ( 3 ) terminal stage , and ( 4 ) do nt know .
multinomial logistic regression was performed to determine related factors for respondents answers . for selection of covariates for adjustment in the multinomial logistic regression model , univariate analysis
was performed for each of the socio - demographic characteristics and answered stages . characteristics with p - values less than 0.1
, college station , tx ) ; p - values less than 0.05 were considered statistically significant .
a total of 2,000 persons ( response rate , 41.2% ) responded to the survey questions .
1,677 respondents ( 70.2% ) graduated high school or above and 1,672 respondents ( 83.6% ) earned more than 2 million korean won per month .
eight hundred thirty - nine participants ( 42.0% ) had relatives or acquaintances diagnosed with cancer .
terminal stage as the stage of the hypothetical case ( n=1,489 , 74.5% ) .
a small proportion of respondents chose early stage ( n=13 , 0.7% ) or do nt know ( n=87 , 4.4% ) . in univariate analysis , marital status ( p=0.019 ) , educational status
( p < 0.001 ) , and income ( p=0.021 ) significantly affected the answer concerning the cancer stage of the hypothetical case .
therefore , marital status , educational status , and income status were included as covariates in multivariate analysis .
as the p - values of sex and ' cancer patients in relatives or acquaintances ' were less than 0.1 in univariate analysis , they were also included as covariates ( table 2 ) .
less frequently than those who had relatives or acquaintances with cancer ( or , 0.77 ; p=0.022 ) .
respondents with educational status of high school ( or , 0.40 ; p=0.005 ) or above ( or , 0.22 ; p < 0.001 ) answered do nt know significantly less frequently than those with educational status below high school .
however , there were no consistent and strong predictors across the choices ( table 3 ) .
terminal stage than early stage ( or , 0.15 ; p=0.015 ) .
in addition , respondents who had relatives or acquaintances with cancer chose advanced stage more frequently than those who had no relatives or acquaintances with cancer ( or , 1.30 ; p=0.022 ) ( table 4 ) .
a total of 2,000 persons ( response rate , 41.2% ) responded to the survey questions .
1,677 respondents ( 70.2% ) graduated high school or above and 1,672 respondents ( 83.6% ) earned more than 2 million korean won per month .
eight hundred thirty - nine participants ( 42.0% ) had relatives or acquaintances diagnosed with cancer .
as shown in fig . 1 , only 411 respondents ( 20.6% ) chose advanced stage .
terminal stage as the stage of the hypothetical case ( n=1,489 , 74.5% ) . a small proportion of respondents chose early stage ( n=13 , 0.7% ) or
in univariate analysis , marital status ( p=0.019 ) , educational status ( p < 0.001 ) , and income ( p=0.021 ) significantly affected the answer concerning the cancer stage of the hypothetical case . therefore , marital status , educational status , and income status were included as covariates in multivariate analysis .
as the p - values of sex and ' cancer patients in relatives or acquaintances ' were less than 0.1 in univariate analysis , they were also included as covariates ( table 2 ) .
less frequently than those who had relatives or acquaintances with cancer ( or , 0.77 ; p=0.022 ) .
respondents with educational status of high school ( or , 0.40 ; p=0.005 ) or above ( or , 0.22 ; p < 0.001 ) answered do nt know significantly less frequently than those with educational status below high school .
however , there were no consistent and strong predictors across the choices ( table 3 ) .
terminal stage than early stage ( or , 0.15 ; p=0.015 ) .
in addition , respondents who had relatives or acquaintances with cancer chose advanced stage more frequently than those who had no relatives or acquaintances with cancer ( or , 1.30 ; p=0.022 ) ( table 4 ) .
to the best of our knowledge , this is the first study assessing the understanding of the definition of advanced versus terminal cancer in the general population using a hypothetical vignette about a case of advanced cancer .
we found that a large proportion ( 74.5% ) of the general population misunderstood the medical definition of terminal cancer , and could not discriminate terminal cancer from advanced cancer with metastasis .
the proportion of inadequate responses was as high as 80% , even higher than that reported in previous survey results ( one - third of participants answered incorrectly ) on the public perception of the definition of terminal cancer in south korea .
although there are several exceptions , many patients with advanced stage cancers can not expect to be cured , and have a poor long - term prognosis . despite the availability of life - prolonging treatment
the results of our survey suggest that most members of the general population seem to not be familiar with the concept of treatment refractoriness and
life expectancy less than 6 months , and may regard advanced stage cancer as terminal cancer .
differences in the correct response rates across different studies can be explained by differences in study design . in a previous study , in which five response options for the definition of terminal cancer were provided to a conceptual question
, many respondents could have chosen the correct option by chance or by avoiding extreme options ( a cancer still resectable and curable or a cancer with a prognosis of death within a few days or weeks ) .
therefore , a continuous effort is required in order to establish common and shared definitions of cancer staging and to increase awareness across the general population .
first , members of the general population can become cancer patients themselves or caregivers of cancer patients in the future .
misunderstanding about the accurate cancer stage can lead to discrepancies in expectation regarding the treatment effect between patients , caregivers , and physicians .
although doctors inform patients of their exact cancer stages , patients and caregivers might not understand the prognostic meaning of the cancer stage .
providing patients and their families with a full understanding of the disease status is very difficult for physicians , and physicians rarely check their patient s understanding of the diagnosis .
therefore , for informed decision - making or shared understanding , it is not sufficient to simply provided information on the stage of the cancer as early , advanced , or terminal . for patients , the perception of terminal cancer can influence decision - making regarding the treatment plan .
this misunderstanding can lead to an undesirable scenario , in which patients forgo treatment when diagnosed with advanced cancer that could prolong survival . for patients with terminal cancers ,
this misunderstanding may lead to precious time being occupied with futile disease - directed treatment options .
when shared understanding about the stage of the cancer is lacking , the decision - making process can not guarantee an appropriate outcome .
therefore , for informed decision - making , it is essential for physicians to not only provide information on the exact cancer stage , but to also explain the meaning of that stage and to confirm the patient s understanding .
second , there was no consistent and strong predictor as to which respondents would answer correctly , as defined in terms of medical context .
interestingly , the educational status of the participants was not significantly associated with choosing the correct answer .
in addition , personal experience with relatives or acquaintances diagnosed with cancer also did not improve the understanding of cancer staging , and even increased the frequency with which respondents chose the response option of terminal cancer .
the experience of cancer in close relationships could affect the perceived knowledge of the general population , resulting in a more sensitive reaction than those who had no relatives or acquaintances with cancer .
although no significant difference in sex was observed between the response groups advanced stage and terminal stage , female tended to choose early stage more frequently .
as the hypothetical case in our vignette was a breast cancer patient , females might react with more emotion .
however the result is limited to those who chose early stage ( n=13 ) .
in addition , marital status and respondents income were not consistently associated with their choices .
these results suggest that physicians should verify the understanding of cancer stages during communication with cancer patients and their caregivers regardless of their socio - demographic characteristics and personal experiences . finally , many studies based on survey data of the general population regarding terminal disease relied on the correct perception of terminal cancer .
some of the surveys did not inform participants of the exact definition of terminal disease , which would be sufficient to ensure that patients have the same understanding as the researchers .
however , considering the high proportion of the general population lacking accurate knowledge of the medical definition of terminal cancer , the reliability and validity of those surveys might be compromised .
for example , in surveys on topics such as the recently issued legalization of euthanasia , accurate understanding of terminal disease is required for the response to be valid .
therefore , our study suggests that more care should be taken to ensure that respondents of such surveys have a shared understanding of the exact definition of advanced or terminal disease .
the strengths of this study include systematic sampling methods and the large sample size , ensuring the generalizability of these findings across the korean population .
therefore , the practical understanding of the respondents can be better assessed using clinical vignettes based on a hypothetical case than by using questions based on conceptual knowledge .
many of the respondents had not experienced advanced or terminal cancer , and these participants may have less knowledge about cancer than those affected by cancer .
however , the proportions of the correct answer between the general population and patients or family caregivers were not significantly different in a previous study .
in addition , personal experiences with cancer did not significantly affect the response rate in our study , suggesting that a potential bias would not be significant .
moreover members of the general population can become patients themselves and their close caregivers or friends may influence medical decisions . therefore ,
understanding of cancer staging of the general population is relevant from a clinical and research perspective .
development of a communication strategy for end - of - life discussions would also be important .
second , the staging terms for cancer are defined based on the viewpoint of medicine .
thus , most of the general population might not perceive the exact definitions of the terms for cancer staging .
however , as mentioned above , clinical vignettes do not require in - depth knowledge of the study topics .
therefore the results of our study might reflect the understanding of the definition of cancer stages in the naive general population .
however , if we asked about the possibility of complete cure , necessity for aggressive chemotherapy for life prolongation , or expected survival time of the hypothetical patient in the vignette , more of the general population might have answered correctly , as defined in terms of medical context .
also , a well - designed study comparing before and after adequate education using patient decision aids regarding the definition of the cancer stages may be needed for more sensitive evaluation of the knowledge in the general population .
in conclusion , we found that a large proportion of the general population could not differentiate advanced cancer from terminal cancer , as defined in terms of medical context .
continuous effort is required in order to establish common and shared definitions of cancer stages and to increase the shared understanding of cancer staging for patients , caregivers , and the general population .
in addition , a detailed explanation concerning the stage of the cancer and its clinical meaning is essential for shared understanding and informed decision - making between cancer patients and their families and physicians .
researchers need to ensure that respondents to surveys and studies have a shared understanding of the definition of advanced or terminal disease . | purposemany end - of - life care studies are based on the assumption that there is a shared definition of language concerning the stage of cancer . however , studies suggest that patients and their families often misperceive patients cancer stages and prognoses .
discrimination between advanced cancer and terminal cancer is important because the treatment goals are different . in this study
, we evaluated the understanding of the definition of advanced versus terminal cancer of the general population and determined associated socio - demographic factors.materials and methodsa total of 2,000 persons from the general population were systematically recruited .
we used a clinical vignette of a hypothetical advanced breast cancer patient , but whose cancer was not considered terminal . after presenting the brief history of the case , we asked respondents to choose the correct cancer stage from a choice of early , advanced , terminal stage , and do nt know .
multinomial logistic regression analysis was performed to determine sociodemographic factors associated with the correct response , as defined in terms of medical context.resultsonly 411 respondents ( 20.6% ) chose advanced , while most respondents ( 74.5% ) chose terminal stage as the stage of the hypothetical patient , and a small proportion of respondents chose early stage ( 0.7% ) or do nt know ( 4.4% ) .
multinomial logistic regression analysis found no consistent or strong predictor.conclusiona large proportion of the general population could not differentiate advanced cancer from terminal cancer .
continuous effort is required in order to establish common and shared definitions of the different cancer stages and to increase understanding of cancer staging for the general population . |
acquired perforating dermatoses ( apd ) is a group of rare chronic persisting dermatological disorders with poor treatment outcomes , characterized by transepidermal elimination in which collagen , elastic tissue or necrotic connective tissue from the dermis is extruded to the exterior with minimal disruption of surrounding structures .
it is most commonly seen in patients with diabetes and on hemodialysis . it has also been reported in hypothyroidism , hepatitis , lymphoma , periampullary carcinoma , sclerosing cholangitis , scabies , a variety of malignancies ( colon cancer , prostate cancer , hepatocellular carcinoma , breast cancer ) and acquired immunodeficiency syndrome ( aids ) .
an association of this disease with systemic mycosis has never been reported to the best of our knowledge .
we present for the first time the occurrence of apd in a patient of addison s disease due to disseminated histoplasmosis , which resolved spontaneously with amphotericin - b and itraconazole therapy .
a 60 y gentleman presented to endocrinology clinic with reportts intermittent low grade fever , nausea , vomiting , anorexia , pain abdomen , weight loss and increased pigmentation for 4 mo and multiple , discrete , pigmented , intensely pruritic nodules with history of extrusion of material from these nodules , over extensor aspect of both lower extremities of 1 mo duration ( fig.1a and b ) .
examination was significant for low blood pressure ( 80/60 mm of hg ) , tachycardia , oral mucosal hyperpigmentation , hepatosplenomegaly and multiple hyperpigmented nodules with central keratinous plug over the extensor surface of both lower limbs , especially over the thighs .
investigations revealed hyponatremia ( 117 mmol / l ; normal : 135145 mmol / l ) , hyperkalemia ( 5.3 mmol / l ; normal : 35 - 5.5 mmol / l ) , normal renal ( creatinine-0.8 mg / dl ; normal : 0.21.2 mg / dl ) and hepatic function , low 8 a.m. cortisol ( 2.3 g / dl ; normal : 534 g / dl ) and elevated plasma acth ( 68 pg / ml ; normal : 515 pg / ml ) . contrast enhanced ct ( cect ) scan of abdomen revealed mild hepatosplenomegaly with bilateral heterogeneously enhancing enlarged adrenals with areas of necrosis , left adrenal ( 63 36 mm ) larger than the right ( 49 mm 33 mm ) ( fig .
serum igg , iga and igm levels were estimated by nephelometry assay ( dade behring ) and were normal [ igg : 1129 mg / dl ( normal : 7001,600 mg / dl ) ; iga : 232mg / dl ( normal : 70400 mg / dl ) and igm : 218 mg / dl ( normal : 40230 mg / dl ) ] .
usg guided fine needle aspiration ( fna ) from the left adrenal mass and bone marrow aspiration from the iliac crest showed plenty of oval cells on methanamine silver staining suggestive of yeasts of histoplasma capsulatum ( fig .
histopathological examination of punch biopsy specimen of one of the nodules over the left thigh revealed a transepidermal elimination process characterized by invagination of epidermis with extrusion of dermal materials through cup shaped depression ( fig.4a and b ) .
staining for collagen and elastin revealed collagen bundles extruding from the epidermis ( figs . 5 and 6 ) . a diagnosis of acquired perforating dermatosis , addison s disease with disseminated histoplasmosis was made .
( c ) picture of both thighs ( 10 mo after diagnosis ) showing complete resolution of lesions with presence of hyperpigmentation .
ct abdomen showing enlarged left ( 63 36 mm ; black arrow ) and right ( 49 mm 33 mm ; white arrow ) adrenals with areas of necrosis .
( a ) methanamine silver staining of adrenal fine needle aspiration showing numerous oval yeast cells suggestive of histoplasma capsulatum ( 400 magnification ) .
( b ) hematoxylin and eosin staining of skin biopsy showing transepidermal elimination of dermal contents ( 100 ) .
( d ) masson s trichrome staining of skin biopsy showing intensely blue staining of collagen within the epidermifigs ( 400 ) .
( e ) verhoeff van gieson staining of skin biopsy showing brown staining for elastin sparing the epidermal channel ( 100 ) .
he initially received injection hydrocortisone 50 mg thrice daily , which was gradually tapered and converted to oral hydrocortisone 15 mg on getting up from sleep in the morning and 10 mg at 3 p.m. he received amphotericin deoxycholate ( fungisome , lifecare innovations ) 485 mg over 14 d , which was then stopped due to rising creatinine and hypokalemia .
itraconazole ( sporanox , 100 mg capsule , johnson and johnson ) was started at 200 mg per day .
patient showed clinical improvement with cessation of fever , improvement in appetite and weight gain .
after 3 mo of itraconazole therapy , the cutaneous lesions over the thighs started regressing .
last evaluated 10 mo after the initial diagnosis , he was doing well on hydrocortisone supplementation and itraconazole 200 mg / day .
the lesions over the thighs had resolved completely with presence of residual pigmentation ( fig .
a 60 y gentleman presented to endocrinology clinic with reportts intermittent low grade fever , nausea , vomiting , anorexia , pain abdomen , weight loss and increased pigmentation for 4 mo and multiple , discrete , pigmented , intensely pruritic nodules with history of extrusion of material from these nodules , over extensor aspect of both lower extremities of 1 mo duration ( fig.1a and b ) .
examination was significant for low blood pressure ( 80/60 mm of hg ) , tachycardia , oral mucosal hyperpigmentation , hepatosplenomegaly and multiple hyperpigmented nodules with central keratinous plug over the extensor surface of both lower limbs , especially over the thighs .
investigations revealed hyponatremia ( 117 mmol / l ; normal : 135145 mmol / l ) , hyperkalemia ( 5.3 mmol / l ; normal : 35 - 5.5
mmol / l ) , normal renal ( creatinine-0.8 mg / dl ; normal : 0.21.2 mg / dl ) and hepatic function , low 8 a.m. cortisol ( 2.3 g / dl ; normal : 534 g / dl ) and elevated plasma acth ( 68 pg / ml ; normal : 515 pg / ml ) . contrast enhanced ct ( cect ) scan of abdomen revealed mild hepatosplenomegaly with bilateral heterogeneously enhancing enlarged adrenals with areas of necrosis , left adrenal ( 63 36 mm ) larger than the right ( 49 mm 33 mm ) ( fig .
serum igg , iga and igm levels were estimated by nephelometry assay ( dade behring ) and were normal [ igg : 1129 mg / dl ( normal : 7001,600 mg / dl ) ; iga : 232mg / dl ( normal : 70400 mg / dl ) and igm : 218 mg / dl ( normal : 40230 mg / dl ) ] .
usg guided fine needle aspiration ( fna ) from the left adrenal mass and bone marrow aspiration from the iliac crest showed plenty of oval cells on methanamine silver staining suggestive of yeasts of histoplasma capsulatum ( fig .
histopathological examination of punch biopsy specimen of one of the nodules over the left thigh revealed a transepidermal elimination process characterized by invagination of epidermis with extrusion of dermal materials through cup shaped depression ( fig.4a and b ) .
staining for collagen and elastin revealed collagen bundles extruding from the epidermis ( figs . 5 and 6 ) . a diagnosis of acquired perforating dermatosis , addison s disease with disseminated histoplasmosis was made .
( c ) picture of both thighs ( 10 mo after diagnosis ) showing complete resolution of lesions with presence of hyperpigmentation .
ct abdomen showing enlarged left ( 63 36 mm ; black arrow ) and right ( 49 mm 33 mm ; white arrow ) adrenals with areas of necrosis .
( a ) methanamine silver staining of adrenal fine needle aspiration showing numerous oval yeast cells suggestive of histoplasma capsulatum ( 400 magnification ) .
( b ) hematoxylin and eosin staining of skin biopsy showing transepidermal elimination of dermal contents ( 100 ) .
( d ) masson s trichrome staining of skin biopsy showing intensely blue staining of collagen within the epidermifigs ( 400 ) .
( e ) verhoeff van gieson staining of skin biopsy showing brown staining for elastin sparing the epidermal channel ( 100 ) .
he initially received injection hydrocortisone 50 mg thrice daily , which was gradually tapered and converted to oral hydrocortisone 15 mg on getting up from sleep in the morning and 10 mg at 3 p.m. he received amphotericin deoxycholate ( fungisome , lifecare innovations ) 485 mg over 14 d , which was then stopped due to rising creatinine and hypokalemia .
itraconazole ( sporanox , 100 mg capsule , johnson and johnson ) was started at 200 mg per day .
patient showed clinical improvement with cessation of fever , improvement in appetite and weight gain .
after 3 mo of itraconazole therapy , the cutaneous lesions over the thighs started regressing .
last evaluated 10 mo after the initial diagnosis , he was doing well on hydrocortisone supplementation and itraconazole 200 mg / day .
the lesions over the thighs had resolved completely with presence of residual pigmentation ( fig .
apd belongs to the group of disorders termed perforating dermatoses , the others members being kyrle 's disease , perforating folliculitis , elastoses perforans serpiginosa ( eps ) and reactive perforating collagenoses ( rpc ) .
they are differentiated by the nature of perforating material : predominantly collagen bundles in rpc , elastin in eps and keratin in kyrle s disease .
lesions of apd usually present as pigmented papules , which may become hyperkeratotic , erythematous plaques or umblicated nodules , commonly seen over the arms and legs .
it is believed that poor blood supply due to microangiopathy in diabetes , accumulation of poorly dialyzable substances in patients on hemodialysis , and trauma ( secondary to itching ) leads to dermal necrosis , alteration of dermal connective tissue protein expression [ increased expression of elastin receptor , transforming growth factor- ( tgf- ) , matrix metalloproteinase-1 ( mmp-1 ) and tissue inhibitor of metalloproteinase-1 ( timp-1 ) ] .
this alteration of dermal connective tissue protein expression may play a key role in the development of perforating dermatoses .
apd is a difficult to treat disorder with the outcomes dependent on the underlying disease .
there have been reports of successful treatment with oral retinoids , doxycycline , clindamycin and allopurinol .
transcutaneous electrical nerve stimulation , uvb , topical cantharidin ( vesicant and keratolytic agent ) has also been tried .
apd was diagnosed in our patient on the basis of the clinical presentation and histopathologic evidence of extrusion of predominantly collagen bundles .
our patient presented with clinical features of adrenal insufficiency with hyperpigmentation ( addison s disease ) , which was confirmed by documentation of low serum cortisol and elevated plasma acth .
histoplasmosis was diagnosed as the cause of addison s disease in view of presence of oval yeast cells on methanamine silver staining of the adrenal fna ( fig .
histoplasmosis is predominantly a disease of the reticuloendothelial system caused by histoplasma capsulatum , a dimorphic fungus , with the yeast form found in the macrophages and phagocytes of humans .
most of the infections in immunocompetent individuals are asymptomatic , mild or self - limiting .
disseminated hispoplasmosis ( dh ) is predominantly a disease of the immunocompromized patients with only 30% occurring in immunocompetent individuals .
dh was diagnosed in our patient who did not have any immunodeficiency , in view of fever , weight loss , hepatosplenomegaly , adrenomegaly with adrenal insufficiency and histopathologic abundance of oval yeast cells on adrenal fna and bone marrow aspiration .
other forms of histoplasmosis are acute pulmonary histoplasmosis , chronic cavitary pulmonary histoplasmosis and central nervous system histoplasmosis .
liposomal amphotericin b is the treatment of choice , and has been observed to be more effective than amphotericin deoxycholate in the treatment of dh in immunocompromized individuals .
also our patient did not have any immunodeficiency and responded well to the combination therapy of amphotericin deoxycholate and itraconazole .
occurrence of apd in the background of addison s disease due to disseminated histoplasmosis has not been reported .
temporal relationship of the occurrence of apd with the clinical course and its complete resolution with the treatment of histoplasmosis establishes the likely causal relationship between disseminated histoplasmosis and apd in our patient .
our patient highlights the observation that treatment of the underlying disease often leads to resolution of apd .
it may be hypothesized that altered structural protein in the dermis as a consequence of disseminated histoplasmosis may have had some role in the development of apd in our patient .
this is most likely an immunological phenomenon in the absence of definitive demonstration of histoplasma yeast cells in the skin biopsy .
one of the limitations of our report is the lack of immunohistochemistry to look for altered protein expression in the dermis of the skin biopsy specimen due to institutional non - availability .
to summarize , this is probably the first reported case of the occurrence of apd in a patient of disseminated histoplasmosis who had presented with adrenal insufficiency , and responded well to hydrocortisone supplementation and amphotericin - b with itraconazole therapy . | acquired perforating dermatosis ( apd ) is a rare disorder characterized by transepidermal elimination of contents from dermis with minimal disruption of surrounding structures , believed to be due to altered expression of dermal proteins .
its occurrence in patients with systemic mycosis has never been reported .
we report a 60-y gentleman who presented with features of adrenal insufficiency ( nausea vomiting , hypotension and increased pigmentation ) for 4 mo , multiple hyperpigmented pruritic nodules with central keratinous plug over extensor surface of both lower limbs along with hepatosplenomegaly of one month duration .
investigations revealed low cortisol ( 2.3 g / dl ; normal : 534 g / dl ) , elevated acth ( 68 pg / ml ; normal : 515 pg / ml ) , enlarged bilateral adrenals with hepatosplenomegaly on ct .
methanamine silver staining of fine needle aspiration from the adrenals and bone marrow aspiration showed numerous oval yeast cells suggestive of histoplasma .
histopathology of biopsy of one of the skin nodules revealed transepidermal elimination process characterized by invagination of epidermis with extrusion of collagen bundles suggestive of apd .
patient improved with hydrocortisone replacement and there was clinical improvement with resolution of skin lesions following amphotericin - b and itraconazole therapy .
this is probably the first reported case of apd in a patient with disseminated histoplasmosis who had presented with addison s disease . |
gross alpha / beta activity measurements are widely applied as a screening technique in many fields ( e.g. environmental monitoring , industrial applications ) .
water intended for drinking purposes has to be analysed first for gross alpha / beta activity according to national and international standards and recommendations .
anticipating the new eu drinking water directive , which includes gross alpha / beta activity screening levels , jrc - irmm organised in 2012 an interlaboratory comparison ( ilc ) to check the fitness for purpose of this method and the performance of european monitoring laboratories .
the eu member state national representatives ( in the art . 3536 expert group ) under the euratom treaty nominated the participants .
the participants submitted signed reports including their results together with their answers to a questionnaire .
the ilc was evaluated on the basis of the measurement values that the 71 participant laboratories reported .
several waters of natural origin were analysed to find representative samples for this ilc . to make the final selection , the activity concentration of the alpha - emitting radionuclides , salinity , chemical composition , directives and recommendations were taken into account .
three water samples ( water a , water b : two commercially available natural mineral waters ; water c : one laboratory - made spiked water ) were selected .
water b was bottled into anonymous 1.5 l polyethylene terephthalate bottles by a mineral water producer company .
two perfluoroalkoxy polymers ( pfa)-lined drums of 550 l were filled with the mineral water and the water was thereafter re - circulated for 24 h at 15 l / min using two inert iwaki bellow pumps . during filling an intermediate polycarbonate buffer tank of 20 l ( nalgene , rochester , ny , usa ) was used and the water was pumped simultaneously from the two tanks into the buffer tank . the buffer tank was placed in a clean bench and the water bottles were filled manually by opening and closing the tap of the buffer tank . prior to filling ,
the buffer tank was rinsed with 2 10 l of type 1 water ( 18.2 m cm , 0.056 s / cm at 25 c and toc < 5 ng / ml from a milli - q advantage system ( millipore , billerica , ma , usa ) and 20 l of mineral water . in this manner
the 1-l bottles were made of high density polypropylene ( hdpe ) with a leak - proof hdpe - screw cap ( nalgene ) .
water c was a spiked type 2 water from a millipore elix-35 system ( > 5 m cm , 0.2 s / cm at 25 c and toc < 30 ng / ml ) with added inorganic salt mixture . during several days
500 l of type 2 water was collected in portions into one pfa - lined drum of 550 l. subsequently 120 g of salt mixture of cacl2 and sr(no3)2 was added .
thereafter 2 l of concentrated nitric acid was added ( ph = 1.2 0.1 ) followed by sr / y and am spike .
the contents were thereafter mixed using the iwaki inert bellows pump of the water handling system for 16 h at 15 l / min .
subsequently 482 of the 1-l hdpe bottles ( nalgene ) were filled as described above .
the reference value determination was done in three independent laboratories where the four most common routine methods were used ( table 1).table 1methods used for the determination of gross alpha / beta reference valuescollaboratormethodbelgian nuclear research centre ( sckcen ) , belgiumevaporation , solid scintillation counting ( iso 10704)the national institute for public health and the environment ( rivm ) , the netherlandsspike addition , evaporation and gas flow proportional counting ( iso 9696/9697)institute for reference materials and measurements ( ec - jrc - irmm ) , belgiumco - precipitation , gas flow proportional counting ( iso 10704 ) and k activity concentration determination by gamma - ray spectrometry
thermal pre - treatment , liquid scintillation counting ( iso 11704 )
complimentary method for iso 10704 co - precipitation approach methods used for the determination of gross alpha / beta reference values
complimentary method for iso 10704 co - precipitation approach the reason for using a complimentary method for the iso 10704 co - precipitation approach was that k activity was not included in the gross beta activity results . during the sample preparation k was precipitated neither as sulphate nor as hydroxide , but it stayed in the solution and eventually the analysts using iso 10704 co - precipitation approach would fail to detect it .
the k activity concentration determination was performed for the natural waters ( water a and b ) .
these samples were measured on the hpge - detector ge-4 in the underground laboratory hades using ultra low - level gamma - ray spectrometry ( ulgs ) [ 5 , 6 ] .
the homogeneity of the samples within the whole batch and the short term stability during the ilc campaign were checked .
the homogeneity of the radionuclides in the matrix was evaluated using the softcrm version 2.0.10 software following the certification principles for reference materials as given in iso / iec guide 35 . for the homogeneity study a random stratified method
eight to ten bottles were randomly selected and analysed using gross measurements and radionuclide specific analysis of the natural origin alpha emitting radionuclides ( water a and b ) .
this included the activity concentration determination of the main contributing alpha - emitting radionuclides to the gross alpha activity concentration ( e.g. in case of water b the activity concentration of ra was determined ) .
the scatter of the results from the gross measurements was larger than that from the homogeneity values from the radionuclide specific analysis , so the former were used in the uncertainty budget . in the case of bottled waters
, the main contribution to their instability was the adsorption of radionuclides to the container wall .
short term stability analysis was done on a monthly basis using gross measurements and the aforementioned radionuclide specific analysis ( water a and b ) .
the first stability measurements were done already 2 months before the beginning of the ilc and the last 1 month after the submission of the last result .
the contribution of uncertainties from the characterization of the material ( uchar ) , homogeneity between bottles ( ubb ) , and the short term stability ( usts , ) are presented in table 2.table 2contribution of uncertainties to the expanded uncertainty of the reference values ( % ) sample
u
char
u
bb
u
sts
gross alpha activity water a19.613.14.8 water b6.01.52.0 water c7.53.46.1gross beta activity water a6.02.05.9 water b8.51.52.0 water c7.42.84.7 contribution of uncertainties to the expanded uncertainty of the reference values ( % ) as shown , the largest part of the uncertainty comes from the characterization followed by the short term stability , except in the case of the gross alpha activity in water a , where the uncertainty contribution from homogeneity is much higher than that from the short term stability .
the combined standard uncertainty of the mean of the measurement results from the reference measurements and is calculated in eq .
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\begin{document}$$ u_{\text{char } } = \frac{{\sqrt { \sum\nolimits_{i = 1}^{n } { ( u_{c , i } ) ^{2 } } } } } { n } $ $ \end{document}uchar=i=1n(uc , i)2n
where : uc , i is the combined standard uncertainty of the laboratory or method result , andn is the number of laboratories considered .
uc , i is the combined standard uncertainty of the laboratory or method result , and n is the number of laboratories considered .
the expanded uncertainty ( uref ) of the reference value is calculated by using eq .
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\begin{document}$$ u_{\text{ref } } = k \times \sqrt { u_{\text{char}}^{2 } + u_{\text{bb}}^{2 } } + u_{\text{sts}}^{2 } $ $ \end{document}uref = kuchar2+ubb2+usts2
where : k is the coverage factor ( k = 2 ) at ~95 % confidence interval , uchar is the combined standard uncertainty of the mean of the measurement results from the laboratories contributing to the reference value , ubb is the uncertainty of the activity concentration between bottles of the same batch , andusts is the uncertainty due to the short - term stability of the samples ( longer than the duration of the comparison exercise ) .
k is the coverage factor ( k = 2 ) at ~95 % confidence interval , uchar is the combined standard uncertainty of the mean of the measurement results from the laboratories contributing to the reference value , ubb is the uncertainty of the activity concentration between bottles of the same batch , and usts is the uncertainty due to the short - term stability of the samples ( longer than the duration of the comparison exercise ) .
table 3 gives the reference values for the three waters analysed and used for the ilc , with their expanded uncertainties.table 3reference activity concentration values ( a
ref ) of the three waters used in the ilc and their expanded uncertainties ( u
ref ) ( coverage factor k = 2)parameterreference values with expanded uncertainty ( a
ref u
ref ; mbq l)water awater bwater cgross alpha activity47.5 22.8434.7 56.6954.5 77.3gross beta activity309.8 57.4190.4 32.61037.3 83.0 reference activity concentration values ( a
ref ) of the three waters used in the ilc and their expanded uncertainties ( u
ref ) ( coverage factor k = 2 )
the evaluation of the participant s results is based on their per cent difference or relative deviation from the reference value eq .
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\begin{document}$$ d_{\% } = 100 \times \frac{{a_{\text{lab } } - a_{\text{ref } } } } { { a_{\text{ref } } } } $ $ \end{document}d%=100alab - arefaref
where : alab is the participant laboratory result ( mean activity concentration),aref is the reference value .
alab is the participant laboratory result ( mean activity concentration ) , aref is the reference value .
the results are considered compatible if they fall within the 30 % range from the reference value and incompatible if they fall outside this range .
the 30 % criterion is arbitrary , based on the estimation that the analysis is feasible within this level of deviation .
besides sending the measurement results , laboratories submitted answers to a questionnaire giving details of their laboratory and routine procedures . from the questionnaire it turned out that 65 laboratories work according to a quality system ( mainly iso 9000 and iso 17025 ) and 58 laboratories are either accredited , authorized , certified or have a combination of these three . in 65 laboratories ,
the same routine analytical procedure was used for the ilc samples as for their regular routine samples .
the amount of water used for the preparation of a single measurement sample ranged from 5 ml up to 5 l. details on the sample preparation and measurement techniques are presented in table 4 .
for the counting efficiency calibration the following radionuclides were used : am , unat , pu , ra , po , u for alpha ; and k , sr / y , cl , cs , pb , c , h for beta . these radionuclides cover a wide alpha / beta energy range ( 18.61175.6 kev ) . furthermore , one laboratory reported to use ra for beta calibration.table 4number of laboratories for sample preparation and measurement techniques used for determining the gross activitiesnumber of laboratoriessample preparation method evaporation to complete dryness36 evaporation and mixing with lsc cocktail16 evaporation to complete dryness , coprecipitation7 other
4 coprecipitation3 evaporation to complete dryness , other
3measurement technique proportional counter42 liquid scintillation counter22 scintillation counter ( solid)10 semiconductor si detector2 i - matic si - det1 grid ionization chamber1
category other not specified by the participants number of laboratories for sample preparation and measurement techniques used for determining the gross activities
category other not specified by the participants the most used sample preparation method was evaporation to dryness with no further sample treatment .
the second most used method was by evaporation ( thermal pre - concentration ) of an aliquot of the sample to a smaller volume and by mixing it with lsc cocktail .
coprecipitation was applied in ten cases and other techniques were used by seven laboratories . among the 49 participants who used other techniques than liquid scintillation counting ( lsc ) , 20 laboratories answered yes and 29 no to the question if they have a procedure for hygroscopic residue .
these 49 laboratories deposit the residue onto the planchet in many different ways as listed , like automatic evaporation , residue homogenization with a solvent , evaporation of the last few ml on the planchet , direct evaporation on filter paper , direct evaporation and mechanical homogenization .
the most popular measurement techniques were proportional counting , lsc and solid state scintillation counting .
few laboratories applied some non - conventional gross counting like semiconductor si detector , i - matic si - det and grid ionization chamber . in the case of lsc , the following sample to cocktail ratios were used : 1:4 , 2:3 , 1:21 , 1:3 and 2:1 . only five out of the 21 laboratories using lsc applied quench correction .
the type of lsc vials used were : polyethylene ( used by 10 laboratories ) , teflon coated ( 9 ) , low potassium glass ( 1 ) , glass ( 1 ) and other ( 1 ) .
the procedures for the determination of background used by the participant laboratories are summarized in table 5.table 5procedures used for gross alpha / beta background determination by the participantsbackground determination procedurenumber of laboratoriesempty
planchette35blank samples7acidified water + ls cocktail5distilled water + ls cocktail5zns(ag ) powder3background sample in nearly the same chemical composition as the water sample2caso4 spread on planchet1filter paper on a planchet1acidified water + radon removal + ls cocktail1no definite answer11 procedures used for gross alpha / beta background determination by the participants as seen , there are nine different approaches for the background determination which may be a reason for biased results .
moreover , there were 11 laboratories that did not provide definite answers but we assume that they might have used one of the nine background determination approaches .
comparing the gross alpha / beta detection limits with the detection limits given in the new drinking water directive ( table 6 ) one can see there are laboratories not complying with the requirements.table 6limit of detection of gross alpha / beta activity concentrations reported by the participant laboratories in mbq l
gross alphagross betalimit of detection reported by the participants ( mbq l ) 1.43400424limit of detection ( mbq l ) from the new drinking water directive 40400 limit of detection of gross alpha / beta activity concentrations reported by the participant laboratories in mbq l
the 71 registered participant laboratories were requested to determine the gross alpha and beta activity concentration of three different water samples .
this means that each participant could submit maximum six independent measurement results with their corresponding expanded uncertainties .
only 44 laboratories reported results for the parameter gross alpha activity concentration in sample water a , while for the same parameter in sample water b , 70 results were reported .
the reported results of gross alpha and beta activity concentration are sorted in ascending order in figs . 1 and 2 .
the error bars represent expanded uncertainties ( k = 2 ) and solid red line represents the reference value , while dashed red lines represent the corresponding expanded uncertainties . for the better visibility of the data points , the identifier laboratory codes are not indicated on the plots.fig .
1results of gross alpha activity concentration sorted in ascending order . error bars represent expanded uncertainties ( k = 2)fig .
error bars represent expanded uncertainties ( k = 2 ) results of gross alpha activity concentration sorted in ascending order .
error bars represent expanded uncertainties ( k = 2 ) results of gross beta activity concentration sorted in ascending order .
error bars represent expanded uncertainties ( k = 2 ) many of the participants results deviate by more than two orders of magnitude from the reference values regardless of the techniques used .
it is interesting to evaluate the ratio of maximum to minimum reported gross activities ( table 7 ) and the percentage of compatible results ( table 8) . the number of compatible results together with the number of laboratories and their identification codes are presented in table 9.table 7ratio of the reported maximum to minimum gross activitiesparameter
a
max / a
min
water awater bwater cgross alpha activity1,01734693gross beta activity3,0502,0803,150table 8percentage of the reported results within 30 % from the reference valueparameterresults within 30 % deviation ( % ) water awater bwater cgross alpha activity363963gross beta activity452761table 9number of laboratories and their ilc identification codes versus the number of reported compatible resultsnumber of compatible resultsnumber of laboratorieslaboratory code613351544717 , 18 , 22 , 34 , 36 , 41 , 483111 , 2 , 5 , 13 , 21 , 25 , 30 , 51 , 57 , 62 , 712203 , 6 , 8 , 10 , 11 , 15 , 23 , 24 , 26 , 27 , 35 , 37 , 40 , 46 , 47 , 52 , 63 , 64 , 66 , 681204 , 7 , 9 , 12 , 14 , 16 , 19 , 28 , 29 , 32 , 39 , 49 , 50 , 55 , 59 , 60 , 65 , 67 , 72 , 7301320 , 31 , 38 , 42 , 43 , 44 , 45 , 53 , 56 , 58 , 61 , 69 , 70 ratio of the reported maximum to minimum gross activities percentage of the reported results within 30 % from the reference value number of laboratories and their ilc identification codes versus the number of reported compatible results as shown in table 9 , only 20 laboratories ( 27 % ) out of 71 reported at least half of the results within the reference range .
furthermore , 13 laboratories ( 18 % ) did not report compatible result at all . among the 20 best
performing laboratories we did not find any of the methods to be superior to the other methods .
of these 20 laboratories , only four laboratories used solid scintillation counting and the others applied lsc or proportional counting . during the evaluation of the ilc results ,
they were sorted by counting technique , sample preparation , radionuclides used for calibration and the time delay between sample preparation and counting .
3results sorted on the basis of a measurement techniques , b sample preparation used and c time delay between sample preparation and measurementfig .
4results sorted on the basis of the radionuclides used for a alpha and b beta counting efficiency calibration results sorted on the basis of a measurement techniques , b sample preparation used and c time delay between sample preparation and measurement results sorted on the basis of the radionuclides used for a alpha and b beta counting efficiency calibration comparing the different groups of sorted results , no significant differences between those groups are observed .
however , for some groups the available data are limited ( e.g. for group other ) .
it is worth to mention that laboratories using the same radionuclide for calibration , as was added as spike in water c , did not perform better than the laboratories using other radionuclides .
as mentioned above , only 27 % of the labs reported at least half of the results within the reference range , while 18 % reported incompatible results only .
the present situation is far from satisfactory knowing that these screening methods are very likely to be used for testing drinking water as foreseen by the drinking water directive and will lead to different decisions seen the large spread in the data .
the large spread of the results may be due to influencing factors during both the sample preparation and the measurement process [ 10 , 11 ] .
these influences can not generally be predicted and it is already difficult to define the measurand for gross activity analysis since the radionuclide composition of the sample is a priory not known .
additionally , the activity of the sample may substantially change with time as some radionuclides decay and others grow in during the measurement time .
for drinking water although a few decay processes are very likely to occur and should be accounted for in the measurement process . for these reasons ,
we recommend following strictly accepted common procedures for sample preparation and measurement , to be aware of all decay processes that may affect the measurement , to test procedures for robustness and to set up realistic uncertainty budgets .
the outcome of the analysis may be influenced by the proficiency and training of laboratory personnel too .
at least in two european countries ( austria , switzerland ) no gross methods are used for drinking water qualification due to their drawbacks and unreliability . as long as gross activity parameters are included in the european drinking water directive
, this interlaboratory comparison should be repeated with pre - defined guideline procedures to be followed . | an interlaboratory comparison was organised by jrc - irmm among environmental radioactivity monitoring laboratories for the determination of gross alpha / beta activity concentration in drinking water .
independent standard methods were used for the reference value determination .
the performance of participating laboratories was evaluated with respect to the reference values using relative deviations .
sample preparation and measurement methods used by the participating laboratories are detailed , in particular in the view of method - dependency of the results .
many of the participants results deviate by more than two orders of magnitude from the reference values regardless of the techniques used .
this suggests that gross methods need revision . |
recently , the health care community has recognised the importance of using qol measurement as an essential component of a treatment modality 's efficacy . at every stage of disease
, treatment choices may involve modalities that differ in side effects and impact upon qol .
quality - of - life assessment can be helpful in weighting the risks and benefits of treatment options , particularly when differences in survival among the options are small or non - existent .
quality of life is subjective in nature , therefore there is a wide agreement that health - related quality of life should be conceptualised as a multidimensional construct .
physical functioning , disease - and treatment - related symptoms , psychological / emotional well being , and social interactions are critical domains that are included in most efforts to measure overall quality of life .
when considering quality of life in advanced cancer patients one of the treatment choices is a palliative one , therefore we consider of great importance the inclusion of a new dimension when measuring quality of life in such a population , that of " choice of treatment " .
recent studies and new articles clearly indicate that physicians must be educated to routinely ask patients about their wishes for medical care and to recognise that they are legally and morally bound to honour those requests .
such communication is especially pressing in the context of advanced illness , when the achievement of a peaceful death assumes priority over inappropriate prolongation of dying .
many valid assessment instruments have been developed that measure qol such as eortc , the functional assessment of cancer treatment ( fact ) . in 1986 ,
the european organisation for research and treatment initiated a research program to develop an integrated , modular approach for evaluating the quality of life of patients participating in international clinical trials .
eortc with its clinical focus and its multicultural orientation provides a rather unique context for developing and testing quality of life questionnaires .
the aim of our study was to assess the psychometric properties of a new quality of life instrument on a hellenic sample of terminally ill cancer patients receiving only palliative treatment , which is called pqli ( palliative care quality of life instrument ) .
it was found to be concise , quantitative and easily used ; it has been designed primarily for use by the patients themselves ; it was based on the existing literature [ 7,9 - 11 ] and the items that the patients consider as most important to what they perceive as " quality of life " , the latter was elicited by means of qualitative research .
it became evident from the qualitative assessment on the patients ' description on their qol that there is a need to participate in the treatment process ; this would give them a sense of control over their fatal disease .
patients want a voice in their end - of - life care , and participation in treatment choices encompasses the psychosocial outcomes that these may have in their lives .
when considering quality of life in advanced cancer patients the treatment choice is a palliative one , therefore we consider of importance the inclusion of a new dimension when measuring quality of life in such a population , that of " choice of treatment " .
the lack of a questionnaire that examines the quality of life specifically in a palliative care setting , the individuals that form their support system , as well as the unique needs that these patients have , was the driving force for a measurement like this to be developed .
patients with symptomatic incurable cancer disease were selected for study by means of palliative treatment modality .
no restrictions were placed with regard to histologic type of cancer , age , or performance status .
all patients attended the " pain relief and palliative care unit " of areteion hospital where the study took place , between january 2002 and october 2002 .
criteria for inclusion were : age > 18 years , no cerebral metastases , no known psychiatric disorder , to be cognitively capable of filling in the questionnaire , fluent in the hellenic language , and off anticancer treatment for 3 months . from 630 cancer and non - cancer patients that were treated in the unit that period ,
144 advanced cancer patients were drawn using stratified random sampling , according to the performance status , and were judged eligible to enter the study . from them ,
24 patients ( 16.7% ) were excluded due to refusal to participate in the study .
the final sample was consecutive and consisted of 120 responding patients from whom written informed consent was obtained .
demographic and clinical data of sample the pqli is a 28-item questionnaire , composed of six multi - item scales ( 2 functional scales , 1 symptom scale , 1 choice of treatment scale , 1 psychological scale ) and a single item scale ( overall quality of life ) . each scale was accompanied by a title .
the questionnaire development involved the following phases : first , literature search that identified the relevant qol issues , and the existing questionnaires , second , a provisional list of items was presented to 3 experts for feedback on appropriateness of content and breadth of coverage , third , the list was administered to patients from the target group to determine the extent to which they have experienced these problems , while they were asked to choose a number of issues that troubled them the most ( figure 1 ) .
next , the resulting list of items was reviewed for clarity and overlap by other experts , and finally , the questionnaire was pretested by administering it to some patients ( n = 20 ) from the target population , and through structured interviews with each patient individually after the completion of the questionnaire . in these interviews , the patients rated the questionnaire scales within a range of " 1 " ( i.e. first choice ) to " 7 " ( i.e. seventh choice ) .
the researchers , then , evaluated whether scores from the resulting pqli corresponded well with those independently obtained ratings .
variables , which -according to patients -affect their quality of life . in the final questionnaire format six of the scales were presented into three optional statements to be scored 1 , 2 and 3 respectively . in " choice of treatment "
scale the patients were asked to choose the item that is " most " important in the choice of treatment and rate it 1 ; then choose the item that is " next " important and rate it 2 ; and so on , the last item which is the " least " important , the patients were asked to rate it as 5 .
the single item scale " overall quality of life " has the form of a bi - polar numerical scale .
this rather short interval was chosen because of the imminent risk of sudden changes in their health status .
the instrument was designed primarily to be a self - assessment but where the patient 's condition would not permit it the researcher assisted him / her .
{ in the appendix - see additional file 1 ] is presented the questionnaire in the english language .
two independent translators translated the pqli in english and then another two independent translators translated it back to greek .
the same translation method has already been used in the validity and reliability of the eortc qlq c-30 ( v.3 ) in greek .
a range of analyses was carried out to establish scale reliability , and to evaluate empirically the validity of the questionnaire scales .
higher mean scores from 0 to 100 represent a better level of functioning and qol on the scales of " activity " , " self - care " , " support " , " communication " , " psychological affect " , and " overall quality of life " , and higher mean values on the health status scale , represent more symptomatology and worse quality of life .
the current procedures for scoring the pqli reflect the multidimensionality of the quality of life domain .
internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally .
cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest .
test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) .
the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature
, the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test .
five indirect methods to evaluate validity were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test .
second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects .
third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli .
as a fourth process , correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) .
the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid .
in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions .
it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales .
concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) .
accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice .
the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings
, we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview .
we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements . initially
, all possible predictive paths were included simultaneously and non - significant paths were dropped gradually .
this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) .
the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) .
the pqli is a 28-item questionnaire , composed of six multi - item scales ( 2 functional scales , 1 symptom scale , 1 choice of treatment scale , 1 psychological scale ) and a single item scale ( overall quality of life ) .
the questionnaire development involved the following phases : first , literature search that identified the relevant qol issues , and the existing questionnaires , second , a provisional list of items was presented to 3 experts for feedback on appropriateness of content and breadth of coverage , third , the list was administered to patients from the target group to determine the extent to which they have experienced these problems , while they were asked to choose a number of issues that troubled them the most ( figure 1 ) .
next , the resulting list of items was reviewed for clarity and overlap by other experts , and finally , the questionnaire was pretested by administering it to some patients ( n = 20 ) from the target population , and through structured interviews with each patient individually after the completion of the questionnaire . in these interviews , the patients rated the questionnaire scales within a range of " 1 " ( i.e. first choice ) to " 7 " ( i.e. seventh choice ) .
the researchers , then , evaluated whether scores from the resulting pqli corresponded well with those independently obtained ratings . variables , which -according to patients -affect their quality of life . in the final questionnaire format six of the scales were presented into three optional statements to be scored 1 , 2 and 3 respectively . in " choice of treatment "
scale the patients were asked to choose the item that is " most " important in the choice of treatment and rate it 1 ; then choose the item that is " next " important and rate it 2 ; and so on , the last item which is the " least " important , the patients were asked to rate it as 5 .
the single item scale " overall quality of life " has the form of a bi - polar numerical scale .
this rather short interval was chosen because of the imminent risk of sudden changes in their health status .
the instrument was designed primarily to be a self - assessment but where the patient 's condition would not permit it the researcher assisted him / her .
{ in the appendix - see additional file 1 ] is presented the questionnaire in the english language .
two independent translators translated the pqli in english and then another two independent translators translated it back to greek .
the same translation method has already been used in the validity and reliability of the eortc qlq c-30 ( v.3 ) in greek .
a range of analyses was carried out to establish scale reliability , and to evaluate empirically the validity of the questionnaire scales .
higher mean scores from 0 to 100 represent a better level of functioning and qol on the scales of " activity " , " self - care " , " support " , " communication " , " psychological affect " , and " overall quality of life " , and higher mean values on the health status scale , represent more symptomatology and worse quality of life .
the current procedures for scoring the pqli reflect the multidimensionality of the quality of life domain .
internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally .
cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest .
test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) .
the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature
, the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test . five indirect methods to evaluate validity
were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test .
second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects .
third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli . as a fourth process
, correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) .
the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid .
in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions .
it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales .
concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) .
accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice .
the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings
, we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview .
we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements .
initially , all possible predictive paths were included simultaneously and non - significant paths were dropped gradually .
this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) .
the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) .
internal consistency of the questionnaire before and during palliative treatment was assessed by cronbach 's alpha and was considered acceptable for group comparisons if the coefficient exceeded 0.70 , as recommended by nunnally .
cronbach 's alpha tests whether the items in a questionnaire have a homogeneous content with respect to the construct of interest .
test - retest reliability of patients ' ( n = 120 ) responses was evaluated by comparing the scores recorded on two occasions , an average of seven days apart ( spearman - rho test ) .
the patient 's clinical stage did not change between the first and the second completion , and the status of the patients was stable between test and retest . due to its hierarchical nature
, the intertest reliability of the ranking statements ( choice of treatment ) was established by using the " kendall's - w " test .
five indirect methods to evaluate validity were adopted : first , by comparing the scale scores with patients with poorer and better eastern cooperative oncology group ( ecog - the clinical variable assessed ) performance status using the mann - whitney u non - parametric test .
second , by assessing the statistical difference of the questionnaire - scales before and during treatment in terms of wilcoxon rank test between scales for related subjects .
third , by exploratory factor analysis , using principal components with non - orthogonal ( direct oblimin ) rotation , was used to assess the validity of the pqli . as a fourth process
, correlations were calculated between pqli items and those of two others instruments , the assessment of quality of life in palliative care ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) .
the aqel is focusing in patients undergoing palliative treatment , includes 22 items in seven scales , from physical , psychological , social and existential domains , while it has proven to be reliable and valid .
in addition , the eortc qlq - c30 is a widely used second - generation questionnaire designed to measure cancer patients ' physical , psychological and social functions .
it is a psychometrically established 30-item questionnaire , incorporated in nine multi - item , and several single - item scales .
concurrent related validity was performed with correlations among the scales of pqli ( inter - scale correlations ) . accordingly , the seven factors obtained from the interview from the 120 patients were rated and coded from 1 to 7 according to the patients ' choice : 1 , 2 , 3 , 4to 7choice .
the closeness of the hypothetical model of pqli to the empirical data of interviewing rating scores is evaluated statistically through gamma test . to evaluate whether scores from the pqli instrument corresponded well with those independently obtained ratings
, we first performed a factor analysis in which the seven latent variables from the pqli form were intercorrelated with the seven forms from the interview .
we then tested a predictive model to observe whether constructs from the pqli instrument could predict analogous measured constructs from the interview measurements . initially , all possible predictive paths were included simultaneously and non - significant paths were dropped gradually .
this procedure was a test of both the convergent and discriminative validity of the pqli instrument ( i.e. , variables on the pqli should be related to corresponding variables on the interview and not to non - corresponding variables ) and the criterion - related validity of the pqli form ( i.e. , the ability of the pqli to predict an independent criterion variable ) .
the whole statistical analysis was conducted using the spss version 8.0 statistical package ( spss inc , chicago , il ) .
the 28 items were all acceptable to the participants . they encompassed physical , social , health , and psychological aspects of life .
the 61.7% of the participants regarded as most important the variable of long - term quality of life , while the 76.7% of the respondents regarded as least important the variable " effects on sexual life " .
the distributions of the respondents in each category , for example , were 51.7% of the respondents are not working , a 40% can fully care for themselves , a 66.7% reported pain , 66.7% of the patients reported support from their friends and relatives , the 64.2% stated that they do not discuss their family problems with their doctor , and a 49.2% answered that they do not feel fear of death . from this figure the clinical profile of patients can be seen .
although restricted to a limited cultural setting , this data was considered quite interesting for clinicians .
table 2 shows the means and standard deviations for the multi - item measures , before and during treatment . from the descriptive statistics matrix ,
the cronbach 's alpha for each scale was found to be greater than the critical value of 0.70 , while the overall cronbach 's alpha was 0.787 . the test - retest reliability ( table 3 ) of scales and items as well showed that all the coefficients of agreement were greater than 0.82 ( p < 0.001 in all cases ) . due to the nature of the " choice of treatment "
scale the reliability was calculated by performing the kendall 's coefficient of concordance and was found 0.353 with a p - value < 0.0001 .
the correlation matrix of the scales within the pqli - pre - and - on - treatment is displayed in table 4 .
the agreements are strong , consistent and statistically significant at the 0.005 or 0.001 levels . as expected , the strongest correlations were observed between the " activity " , " self - care " , " health status " , and " choice of treatment " .
the " overall quality of life " ( oqol ) , correlated substantially with " activity " , " health status " , " self - care " , " choice of treatment " , " communication " , " support " , and " psychological affect " .
correlations among the pqli scales . * before treatment under the diagonal ; during treatment above the diagonal .
exploratory non - orthogonal factor analysis ( principal axis factoring extraction with direct oblimin rotation ) was carried out to further explore the validity of the pqli instrument .
the bartlett test of sphericity was 3042.7 and it was significant ( p < 0.0001 ) .
the kaiser - meyer - olkin measure of sampling adequacy was equal to 0.81 showing that the data is suitable for factor analysis .
principal axis factoring extraction was used to analyse the underlying structure of the questionnaire , yielding seven independent factors accounting for 79.7 % of the variance .
this seven - factor solution was deemed appropriate by examining the magnitude and rate of change in eigenvalues .
based on the rule that meaningful factors should be associated with eigenvalues greater than 1.0 and a marginal change occur after seven factors ( scree test ) , the seven - factor solution is appropriate . for the interpretation of the factor solution direct oblimin rotation
the variables constituting the seven factors are marked in bold fonts . by performing an orthogonal rotation using varimax ,
the same 7 factors were identified without any material difference confirming the results from the non - orthogonal rotation .
factor 1 : activity ( keep working , house chores , enjoyment , hobbies ) , factor 2 : self - care ( driving or transportation , self - sufficient ) , factor 3 : health status ( pain , nausea / vomiting , lack of appetite , weak / tired , dyspnoea , stool disturbances , sleep disturbances ) , factor 4 : choice of treatment ( like to choose , able to choose ) , factor 5 : support ( relatives / friends , health care team , nursing stuff ) , factor 6 : communication ( social relationships , economic / professional problems , family problems ) , and factor 7 : psychological affect ( calm , optimistic , blue , control of the situation , fears of death ) .
as shown in table 3 , the correlations under the diagonal among the scales of pqli were significantly associated , giving evidence of concurrent related validity .
following , the pqli model was significantly associated with the empirical model deriving from the patients ' interview ( gamma = 0.78 , se = 0.11 , p < 0.001 ) .
correlations between the ratings derived from the interview and the pqli factors are reported in table 6 .
we allowed covariances ( correlations ) among the predictor variables and significant covariances among the error residuals of the outcome variables .
the fit indices for this final path model reflected an excellent fit ( p < 0.001 , chitest ) .
we found that the pqli constructs significantly predicted analogous interviewing scores by the patients . in most cases
, there was considerable discriminative validity between similar observed and reported variables , except that pqli " health " also predicted " activity " from the interview rating . to refine these results , we needed to determine whether the path from the pqli " health " factor to the interviewing " health " rating was significantly larger than the path from pqli " health " to the " activity " interviewing rating variable . therefore , we ran a model that constrained these paths to equivalence and then examined the chi - difference test between these nested models .
the difference test revealed that the paths were significantly different in magnitude ( p < 0.01 ) , thereby providing additional evidence of the discriminant validity of the pqli .
correlations between pqli factors and interview scores for the 120 patients ( criterion related validity ) .
significant regression paths among latent variables pqli model predicting interviewing ratings ( n = 120 ) .
regression coefficients are standardized ( p < 0.001 , p < 0.01 , p # 60 ; 0.05 ) between the pqli and the assessment of quality of life in palliative care instrument ( aqel ) the correlations were , generally , strong in all the scales , ranging from 0.44 to 0.94 .
the strongest correlations were found between the items of " insomnia " and " sleep disturbances " ( 0.94 ) and also in the items of " pain " ( 0.93 ) ( table 7 ) .
moreover , there were significant correlations between the eortc qlq c30 and the relevant items of pqli , ranging from 0.79 to 0.97 , especially between the items of " pain " ( 0.97 ) , and " lack of appetite " ( 0.97 ) ( table 8) .
there were significant correlations between the scales of pqli and the relevant scales of aqel and eortc qlq c30 , as shown in table 9 .
the distinction between patients with low or high ecog performance status showed significant relationship between ecog scores and instrument scale scores . as we see in table 10 , patients with a better ecog performance status reported significantly higher scores in all the scales of the instrument .
correlations between the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) and pqli items .
correlations between relevant scales of the pqli and the scales of aqel as well as the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) .
scores by ecog status , combined validity samples ( n = 120 ) . * mann - whitney u test
table 2 shows the means and standard deviations for the multi - item measures , before and during treatment . from the descriptive statistics matrix ,
the cronbach 's alpha for each scale was found to be greater than the critical value of 0.70 , while the overall cronbach 's alpha was 0.787 . the test - retest reliability ( table 3 ) of scales and items as well showed that all the coefficients of agreement were greater than 0.82 ( p < 0.001 in all cases ) . due to the nature of the " choice of treatment "
scale the reliability was calculated by performing the kendall 's coefficient of concordance and was found 0.353 with a p - value < 0.0001 .
descriptive statistics and scale reliability before and during treatment . test retest correlations for all pqli scales and items .
the correlation matrix of the scales within the pqli - pre - and - on - treatment is displayed in table 4 .
the agreements are strong , consistent and statistically significant at the 0.005 or 0.001 levels .
as expected , the strongest correlations were observed between the " activity " , " self - care " , " health status " , and " choice of treatment " .
the " overall quality of life " ( oqol ) , correlated substantially with " activity " , " health status " , " self - care " , " choice of treatment " , " communication " , " support " , and " psychological affect " .
correlations among the pqli scales . * before treatment under the diagonal ; during treatment above the diagonal .
exploratory non - orthogonal factor analysis ( principal axis factoring extraction with direct oblimin rotation ) was carried out to further explore the validity of the pqli instrument .
the bartlett test of sphericity was 3042.7 and it was significant ( p < 0.0001 ) .
the kaiser - meyer - olkin measure of sampling adequacy was equal to 0.81 showing that the data is suitable for factor analysis .
principal axis factoring extraction was used to analyse the underlying structure of the questionnaire , yielding seven independent factors accounting for 79.7 % of the variance .
this seven - factor solution was deemed appropriate by examining the magnitude and rate of change in eigenvalues .
based on the rule that meaningful factors should be associated with eigenvalues greater than 1.0 and a marginal change occur after seven factors ( scree test ) , the seven - factor solution is appropriate . for the interpretation of the factor solution direct oblimin rotation
the variables constituting the seven factors are marked in bold fonts . by performing an orthogonal rotation using varimax ,
the same 7 factors were identified without any material difference confirming the results from the non - orthogonal rotation .
factor 1 : activity ( keep working , house chores , enjoyment , hobbies ) , factor 2 : self - care ( driving or transportation , self - sufficient ) , factor 3 : health status ( pain , nausea / vomiting , lack of appetite , weak / tired , dyspnoea , stool disturbances , sleep disturbances ) , factor 4 : choice of treatment ( like to choose , able to choose ) , factor 5 : support ( relatives / friends , health care team , nursing stuff ) , factor 6 : communication ( social relationships , economic / professional problems , family problems ) , and factor 7 : psychological affect ( calm , optimistic , blue , control of the situation , fears of death ) .
as shown in table 3 , the correlations under the diagonal among the scales of pqli were significantly associated , giving evidence of concurrent related validity .
following , the pqli model was significantly associated with the empirical model deriving from the patients ' interview ( gamma = 0.78 , se = 0.11 , p < 0.001 ) .
correlations between the ratings derived from the interview and the pqli factors are reported in table 6 .
we allowed covariances ( correlations ) among the predictor variables and significant covariances among the error residuals of the outcome variables .
the fit indices for this final path model reflected an excellent fit ( p < 0.001 , chitest ) .
in most cases , there was considerable discriminative validity between similar observed and reported variables , except that pqli " health " also predicted " activity " from the interview rating . to refine these results
, we needed to determine whether the path from the pqli " health " factor to the interviewing " health " rating was significantly larger than the path from pqli " health " to the " activity " interviewing rating variable .
therefore , we ran a model that constrained these paths to equivalence and then examined the chi - difference test between these nested models . the difference test
revealed that the paths were significantly different in magnitude ( p < 0.01 ) , thereby providing additional evidence of the discriminant validity of the pqli .
correlations between pqli factors and interview scores for the 120 patients ( criterion related validity ) .
significant regression paths among latent variables pqli model predicting interviewing ratings ( n = 120 ) .
regression coefficients are standardized ( p < 0.001 , p < 0.01 , p # 60 ; 0.05 )
between the pqli and the assessment of quality of life in palliative care instrument ( aqel ) the correlations were , generally , strong in all the scales , ranging from 0.44 to 0.94 .
the strongest correlations were found between the items of " insomnia " and " sleep disturbances " ( 0.94 ) and also in the items of " pain " ( 0.93 ) ( table 7 ) .
moreover , there were significant correlations between the eortc qlq c30 and the relevant items of pqli , ranging from 0.79 to 0.97 , especially between the items of " pain " ( 0.97 ) , and " lack of appetite " ( 0.97 ) ( table 8) .
there were significant correlations between the scales of pqli and the relevant scales of aqel and eortc qlq c30 , as shown in table 9 .
the distinction between patients with low or high ecog performance status showed significant relationship between ecog scores and instrument scale scores . as we see in table 10 , patients with a better ecog performance status reported significantly higher scores in all the scales of the instrument .
correlations between the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) and pqli items
. correlations between relevant scales of the pqli and the scales of aqel as well as the european organization for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0 ) .
scores by ecog status , combined validity samples ( n = 120 ) . * mann - whitney u test
the purpose of this study was to design and evaluate a method of collecting information about the quality of life of advanced ill cancer patients receiving palliative care treatment .
although the primary intent of this project was to establish the basic reliability and validity of the pqli measurement system , we also hoped to demonstrate the sensitivity to change of this instrument by incorporating it into a clinical trial using repeated measures design .
the questionnaire was simple to administer and score , and was well accepted by the responding patients . in average , it required 8 minutes to complete and , in most cases , could be filled with little or no assistance .
this is a further proof that the proposed instrument is appropriate for this patient population , bearing in mind that one of the reasons for developing qol tools for palliative care is to meet the needs of patients who can not use traditional measures because they are frail and very ill .
patients seemed to be genuinely pleased that an interest in their quality of life was a component of their overall care .
two aspects of this instrument development are worth noting : the opportunity given to the patients to actively participate in this process , and the incorporation , for the first time , of the patients ' beliefs in their involvement in treatment decision and what this encompasses .
the latter consists a major advantage of the pqli over the already existing ones in quality of life .
the internal consistency coefficients for the pqli subscales are all beyond the acceptable level required for making group comparisons when evaluating changes in scores over time .
the scales that showed too significant associations were those of " activity " , " self - care " , and " health status " .
the careful construction of the test deriving from the patients ' interview as well as the setting of pass / fail scores through the exploratory factor analysis enhance the content and construct validity of the instrument .
the correlation between the latent factors of pqli and the empirical factors of patients ' interview ( table 6 ) provide empirical evidence for concurrent criterion - related validity .
additionally , the latter was strongly indicated by the significant regression coefficients as shown in figure 2 .
although the pqli was related to both " health " and " activity " item of the interviewing rating score , this was not unexpected .
indeed , the high intercorrelation between the " health " and " activity " items supports the clinical observation that a regressive health status often occurs in the context / of regressive activity .
moreover , the significant correlation among the variables of pqli indicates that performance is related across test components as might be expected .
furthermore , the factor analysis showed that the various components do assess unique and independent domains , confirming the discriminative validity of the measures .
an important aspect that arises from this study concerns the doctor - patient communication and the relations of patients with their families and the important role of the latter . concealing a diagnosis of cancer from patients themselves , unfortunately ,
is still common practice in greece . in " psychological affect " , and specifically in the item concerning the " fear of death " , 56% of the patients said that they do n't feel fear of death , while reporting that many have been kept in the dark about the details of their illness diagnosis and prognosis .
while most greek doctors nowadays favour disclosing a cancer diagnosis and prognosis to the patient directly , the relatives often veto this decision , since the role of the family is very powerful in the greek culture . nevertheless , they reported a high percentage ( 84.2% ) on the item of support .
regarding the nature of their communication with their physicians on issues like social relationships , financial , and family relationships were very low ( 21.7% , 5.0% , and 12.5% respectively ) .
quality of life research can provide the researcher and the clinician with a clearer view of the impact that a cancer treatment has on a patient 's life .
this is clearly shown on this instrument from the scale that examines the attitude of patients ' toward treatment selection .
the participant patients were receiving palliative treatment , which aims are psychological support and symptom relief both in short and long term , so the findings are consistent with the nature of the treatment they are receiving .
moreover , another important aspect that arises from this study is the participation of the patients in the treatment choice .
half of the responding population ( 50% ) reported that they want to choose the treatment they are going to receive , while a 47.5% said that they actually do choose their treatment .
the latter is quite a large percentage considering they had no information on their diagnosis and prognosis .
the psychometric testing of the pqli provided evidence that the elements of the measurement appropriately reflect quality of life in terminal cancer patients since the tests of validity and reliability yielded consistent results .
indeed , research initiatives must rely on established quality of life instruments with proved records of statistical reliability and validity administered by objective parties .
these results must be confirmed in larger multicenter trials to decrease any possible selection bias .
moreover , when conducting a study in quality of life , since it is subjective in nature , we must remain cautious in our findings , since numbers may be inappropriate to adequately describe all aspects of patients ' lives .
inclusion of health related quality of life measures in current and future prospective studies would be necessary to provide a database that is richer and more useful to patients and physicians . taken together , these results lend considerable support to the clinical validity of the pqli , and could be used as an audit in clinical studies involving patients with advanced stage cancer . in future studies
we will continue to examine the validity of quality of life measurements obtained with the pqli using a variety of research strategies including corroboration of additional sources of independent evidence .
| backgroundthis paper describes the development of a new quality of life instrument in advanced cancer patients receiving palliative care.methodsthe palliative care quality of life instrument incorporates six multi - item and one single - item scale .
the questionnaire was completed at baseline and one - week after .
the final sample consisted of 120 patients.resultsthe average time required to complete the questionnaire , in both time points , was approximately 8 minutes .
all multi - item scales met the minimal standards for reliability ( cronbach 's alpha coefficient .70 ) either before or during palliative treatment .
test - retest reliability in terms of spearman - rho coefficient was also satisfactory ( p < 0.05 ) .
validity was demonstrated by inter - item correlations , comparisons with ecog performance status , factor analysis , criterion - related validation , and correlations with the assessment of quality of life in palliative care instrument ( aqel ) , and the european organisation for research and treatment of cancer ( eortc ) core quality of life questionnaire ( qlq - c30 , version 3.0).conclusionthe pqli is a reliable and valid measure for the assessment of quality of life in patients with advanced stage cancer . |
methicillin - resistant staphylococcus aureus ( mrsa ) is a common and important cause of infection in the intensive care unit ( icu ) setting .
the colonizing bacterial strains may serve as endogenous reservoirs for overt clinical infections or may spread to other patients .
several studies have demonstrated a link between s. aureus carriage and subsequent infection in continuous peritoneal dialysis patients , nonsurgical patients , critical neonates , and medical icu patients .
the routine mrsa surveillance to prevent mrsa infections among icu patients , however , is still a controversial policy .
it is important to confirm the linkage between nasal carriage and clinical mrsa isolates to develop the best strategy to avoid systemic infection by decolonization methods . in 2000 , 53% to 83% of s. aureus isolates attributed to nosocomial infections in 12 taiwanese major hospitals were resistant to methicillin . in our adult icus ,
mrsa accounted for 77% of nasal s. aureus isolates with a high colonization rate ( up to 32% ) during a surveillance study in 2010 .
a prospective cohort observational study of medical icu patients was undertaken to examine mrsa nasal colonization status and the development of mrsa infection .
our research goals were to determine the clinical association between nasal carriage of mrsa and subsequent mrsa infections and to identify additional risk factors associated with mrsa infection .
the relationship between nasal and clinical isolates was also investigated using pulsed - field gel electrophoresis ( pfge ) and multilocus sequence typing ( mlst ) analysis .
chang - gung memorial hospital , lin - kuo branch , is a university - affiliated 3700-bed tertiary teaching hospital in northern taiwan that provides healthcare ranging from primary to tertiary care .
a prospective cohort observational analysis of consecutive patients admitted to the 2 medical icus ( 44 beds ) between november 2008 and may 2010 was performed .
the institutional review board of chang - gung memorial hospital reviewed and approved the study ( irb no .
: 960104b ) and the requirement for written informed consent was waived . for patients with multiple medical icu admissions ,
the specimens from the nares were obtained within 3 days of admission to the icu and again 1 week following admission to the icu .
methicillin - resistant s. aureus isolates recovered from clinical diagnostic samples ( beyond survey culture specimens ) submitted to the clinical microbiology laboratory were defined as clinical isolates .
pneumonia required a positive respiratory culture , a compatible chest radiograph with symptoms and signs of lower respiratory tract infection and a decision to treat .
urinary tract infection required a positive urine culture and either a decision to treat or the growth of > 100,000 cfu / ml plus at least 50 leukocytes per high - power field .
all other sites , including pleural effusion , ascites , skin , and soft tissue required a positive culture and a decision to treat . to identify potential risk factors for mrsa infection ,
the following data were collected from each patient : baseline demographics , characteristics , underlying and current diseases , clinical covariates , dates of previous and current hospitalizations , date of icu admission , previous mrsa infection , already present mrsa infection , and subsequent mrsa infection .
previous infection was defined as mrsa infection diagnosed at least 2 weeks before the current hospitalization .
already present infection was defined as mrsa infection diagnosed during this hospitalization but before admission to medical icu or within 24 hours after icu admission .
nasal and clinical isolates from each study patient with mrsa infection were genotyped and compared .
survey specimens for mrsa culture were obtained with a cotton swab , placed in transport medium ( venturi transystem , copan innovation ltd , limmerick , ireland ) , and processed in the microbiology laboratory within 4 hours .
coagulase tests were carried out by using rabbit plasma to make sure the identification of s. aureus after incubation and subcultivation . based on the recommendation of clinical and laboratory standards institute ( clsi ) ,
cefoxitin test was used to differentiate the mrsa from methicillin - susceptible s. aureus ( mssa ) .
molecular genotyping analysis was performed on a total of 65 clinical isolates and 43 nasal isolates from 36 randomly selected patients with mrsa infections .
genotypes were allocated in alphabetical order , as in our prior studies ; any new genotype , if identified , was allocated consecutively .
pulsed - field gel electrophoresis patterns with less than 4 band differences from an existing genotype were defined as subtypes of that genotype .
two isolates were regarded to be indistinguishable , highly related , or distinct if they had the similar subtype ( no band difference ) , the same genotype ( less than 4-band differences ) , or a different type ( 4-band differences ) , respectively . according to the measures described previously , staphylococcal chromosome cassette mec ( sccmec ) type and presence of pantone - valentine leukocidin ( pvl )
multilocus sequence typing was performed for selective strains of representative pfge patterns as described elsewhere .
statistical analyses were performed using spss for windows software version 21.0 ( ibm , armonk , n y ) .
continuous data obtained were expressed as the mean standard deviation ( sd ) and frequencies were calculated for categorical data .
relative risk ( rr ) and 95% confidence interval ( ci ) were calculated using poisson regression .
potential predictors of colonization with p < 0.05 in univariate analysis were further analyzed with a multivariate poisson regression model in spss .
chang - gung memorial hospital , lin - kuo branch , is a university - affiliated 3700-bed tertiary teaching hospital in northern taiwan that provides healthcare ranging from primary to tertiary care .
a prospective cohort observational analysis of consecutive patients admitted to the 2 medical icus ( 44 beds ) between november 2008 and may 2010 was performed .
the institutional review board of chang - gung memorial hospital reviewed and approved the study ( irb no .
: 960104b ) and the requirement for written informed consent was waived . for patients with multiple medical icu admissions ,
the specimens from the nares were obtained within 3 days of admission to the icu and again 1 week following admission to the icu .
methicillin - resistant s. aureus isolates recovered from clinical diagnostic samples ( beyond survey culture specimens ) submitted to the clinical microbiology laboratory were defined as clinical isolates .
pneumonia required a positive respiratory culture , a compatible chest radiograph with symptoms and signs of lower respiratory tract infection and a decision to treat .
urinary tract infection required a positive urine culture and either a decision to treat or the growth of > 100,000 cfu / ml plus at least 50 leukocytes per high - power field .
all other sites , including pleural effusion , ascites , skin , and soft tissue required a positive culture and a decision to treat . to identify potential risk factors for mrsa infection ,
the following data were collected from each patient : baseline demographics , characteristics , underlying and current diseases , clinical covariates , dates of previous and current hospitalizations , date of icu admission , previous mrsa infection , already present mrsa infection , and subsequent mrsa infection .
previous infection was defined as mrsa infection diagnosed at least 2 weeks before the current hospitalization .
already present infection was defined as mrsa infection diagnosed during this hospitalization but before admission to medical icu or within 24 hours after icu admission .
nasal and clinical isolates from each study patient with mrsa infection were genotyped and compared .
survey specimens for mrsa culture were obtained with a cotton swab , placed in transport medium ( venturi transystem , copan innovation ltd , limmerick , ireland ) , and processed in the microbiology laboratory within 4 hours .
coagulase tests were carried out by using rabbit plasma to make sure the identification of s. aureus after incubation and subcultivation . based on the recommendation of clinical and laboratory standards institute ( clsi ) ,
cefoxitin test was used to differentiate the mrsa from methicillin - susceptible s. aureus ( mssa ) .
molecular genotyping analysis was performed on a total of 65 clinical isolates and 43 nasal isolates from 36 randomly selected patients with mrsa infections .
genotypes were allocated in alphabetical order , as in our prior studies ; any new genotype , if identified , was allocated consecutively .
pulsed - field gel electrophoresis patterns with less than 4 band differences from an existing genotype were defined as subtypes of that genotype .
two isolates were regarded to be indistinguishable , highly related , or distinct if they had the similar subtype ( no band difference ) , the same genotype ( less than 4-band differences ) , or a different type ( 4-band differences ) , respectively . according to the measures described previously , staphylococcal chromosome cassette mec ( sccmec ) type and presence of pantone - valentine leukocidin ( pvl ) genes
multilocus sequence typing was performed for selective strains of representative pfge patterns as described elsewhere .
statistical analyses were performed using spss for windows software version 21.0 ( ibm , armonk , n y ) .
continuous data obtained were expressed as the mean standard deviation ( sd ) and frequencies were calculated for categorical data .
relative risk ( rr ) and 95% confidence interval ( ci ) were calculated using poisson regression .
potential predictors of colonization with p < 0.05 in univariate analysis were further analyzed with a multivariate poisson regression model in spss .
a total of 1461 patients admitted to the medical icus were evaluated . excluding 195 patients who had already present mrsa infections , 1266 patients were enrolled in this study . of these 1266 study patients ,
the initial mrsa nasal colonization rate was 16.4% ( 207/1266 ) , and the rate of subsequent mrsa infections was 39.1% ( 81/207 ) ( figure 1 ) .
the subsequent mrsa infection rate of non - mrsa colonization patients was significantly lower than mrsa colonization patients ( 14.7% versus 39.1% , or : 3.72 ; 95% ci : 2.685.16 ; p < 0.001 ) .
for the patients with mrsa nasal colonization , true infection occurred 12.3 days ( median , 7 days ; interquartile range , 315 days ) after colonization , and 16.9 days ( median , 12 days ; interquartile range , 720 days ) after icu admission , respectively . there was a significant difference in the meantime from icu admission to the true mrsa infection between patients with mrsa colonization ( 16.9 days ) and those without colonization ( 24.3 days ) ( p = 0.002 ) .
of these 1266 study patients , 939 patients ( 74.2% ) were experiencing respiratory failure with mechanical ventilation .
there were 119 patients ( 9.4% ) who underwent tracheostomy , which were all done before icu admission , and all need ventilator support .
schematic of analysis for medical intensive care unit patients by either mrsa infection or methicillin - resistant s. aureus nasal colonization .
table 1 shows the characteristics and risk factors of nasal mrsa colonization in these enrolled patients .
the colonized mrsa patients were older than noncolonized patients ( 71.8 15.7 versus .
in addition , the nasal mrsa patients had higher instances of hospitalization within the previous year ( p = 0.029 ) , old cerebrovascular accidents ( p = 0.047 ) , and mrsa infections previous to this admission ( p < 0.001 ) .
furthermore , multivariate poisson regression analysis demonstrated that previous mrsa infections ( rr : 2.19 ; 95% ci : 1.563.08 ; p <
risk factors associated with nasal mrsa colonization ( n = 1266 ) table 2 shows that nasal mrsa colonization was an independent predictor for subsequent mrsa infections .
patients with nasal mrsa colonization had significantly higher rates of subsequent mrsa infections than those without colonization ( rr : 2.50 ; 95% ci : 1.903.27 ; p
history of tracheostomy ( rr : 0.38 ; 95% ci : 0.180.79 ; p = 0.010 ) , however , was an independent protective predictor for subsequent mrsa infection .
methicillin - resistant s. aureus nasal colonization was noted for 81 ( 34.2% ) of 237 patients with mrsa infections .
methicillin - resistant s. aureus nasal colonization was also noted for 93 ( 65.5% ) of 142 patients with previous mrsa infections . in patients with mrsa nasal colonization ,
subsequent mrsa infections decreased significantly only with the history of tracheostomy ( rr : 0.22 ; 95% ci : 0.550.91 ; p = 0.037 ) ( table 3 ) .
risk factors for patients with subsequent mrsa infection risk factors for mrsa infection in patients with nasal mrsa colonization ( n = 207 ) molecular genotyping analysis was performed on a total of 65 clinical isolates and 43 nasal isolates from 36 patients with true mrsa infections ( table 4 ) . of these 36 patients , 14 patients ( 38.9% )
the source of the 65 clinical isolates included bloodstream ( 46 isolates ) , sputum ( 14 isolates ) , pleural effusion ( 3 isolates ) , and ascites ( 2 isolates ) .
a total of 11 pfge patterns were identified and 2 major patterns were type a and type f. within 65 clinical isolates , the ratios of type a and type f are 48% and 25% , respectively .
of the 43 nasal isolates , types a and f accounted for 37.2% and 30.2% of the patterns , respectively .
seventeen isolates with various pfge patterns were selected for mlst analysis and 2 sequence types ( st 239 and st 5 ) were identified .
most isolates with pfge type a and b sequenced as st 239 , whereas those with pfge type f sequenced as st 5 .
overall , most mrsa isolates belonged to 1 of 2 major clones characterized as st239/pfge a / sccmec iii or iiia / pvl negative ( 43.5% ) , and st5/pfge f / sccmec ii / pvl negative ( 26.9% ) .
molecular genotyping of 65 clinical and 43 nasal colonized mrsa isolates from 36 patients with mrsa infections the genetic correlation between clinical and nasal colonized isolates was analyzed in 32 patients with mrsa infections after excluding 4 patients who had multiple nasal mrsa colonization or clinical isolates with different genotypes ( table 5 ) .
the infection sources in these 32 patients were : bacteremia ( 19 ) , pneumonia ( 8) , thoracic empyema ( 2 ) , peritonitis ( 1 ) , combined thoracic empyema with bacteremia ( 1 ) , and combined pneumonia with peritonitis ( 1 ) .
the nasal and clinical isolates were indistinguishable in 24 patients ( 75.0% ) , highly related in 6 patients ( 18.8% ) , and distinct in 2 patients ( 6.2% ) based on pfge and mlst analyses .
in this study of medical icus patients , we found the subsequent mrsa infection rate of patients with nasal mrsa colonization to be significantly higher than that of patients without nasal mrsa colonization .
history of hospitalization within 1 year , old cerebrovascular accidents , and previous mrsa infections were risk factors for nasal mrsa colonization .
molecular study revealed that most mrsa isolates were st239/pfge a / sccmec iii or iiia / pvl ( ) and st5/pfge f / sccmec ii / pvl ( ) , which have been classified as healthcare - associated clones .
nosocomial s. aureus bacteremia is 3 times more frequent in s. aureus nasal carriers than in noncarriers ( 1.2% versus 0.4% ) .
methicillin - resistant s. aureus colonization was the leading risk factor for subsequent mrsa infection in patients with chronic skin ulcers during the same admission .
in general , nasal mrsa colonization , either present at admission to the hospital or acquired during hospitalization , increases the risk of mrsa infection compared with patients without nasal colonization ( 25% versus 2% ) .
furthermore , the impact of colonization leading to infection may be prolonged up to 18 months after hospital discharge . in our study patients , we also found that patients with nasal mrsa colonization had higher rates of subsequent mrsa infections than patients without nasal colonization ( 39.1% versus 14.7% ) .
although 1 study showed that nasal mrsa colonization was a poor predictor of icu - acquired mrsa infections requiring antibiotic treatment , a recent study observed that active surveillance cultures of mrsa yielded high specificity and negative predictive values for ventilator - associated pneumonia . in this study , we also found nasal mrsa colonization was 1 important risk factors of mrsa infection .
one possible explanation for these different observations is that different icu patient groups respond differently to the same strategy .
furthermore , we found that tracheostomy , but not translaryngeal endotracheal tubes , was associated with lower risk for mrsa infection with or without previous nasal mrsa colonization .
previous studies have also found that early tracheostomy patients had significantly lower rates of mortality and pneumonia compared with patients with prolonged translaryngeal endotracheal tubes .
translaryngeal endotracheal tubes keep the vocal cords open and that the bacteria from the back of the throat pass through the cords and then pass the cuff , get into the airway , reflux into the tube infect the biofilm in the endotracheal tubes tube , and finally translocate into the lungs .
tracheostomy is protective against nosocomial infection acquired from upper airway because of better hygiene and oral care .
therefore , tracheostomy , rather than a translaryngeal endotracheal tube to bypass the upper airway , may potentially decrease the risk for bacteria colonization and subsequent infection . in this study ,
the rate of subsequent mrsa infection in patients with nasal mrsa colonization on admission was 39.1% .
clinical infection isolates were genotypically indistinguishable from nasal colonized isolates in 75.0% ( 24 in 32 ) of the cases based on pfge patterns and mlst analysis .
we demonstrated a significant association between nasal mrsa colonization and subsequent infections based on genotyping by pfge in medical icu patients . in other groups , such as mixed icu and general ward patients , continuous peritoneal dialysis patients , surgical patients and neonates , the association rate ranged from 67% to 84.6% .
one study also demonstrated that s. aureus nasal carriage and clinical isolates belong to similar genetic clusters using mlst , despite differences in sequence type assignments .
the cochrane database of systematic reviews showed that application of intranasal mupirocin reduced infection rates only in dialysis and surgical patients .
bode et al showed that rapid screening to identify nasal carriers , followed by decolonization with both nasal mupirocin nasal ointment and chlorhexidine soap , could prevent hospital - acquired infection with s. aureus in surgical patients .
notably , a recent large - scale cluster - randomized trial among 74,256 patients concluded that universal decolonization was superior to both targeted decolonization and screening and isolation in both reducing rates of mrsa clinical isolates and bloodstream infection from any pathogen .
although this practice benefited from infection rate control and obviated the need for mrsa surveillance tests , further surveillance for mupirocin and chlorhexidine resistance remains an essential issue if the universal decolonization practice implemented widely .
first , although this was a large study , these results reflected the association between nasal colonization and infection at a single medical center , and may not be generalized to other hospitals .
second , we did not obtain colonization specimens from anatomic sites other than nostrils , which may also contribute to subsequent mrsa infections .
differences in compliance to infection - control programs to prevent horizontal transmission of mrsa in these 2 icus may affect the occurrence of nasal mrsa colonization and subsequent infections .
finally , molecular genotyping analysis was performed randomly on a subset of the total strains , which could not represent all the strains . in conclusion ,
previous mrsa infection was associated with subsequent mrsa infections , and history of tracheostomy could reduce this risk .
molecular and genetic studies determined that most mrsa isolates were healthcare - associated strains with significant correlations between colonized nasal and clinical isolates .
in future studies , this information may support a decolonization strategy for the medical icu patients . | abstractmethicillin - resistant staphylococcus aureus ( mrsa ) is a common and important cause of colonization and infection in medical intensive care units ( icu ) .
the aim of this study was to assess association factors between mrsa nasal colonization and subsequent infections in medical icu patients by clinical investigation and molecular genotyping.a prospective cohort observational analysis of consecutive patients admitted to medical icus between november 2008 and may 2010 at a tertiary teaching hospital were included . to detect mrsa colonization ,
the specimens from the nares were obtained within 3 days of admission to the icu and again 1 week following admission to the icu .
genetic relatedness for colonized and clinical isolates from each study patient with mrsa infection were analyzed and compared.a total of 1266 patients were enrolled after excluding 195 patients with already present mrsa infections .
subsequent mrsa infection rates were higher in patients with nasal colonization than in those without ( 39.1% versus 14.7% , respectively ) .
multivariate poisson regression analysis demonstrated that nasal mrsa colonization ( relative risk [ rr ] : 2.50 ; 95% confidence interval [ ci ] : 1.903.27 ; p <
0.001 ) was independent predictors for subsequent mrsa infections .
history of tracheostomy , however , was a protective predictor in all patients ( rr : 0.38 ; 95% ci : 0.180.79 ; p = 0.010 ) and in patients with mrsa nasal colonization ( rr : 0.22 ; 95% ci : 0.550.91 ; p = 0.037 ) .
molecular genetics studies revealed that most mrsa isolates were healthcare - associated clones and that nasal and clinical isolates exhibited up to 75% shared identity.methicillin-resistant s. aureus nasal colonization was significantly associated with subsequent mrsa infection among medical icu patients .
previous mrsa infection was associated with subsequent mrsa infections , and history of tracheostomy associated with reducing this risk .
most mrsa isolates were healthcare - associated strains that were significantly correlated between nasal and clinical isolates . |
extended surgery is necessary to perform curative resection of gastric cancer with hepatic or / and pancreatic invasion .
the value of extended organ resection for advanced gastric cancer has been debated for many years .
the potential advantage of extended resection for t4 gastric cancer is necessary to improve the r0 resection rate of these lesions .
arguments against this approach are based on an observed increase in the morbidity and mortality rates , with little objective benefit in survival .
the complication rates of additional organ resection with gastrectomy have consistently been reported to be higher when compared to patients undergoing gastrectomy alone .
several new perioperative adjunctive approaches ( neoadjuvant and/or adjuvant ) for highly advanced gastric cancer have been explored [ 1 , 2 , 3 , 4 ] . a randomized phase iii trial of surgery plus neoadjuvant chemotherapy compared with surgery alone for type 4 and large type 3 gastric cancer by the japan clinical oncology group ( jcog 0501 ) is being performed .
although preoperative chemotherapy might be useful as a standard procedure for advanced gastric cancer after the completion of the trial jcog 0501 , the result is not yet available .
we herein present a long - term survival case of advanced gastric cancer with hepatic and pancreatic invasion with extended surgery performed .
the boundary between the stomach and the lateral segment of the liver or pancreas was ambiguous , so we suspected the tumor to be invading these tissues ( fig .
1 ) . gastrointestinal fiberscopy showed a type 5 tumor in the upper to lower stomach showing easy bleeding ( fig .
it required preoperative transfusion of a total of 1,000 ml of red cell concentrate ( mannitol adenine phosphate erythrocytes ) .
preoperative biochemistry showed that the total bilirubin level was 0.2 mg / dl , 15-minute clearance of indocyanine green was 7.5% , carcinoembryonic antigen was 2.2 ng / ml ( normal < 5.0 ng / ml ) , gastrointestinal cancer antigen level was 1.7 u / ml ( normal < 37 u / ml ) , and alpha - fetoprotein was 3.4 ng / ml ( normal < 20 ng / ml ) .
an upper midline incision extending from the xiphoid process to above the umbilicus was made .
on entering the peritoneal cavity , a small amount of serous peritoneal fluid was found to have no malignancy after cytological examination .
since it was difficult to identify the whole aspect of the tumor from just the abdominal upper midline incision , an additional oblique incision and left thoracotomy was carried out .
a tumor involving almost all of the gastric wall from the cardia to the antrum region was seen .
the tumor showed general expanding growth and invasion through the lateral segment of the liver and pancreas , however , it was considered to be resectable . at first
, mobilization of the stomach with the distal pancreas and spleen from the retroperitoneum was performed .
the abdominal esophagus was dissected with merger excision of a part of the esophageal hiatus .
three drainage tubes were inserted into the left thoracic cavity , winslow 's foramen and left subphrenic space .
total gastrectomy and combined resection of the distal pancreas , spleen and left segment of the liver was completed .
macroscopically , the resected tumor was a 18.5 14.0 cm mass , ulcerated without definite limits , infiltrating into the surrounding wall ( fig .
microscopically , the tumor comprised moderately differentiated tubular adenocarcinoma showing hepatic and pancreatic infiltration ( fig .
lymph node metastasis was proved ( lymph nodes along the lesser curvature 1/1 , lymph nodes along the short gastric vessels 4/9 , lymph nodes along the left gastric artery 1/1 ) .
he received adjuvant tegafur / gimeracil / oteracil chemotherapy which had to be stopped for two cycles because of grade 3 skin side effect ( severe rash ; hand - foot skin reaction ) .
he is in good health now without sign of recurrence 7 years and 8 months after therapy .
the two main staging systems for gastric cancer are the tnm staging system of the international union against cancer ( uicc ) and the japanese classification of gastric carcinoma by the japanese gastric cancer association ( jgca ) . in japan
, the jgca classification has been used widely for clinical practice and research . according to this classification ,
the present case was pathological t4 ( tumor invasion of adjacent structures ) , n2 ( metastasis to group 2 lymph nodes ) , m0 ( no other distant metastases ) , stage iv .
the 5-year survival rate of stage iv gastric cancer with spreading through all gastric domains after surgery is reported to be 11.0% .
our present result seems to be good that the patient has been living with no recurrence for 7 years and 8 months . in recent years ,
discussion about the medical treatment guideline , which shows the method that seems to be the most appropriate among the various ones , has been actively done .
gastric cancer treatment guidelines , published by the jgca in april 2004 [ 7 , 8 ] , suggest that extended surgery , reduction surgery , chemotherapy or hyperthermochemotherapy may be performed in stage iv gastric cancer .
extended surgery is defined as gastrectomy more than standard surgery , including combined resection of involved organs .
the principle of indication of extended surgery is tumor directly invading the surrounding organs , in which combined resection is the only treatment for cure [ 7 , 8 ] . in the present case ,
an immediate treatment was necessary , so it was thought that neoadjuvant chemotherapy did not have to adjust .
total gastrectomy and combined resection of surrounding organs is considered to be the almost only treatment for completely cure .
there are various opinions on whether extended surgery of t4 gastric cancer is clinically meaningful .
the most frequent invasion organs of t4 gastric cancer are the transverse colon , pancreas and liver . in case surgical treatment of t4 gastric cancer
is performed , the following two points should be considered : ( 1 ) postoperative complications accompanying excision of the adjacent organs and ( 2 ) whether improvement of survival rate is obtained or not .
reported that the incidence of complications is 31.2% and the mortality rate resulting from complications is 13.2% .
reported that in t4 gastric cancer , especially tumors more than 10 cm in diameter , local complications such as ruptured suture or intraperitoneal infection increase .
proposed that the best indication for en bloc combined organ resection was invasion limited to only one other organ . on the other hand , shchepotin et al .
reported that patients who present with t4 gastric cancer ( about 20% of the patient population ) will benefit from aggressive en bloc surgical resection and should not be considered unresectable .
concluded that pancreas - invasive gastric cancer cells are likely to advance via lymphatic and vascular routes and survival time is shorter , but curative resection can improve the survival rate , and perioperative treatment should be appropriately designed . in the present case , there seemed perioperatively to be no direct invasion of the tumor into the common hepatic artery or no massive metastasis in lymph nodes along the common hepatic artery .
appleby procedure ( ap ) , that is celiac trunk and common hepatic artery resection without arterial reconstruction , could be avoided .
ap should be performed as rarely as possible . left upper abdominal evisceration ( luae ) might be chosen for the present case , that is the en bloc resection of the left side upper abdominal organs , including the whole stomach , omentum , body and tail of the pancreas , spleen , and transverse colon . however
there has been little evidence on significant survival benefit in the clinical trials of chemotherapy for prevention of recurrence from microresidual tumor after curative resection . at present , the single or multiple agents for postoperative adjuvant chemotherapy most frequently used are the following : 5-fu , cddp , cpt-11 ( irinotecan ) , docetaxel , paclitaxel , uft ( tegafur / uracil ) and s-1 ( tegafur / gimeracil / oteracil ) . in the present case ,
s-1 was administered , but the chemotherapy had to be given up for two cycles because of side effects .
it seems that in the present case , surgery rather than chemotherapy greatly contributed to the long - term survival after resection of advanced gastric cancer . in conclusion , although the prognosis for patients with stage iv gastric cancer involving the liver or / and pancreas remains poor , a long - term survival case among the complete resection cases occasionally exists .
we performed not excessive but extended surgery of advanced gastric cancer with hepatic and pancreatic invasion , and long - term survival was achieved . | a 64-year - old man was transferred to our division with a suspicion of gastric cancer .
computed tomography showed widespread irregular thickening of the stomach walls close to the liver and pancreas .
gastrointestinal fiberscopy showed a type 5 tumor in the upper to lower stomach , histologically diagnosed as tubular adenocarcinoma . gastric cancer with hepatic and pancreatic invasion was diagnosed .
distant metastasis was not proven and complete resection was planned . at laparotomy , the tumor showed general expanding growth and invasion through the lateral segment of the liver and pancreas .
total gastrectomy and combined resection of the distal pancreas , spleen and left segment of the liver were performed .
hepatic and pancreatic invasion and lymph node metastasis were microscopically proven .
pancreatic fistula occurred postoperatively .
on postoperative days 40 , he was discharged .
he received two cycles of adjuvant tegafur / gimeracil / oteracil chemotherapy .
he has had no sign of recurrence for 7 years and 8 months . |
giemsa dark and light bands are generally thought to correspond to gc - poor and gc - rich regions , respectively .
giemsa light bands are gene - rich and contain most housekeeping genes as well as a large number of cpg islands , whereas giemsa dark bands are gene - poor and preferentially contain tissue - specific genes .
hence , light bands are transcriptionally more active when compared to dark bands and also have an open chromatin configuration which together with the high content in gc can have an important role in provirus integration .
for example , dna replication timing during cell cycle differs between both ; light bands are early - replicating , whereas dark bands are late replicating .
giemsa bands are also related to chromatin structures as the chromatin in dark bands is more condensed than in light bands during both metaphase and interphase .
another difference between these two giemsa bands is that the dna of giemsa dark bands are located at the nucleus periphery while the dna of giemsa light bands is in the interior of the nucleus .
fss are hereditary loci of human chromosomes susceptible to occurrence of breaks , gaps , or rearrangements when under stress conditions or treated with specific chemical agents [ 57 ] .
according to the frequency of their distribution in the human population , fss can be divided in two distinct groups : common fragile sites ( cfss ) present in all individuals and rare fragile sites ( rfss ) that are present in less than 5% of the population , and these two groups can also be subdivided according to the inducing agent [ 810 ] .
both types of fss have the capacity to form secondary structures that can interfere with elongation in replication or even cause failure in chromatin condensation .
fss are also involved in sister chromatid exchanges translocations and deletions and in intrachromosomal gene amplifications .
cfss are very unstable regions because they contain sequences of high flexibility and are regions of late replication and also correspond to transition regions in replication timing .
several authors have shown that fragile sites are preferential integration targets for some viruses , for example , epstein - barr virus and human papillomaviruses hpv16 and hpv18 .
recently , christiansen et al . showed that transcriptionally active regions and fss are the preferred targets for chromosomal hpv integration in cervical carcinogenesis . the human immunodeficiency virus ( hiv )
is a retrovirus whose stable integration in the human genome is essential for completing its life cycle [ 20 , 21 ] .
the rna genome of hiv is converted into dna by the reverse transcriptase ( rt ) enzyme that is transported to the nucleus .
viral integration into the dna occurs in three steps , ( i ) processing ; ( ii ) joining ; and ( iii ) postintegration repair . after integration
there are two different types of viruses : hiv-1 , identified first in 1983 , and hiv-2 , later discovered in 1986 .
viral integration in human cells can affect gene expression , leading to molecular and epigenetic alterations , and can even activate oncogenes .
concluded that integration sites of hiv are not randomly distributed in the human genome , but in regional hotspots .
the same group also found that integration sites are related to gene - rich sites which can allow a more efficient expression of the viral genome .
retroviruses can be used as vectors in gene therapy since they can integrate stably in the host genome [ 30 , 32 ] and they have the capacity to introduce genetic material in target cells .
therefore , it is important to understand the integration preferences of hiv since knowledge of the integration sites in the human genome can help , for example , to choose gene - delivery vectors
. the integration preferences of hiv in light or dark bands and in fss are not clear . thus , our main objective was to study the hiv integration preferences in giemsa bands and in fss using bioinformatics and statistical analyses .
more specifically we aimed to understand the integration preferences of hiv-1 isolated from jurkat t cells in giemsa bands and fss by the use of an in silico approach based on statistical analysis which may complement laboratorial studies and predict hiv constraints and preferences of integration in the human genome .
for the hiv-1 isolated from jurkat t cells wang et al . have supplied the exact position of the integration sites .
the positions of giemsa bands used in this study were obtained in silico by niimura and gojobori , available from the center for information biology and dna data bank of japan , national institute of genetics , in http://yosniimura.net/research/coordinates.html .
regarding fss , the human genome was divided in frs and nfrs , according to their positions .
a list of fss was obtained from mrasek et al . and completed with fss from lukusa and fryns .
two consecutive bands associated with fss were grouped to form a fragile region ( fr ) and a region between two frs was considered a nfr [ 35 , 36 ] . the y chromosome was not considered because it does not have well defined fss ; there is only a possibility of existing one fs .
integration sites of hiv-1 were colocated with giemsa dark bands and classified in two groups : yes if they colocalized and no if they did not .
some integrations occurring in the centromeres and short arm of the acrocentric chromosomes were excluded from the total .
thus for hiv-1 we have integrations in 24 chromosomes in a total of 42912 integration sites .
then a measure was calculated to determine in which type of band the virus integrates .
the measure is designated as integration intensity number and is given by(1)idark band = nyesldark band;ilight band = nnollight band , where nyes represent the number of viral integrations in dark bands , ldark band is the length of the dark bands , nno is the number of viral integrations in light bands , and llight band is the length of the light bands .
after the calculations of the measure , one pair ( x , y ) was obtained for each chromosome , where x represents the measurement value in dark bands and y the measurement value in light bands . to compare dark bands with light bands graphical representations for the measure
in order to see the preferences of integration of the virus , in each graphic the line y = x was represented that allows the visualization of the number of chromosomes in which y > x and y < x , in other words , if the virus preferentially integrated in dark or in light bands . in order to statistically verify the results obtained graphically two nonparametric tests were applied that enable us to compare two dependent samples , the sign and the wilcoxon tests . in both tests a significance level of 1% was used to test our hypothesis .
the same methodologies were applied in fss , also classifying the integrations in two groups : yes if they colocalized with a fr and no if they colocalized with a nfr .
integrations of hiv-1 for the 23 chromosomes were obtained , in a total of 44150 integrations sites .
the intensity number for the hiv-1 isolated from jurkat t cells was calculated and the graphical representation is presented in figure 1 for giemsa bands . for the wilcoxon test ,
tobs ( 59 ) was lower than tcritic ( 69 ) , so the hypothesis of equal intensity at 1% level can be clearly rejected .
it means that the intensity of integration is not equal in the two types of bands and it can be concluded that hiv-1 isolated from jurkat t cells integrates with more intensity in giemsa light bands .
our in silico results obtained for giemsa bands indicate that hiv-1 isolated from jurkat t cells integrates preferentially in light bands , which have a high content in gc .
these results are in line with previous studies which reveal that hiv favours integration in transcriptionally active units [ 34 , 39 , 40 ] which are associated with regions of high gc content and high gene density .
when integrated in transcriptionally active regions this ensures viral gene transcription [ 41 , 42 ] .
the virus has a limited time to replicate since t cells infected with hiv have a very short half - time .
regions that are more transcriptionally active allow an efficient maintenance of the replication cycle of the virus since they permit a higher provirus transcription , increasing viral gene expression .
elleder et al . also found that hiv integrates preferentially in giemsa light bands and in regions with open chromatin which favours integration , since the in enzyme of hiv-1 interacts with components of the chromatin remodeling complex .
the interaction with ledgf / p75 accounts for the karyophilic properties and chromosomal targeting of hiv-1 in . nevertheless
, one must note that virus integration in transcriptionally active regions with an open chromatin conformation , that is , light bands , could also be a by - product of the integration of hiv-1 integrase with components of the chromatin remodeling complex , which could impact on our interpretation of hiv integration site preference .
marini et al . also revealed that the cellular genes that are targeted by hiv-1 are enriched in open chromatin marks associated with the nuclear complex pore that are constituted by nucleoporins which participate in hiv-1 transcriptional regulation .
this report supports our results for the integration in giemsa light bands that have a less condensed chromatin than giemsa dark bands .
moreover , the same group of authors also found that the areas of open chromatin that are targeted by the hiv-1 preintegration complex are those proximal to the nuclear pore .
hiv-1 could also have a preference for giemsa light bands because they have active genes ; thus the preference for active gene regions may have been developed to favour hiv gene expression after integration .
another feature that supports our results is that light bands possess high levels of histone acetylation , namely , of histones h3 and h4 , which enable access of transcription factors [ 48 , 49 ] . in this regard wang et al
. demonstrated that the frequency of integration of hiv is associated with epigenetic modifications including h3 and h4 acetylation .
figure 2 shows the result of intensity number for the hiv-1 virus isolated from jurkat t cells in frs . for the sign test
the wilcoxon test resulted in a tobs of 82 and a tcritic of 62 which leads us to accept the hypothesis of equal integration intensity .
thus , hiv-1 isolated from jurkat t cells integrates with equal intensity in frs and nfrs . in
the methodology followed , two consecutive bands associated with fss were grouped to form a fr and a region between two frs was considered a nfr .
we verified that hiv-1 isolated from jurkat cells integrates with equal intensity in frs and in nfrs .
other known viruses , such as hepatitis b , epstein - barr virus , hpv16 , and hpv18 , integrate more in fss . these differences between hiv and the other viruses may be explained by the different phases of the cell cycle in which the virus enters in human cells .
pyeon et al . verified that progression in cell cycle through mitosis is critical to hpv infection while hiv can infect nondividing cells [ 24 , 31 ] . the fact that hiv does not have a tendency to integrate in frs could be related to the structure of the fss which are vulnerable to dna breaks , which may not facilitate viral integration or the conclusion of viral replication .
moreover , fss have the tendency to form secondary structures , which interfere with replication thus hampering viral integration and replication .
genomic instability is another characteristic of fss which does not favour viral integration as it needs to integrate its genome stably in hosts to complete its life cycle .
hiv integrates more in regions with gc content which is not the case of fss that are predominantly constituted by at [ 15 , 52 ] . in both graphics for giemsa bands and for fss we found that there were some chromosomes that differ from the rest , for example , chromosome 17
this chromosome is rich in protein coding genes and has a high cg content which indicates a high gene density , besides being rich in sines .
our aim was to develop and apply an approach intended to predict the conditions and constraints of hiv-1 insertion in the human genome . at the present stage
, our approach seems to adequately predict most of the conditions unravelled by empirical data but is still not exempt from weaknesses .
we concluded that hiv-1 isolated from jurkat t cells integrates with more intensity in giemsa light bands and with equal intensity in frs and nfr .
other factors such as the cell cycle phase and the cellular type that act in vivo could also influence the integration site selection of the virus .
moreover , the distribution of integration sites may be altered in vitro due to repeated cell division and selection for certain clones
. nevertheless , our data are in line with previous reports and may contribute to the understanding of viral integration in hiv disease and gene therapy strategies . | chromosomal fragile sites ( fss ) are loci where gaps and breaks may occur and are preferential integration targets for some viruses , for example , hepatitis b , epstein - barr virus , hpv16 , hpv18 , and mlv vectors . however , the integration of the human immunodeficiency virus ( hiv ) in giemsa bands and in fss is not yet completely clear .
this study aimed to assess the integration preferences of hiv in fss and in giemsa bands using an in silico study .
hiv integration positions from jurkat cells were used and two nonparametric tests were applied to compare hiv integration in dark versus light bands and in fs versus non - fs ( nfss ) .
the results show that light bands are preferential targets for integration of hiv-1 in jurkat cells and also that it integrates with equal intensity in fss and in nfss .
the data indicates that hiv displays different preferences for fss compared to other viruses .
the aim was to develop and apply an approach to predict the conditions and constraints of hiv insertion in the human genome which seems to adequately complement empirical data . |
molecules that target and inhibit
v3 , v5 , and 51 integrins have
generated great interest
because of the role of these receptors in mediating angiogenesis and
metastasis .
attempts to increase the binding affinity and hence the
efficacy of integrin inhibitors by dimerization have been marginally
effective . in the present work
, we achieved this goal by using oxime - based
chemical conjugation to synthesize dimers of integrin - binding cystine
knot ( knottin ) miniproteins with low - picomolar binding affinity to
tumor cells .
a non - natural amino acid containing an aminooxy side
chain was introduced at different locations within a knottin monomer
and reacted with dialdehyde - containing cross - linkers of different
lengths to create knottin dimers with varying molecular topologies .
dimers cross - linked through an aminooxy functional group located near
the middle of the protein exhibited higher apparent binding affinity
to integrin - expressing tumor cells compared with dimers cross - linked
through an aminooxy group near the c - terminus . in contrast , the cross - linker
length had no effect on the integrin binding affinity .
a chemical - based
dimerization strategy was critical , as knottin dimers created through
genetic fusion to a bivalent antibody domain exhibited only modest
improvement ( less than 5-fold ) in tumor cell binding relative to the
knottin monomer .
the best oxime - conjugated knottin dimer achieved
an unprecedented 150-fold increase in apparent binding affinity over
the knottin monomer .
also , this dimer bound 3650-fold stronger and
inhibited tumor cell migration and proliferation compared with cilengitide ,
an integrin - targeting peptidomimetic that performed poorly in recent
clinical trials , suggesting promise for further therapeutic development . |
|
paramedics are frequently required to perform tracheal intubation , a potentially life - saving manoeuvre in severely ill patients , in the prehospital setting .
it is a difficult skill to acquire and to maintain , especially with limited training opportunities .
simulation training is an essential educational strategy for health care systems to improve patient safety ; there is good evidence that procedural simulation improves actual operational performance in clinical settings .
the gold standard device used for intubation is the macintosh direct laryngoscope ( dl ) .
however , failures of tracheal intubation when using macintosh laryngoscope have been reported in up to 30% of intubations by paramedics .
recent studies have shown that novice medical students have low initial success when using a standard laryngoscope .
several devices have been developed as alternatives to dl to aid difficult intubation such as glidescope video laryngoscope ( gvl ) and intubating laryngeal mask airway ( i - lma ) .
it may be used to facilitate intubation and secure the airway in patients with normal and abnormal airways or in emergency situations .
gvl provides a high - grade , indirect close - proximity view of the glottis on a monitor screen without alignment of the oral , pharyngeal and laryngeal axes .
the efficacy of i - lma and gvl , when used by paramedics , is not known , and the relative efficacies of these devices in comparison to the macintosh dl have not been compared in a single study .
we , therefore , wished to evaluate whether gvl and i - lma facilitate and improve the intubation success rate and could be easily learned and performed by paramedic students when compared with macintosh dl in easy and simulated difficult intubation ( a manikin with immobilised cervical spine ) .
following local ethical committee approval , and written informed consent , a total of 100 paramedic students with no prior experience in performing tracheal intubation consented to participate in the study .
all participants were given a 60 min lecture on the principles of airway management , including dl , i - lma facilitated tracheal intubation and intubation with gvl .
each participant was then allowed to practice one intubation with each device , before the commencement of the study .
the design of this study was a randomised crossover trial . for normal airway scenario using laerdal airway management trainer ,
each participant performed tracheal intubation with each device in random order while for simulated difficult airway scenario using simman manikin with cervical immobilisation , each participant performed tracheal intubation with each device in the same order used by him in normal airway scenario .
a number 3 macintosh blade and 7 mm cuffed tracheal tube were used for dl .
a 7 mm cuffed tracheal tube was used for gvl . a size 3 lma fastrach and its special 7 mm cuffed tracheal tube
the primary outcome measure was the duration of successful tracheal intubation attempt which was defined as the time taken from the insertion of the device into the mouth until the endotracheal tube ( ett ) was correctly positioned by each participant .
participants confirmed ett position by visualising its tip passing through the vocal cords and inflating the manikin 's lungs using an ambu bag attached to the tube .
if the participant visualised the ett tip passing through the vocal cords , the attempt was considered complete , but if he was unsure about ett position , the attempt was considered complete after inflating the manikin 's lungs . after each intubation attempt
an unsuccessful ( failed ) intubation attempt was defined as an attempt in which the trachea was not intubated , or when intubation of the trachea required > 120 s to perform .
other primary end points included the rate of successful endotracheal intubation with each device and the number of attempts required .
secondary endpoints included severity of dental trauma , ease of intubation and the number of optimisation manoeuvres required .
the severity of dental trauma was calculated based on the number of audible teeth clicks ( 0 , 1 or 2 ) with the laerdal airway trainer , and based on a grading of pressure on the teeth ( none = 0 , mild = 1 , moderate / severe 2 ) in the simman manikin .
each participant was asked to report the laryngeal view obtained during laryngoscopy after each intubation attempt as the following : most ( > 50% ) of the vocal cords visible ; some ( < 50% ) of the vocal cords visible ; the only epiglottis visible ; or no epiglottis visible .
these were converted into grades 1 - 4 according to the cormack and lehane classification .
the ease of intubation was measured by asking every student to evaluate the ease of his intubation attempt for each device using a linear scale ( 0 = easy , 10 = difficult ) .
the number of optimisation manoeuvres required ( re - adjustment of head position , use of a bougie or stylet , external pressure , second assistant ) to aid tracheal intubation were recorded with yes ( used = 1 ) or no ( not used = 0 ) . at the end of the protocol , each participant scored his confidence with each device ( from 0 = not at all confident to 10 = extremely confident ) .
statistical analysis was carried out by the statistical package for the social sciences ( spss ) 15.0 for windows software ( spss inc . ,
data for the success of tracheal intubation attempts were analysed using the chi - square test .
data for the duration of the first and the successful intubation attempts , the number of intubation attempts , the number of optimisation manoeuvres , the severity of dental trauma , ease of intubation and device confidence scores , were analysed using one - way anova or kruskal - wallis test followed by post hoc test .
parametric data were presented as mean ( standard deviation ) whereas non - parametric data were presented as median ( interquartile range ) .
the study was conducted by 100 paramedic students who consented to participate in the study .
no participant had previously performed tracheal intubation and all participants completed the study . in the normal airway scenario ,
the paramedic students performed successful endotracheal intubation in 80 out of 100 attempts ( 80% ) by dl , in 95 out of 100 attempts ( 95% ) by i - lma , and in 94 out of 100 attempts ( 94% ) by gvl ( p < 0.001 ) [ table 1 ] .
14 attempts from the failed attempts were oesophageal in dl compared to 0 and 2 oesophageal attempts in i - lma and gvl , respectively . the duration of both the first and the successful tracheal intubation attempts was longer with the dl ( 31.5 { 14 - 49 } s ) compared to i - lma , and gvl ( 20 { 11 - 31 } , 22 { 10 - 34 } s , respectively ) ( p < 0.001 ) [ table 1 ] .
significantly more intubation attempts were required with the dl compared to i - lma , and gvl ( p < 0.001 ) [ table 1 ] .
glottis visualisation and cormack and lehane grades were significantly better with gvl than dl ( p < 0.001 ) [ table 2 ] .
the incidence and severity of dental trauma was significantly more with dl , compared to i - lma , and gvl ( p < 0.001 ) [ table 3 ] .
number of optimisation manoeuvres required to perform tracheal intubation using i - lma , and gvl were fewer , compared to dl ( p < 0.001 ) [ table 3 ] .
intubation time and rate of successful intubation with direct laryngoscopy , intubating laryngeal mask airway and glidescope video laryngoscope in easy and simulated difficult airway in manikins laryngoscope views obtained when using direct laryngoscopy , intubating laryngeal mask airway and glidescope video laryngoscope in easy and simulated difficult airway in manikins dental clicks and optimization manoeuvres incidence during intubation with direct laryngoscopy , intubating laryngeal mask airway and glidescope video laryngoscope in easy and simulated difficult airway in manikins in the difficult airway scenario , the paramedic students performed successful endotracheal intubation in 65 out of 100 attempts ( 65% ) by dl , in 94 out of 100 attempts ( 94% ) by i - lma , and in 94 out of 100 attempts ( 94% ) by gvl ( p < 0.001 ) [ table 1 ] . 20 attempts from the failed attempts were oesophageal in dl compared to 0 and 1 oesophageal attempts in i - lma and gvl , respectively . the duration of both the first and the successful tracheal intubation attempts was longer with the dl ( 40 { 22 - 48 } s ) compared to i - lma , and gvl ( 19 { 12 - 28 } , 21 { 15 - 35 } s , respectively ) ( p < 0.001 ) [ table 1 ] .
significantly more intubation attempts were required with dl compared to i - lma , and gvl ( p < 0.001 ) [ table 1 ] .
glottis visualisation and cormack and lehane grades were significantly better with gvl than dl ( p < 0.001 ) [ table 2 ] .
the incidence and severity of dental trauma were significantly more with dl , compared to i - lma , and gvl ( p < 0.001 ) [ table 3 ] .
number of optimisation manoeuvres required to perform tracheal intubation using i - lma and gvl were fewer , compared to dl ( p < 0.001 ) [ table 3 ] .
paramedic students described dl as more difficult compared to the other devices ( p < 0.05 ) [ table 4 ] . 12 paramedic students ( 12% )
selected dl , 50 paramedic student ( 50% ) selected i - lma and 38 paramedic students ( 38% ) selected gvl as their first preference [ table 4 ] .
the paramedic students were significantly more confident in performing tracheal intubation using i - lma , and gvl compared to dl ( p < 0.05 ) [ table 4 ] .
the macintosh laryngoscope is the most popular device for tracheal intubation and considered by many as the gold standard .
however , failures of tracheal intubation when using macintosh laryngoscope have been reported in up to 30% of intubations by paramedics .
several devices have been developed as alternatives to dl to aid difficult intubation , gvl makes it easier to view the glottis , and facilitates the placement of the tracheal tube through the vocal cords into the trachea by means of a side channel .
i - lma is a supraglottic airway device with unique advantages such as blind intubation and the possibility of ventilation between intubation attempts . in our study , we found higher overall success rates and shorter intubation times with i - lma and gvl compared to dl in both easy and simulated difficult airway scenarios .
specific advantages of i - lma and gvl over dl include fewer intubation attempts , fewer optimization manoeuvres , less dental trauma , and they were less difficult to use . overall , paramedic students described dl as more difficult .
the participants were significantly more confident in performing tracheal intubation using i - lma , and gvl compared to dl .
studies comparing learning and performance of endotracheal intubation using dl , gvl and i - lma have shown varying results .
similar to our study , there are previous studies comparing gvl and i - lma with macintosh dl by paramedic students for endotracheal intubation in easy airway scenario and by medical students who had no prior airway management experience in simulated easy and difficult laryngoscopy scenarios .
they found intubation with either gvl or i - lma to be easier with better intubation success rates , reduced number of intubation attempts , and reduced number of optimisation manoeuvres required compared with dl .
previous studies on the use of i - lma in prehospital care compared with dl showed comparable or superior results . the success rate of the first intubation attempt was higher and the time to ett placement was shorter with the i - lma , especially in the difficult - to - manage airway compared with dl . the gvl is recommended for improving the laryngoscopy view in paediatric patients with a difficult airway compared with dl .
however in contrast to these results , the number of attempts to successful intubation with either gvl or dl did not differ but the time required for intubation with gvl was longer . in the previous study , using a simulation manikin in normal and difficult airway scenarios , the video laryngoscope compared with dl provided an enhanced view of the cords using less time , increased intubation success and decreased the time to intubation . compared to dl ,
glidescope video laryngoscopy is associated with improved glottic visualisation , particularly in patients with potential or simulated difficult airways . in a manikin study comparing force and pressure distribution using macintosh and glidescope laryngoscopes in normal and difficult airways , the glidescope allowed the participants to obtain a successful intubation applying a lower force . a flatter and more uniform pressure distribution , a higher successful rate , and a better glottis view
were observed with the glidescope . in a retrospective observational study of all patients intubated in a single academic emergency department with a level i trauma centre , the gvl had a higher overall success rate , and lower number of oesophageal complications and offered an excellent option to maximise first - attempt success for airway management compared with dl . in a study comparing four different devices , including dl and gvl , the overall success rate for either device was not different .
another study found that the ease of intubation with gvls was similar to conventional laryngoscopy when performed by novice anaesthesiologists .
hence , their results may not be applicable to other , especially channelled vls like gvl , which was used in our study . in their study , malik et al .
compared five different methods , including i - lma and gvl , with dl in the hands of experienced anaesthesiologists and found that the intubation success rate of i - lma ( 100% ) and gvl ( 96.7% ) was superior to dl ( 90% ) .
similarly , gvl and i - lma provided higher success rates compared with dl in our study .
however , they concluded that i - lma had advantages over dl , but gvl did not in contrary to our results .
these studies suggest that intubation with gvl can be easily learned and performed with a higher success rate after a short period of training . in our study
, gvl provided a higher success rate ( 91.7 vs. 78.5% ) and a lower difficulty score ( 1.84 vs. 2.54 ) compared to dl , among our participants who had not used gvl before .
however , paramedic students defined i - lma as easier than gvl , and they mostly preferred i - lma . in their study , malik et al . compared five different methods , including i - lma and gvl , with dl and found that the intubation success rate of i - lma ( 100% ) and gvl ( 96.7% ) was superior to dl , in the novice
they provide better airway management regarding rapidity , success rate and ease of intubation , with less dental trauma and optimisation manoeuvres compared to macintosh dl in both normal and difficult airway scenarios .
similar comparative studies on humans are needed to further delineate the advantages of gvl and i - lma .
this research is totally funded ( including materials and fees for consultation ) by institute of scientific research and revival of islamic heritage - umm al - qurra university .
this research is totally funded ( including materials and fees for consultation ) by institute of scientific research and revival of islamic heritage - umm al - qurra university .
| background and aims : glidescope video laryngoscope ( gvl ) and intubating laryngeal mask airway ( i - lma ) may be used to facilitate intubation and secure the airway in patients with normal and abnormal airways .
the aim of this study was to evaluate whether ( gvl ) and ( i - lma ) facilitate and improve the tracheal intubation success rate and could be learned and performed easily by paramedic students when compared with macintosh direct laryngoscopy ( dl).methods : this study was a prospective , randomised crossover trial that included 100 paramedic students .
macintosh dl , i - lma and gvl were tested in both normal and difficult airway scenarios .
each participant was allowed up to three intubation attempts with each device , in each scenario .
the time required to perform tracheal intubation , the success rate , number of intubation attempts and of optimisation manoeuvres and the severity of dental trauma were recorded .
statistical analysis was performed using chi - square , one - way anova , or kruskal - wallis test as appropriate , followed by post hoc test.results:gvl and i - lma required less time to successfully perform tracheal intubation , showed a greater success rate of intubation , reduced the number of intubation attempts and optimization manoeuvres required and reduced the severity of dental trauma compared to macintosh dl in both normal and difficult airway scenarios.conclusion:gvl and i - lma provide better airway management than macintosh dl in both normal and difficult airway scenarios . |
precise measurement of the anterior chamber is important in many aspects of ophthalmology , such as assessing glaucoma risk [ 1 , 2 ] and surgical planning and intraocular lens ( iol ) power calculation [ 3 , 4 ] .
previous studies have focused on the measurement of anterior chamber depth ( acd ) or the anterior chamber angle ( aca ) .
accurate measurement of anterior chamber volume ( acv ) has been difficult historically due to technology limitations .
recent development of swept - source optical coherence tomography ( ss - oct casia ss-1000 oct , tomey corporation , nagoya , japan ) is a form of fourier - domain oct ( fd - oct ) , which uses a monochromatic tunable fast scanning laser source and a photodetector to detect wavelength - resolved interference signal instead of using a spectrometer as in spectral - domain oct ( sd - oct ) .
thus , with 30,000 a - scans per second , ss - oct allows rapid and precise anterior chamber measurement . as a variation of fd - oct
, ss - oct has advantages of measurement speed and sensitivity over time - domain oct . besides , ss - oct also has advantages over sd - oct , such as higher robustness and capability of separating the real oct image from its mirror image [ 8 , 9 ] .
previous studies comparing ss - oct and sd - oct in posterior segment measurements have proved these advantages .
ss - oct is also superior in detecting details of choroid - scleral interface and choroidal sublayer [ 11 , 12 ] .
studies comparing ss - oct to other octs in observations of anterior segment were rare , yet ss - oct has been widely used in the measurement of anterior chamber [ 1316 ] .
anterior chamber anatomy may vary with aging , thickening of the lens , liquefaction of the vitreous , corneal changes , gender differences , and elongation of the axial length ( axl ) in high myopia [ 18 , 19 ] .
however , few studies have evaluated the effects of all these factors on the volume of the anterior chamber in normal cataract patients . in this study
, we used the ss - oct , to investigate acv in normal cataract patients and to better understand factors that impact acv measurement .
the institutional review board of the eye and ent hospital of fudan university approved this prospective study .
all procedures adhered to the tenets of the declaration of helsinki and were conducted in accordance with the approved research protocol .
ninety - two eyes of consecutive cataract patients at the eye and ent hospital of fudan university , between may 2015 and august 2015 , were enrolled .
exclusion criteria included zonular weakness , corneal disease , glaucoma , previous trauma , or any ocular surgical history .
posterior vitreous detachment ( pvd ) was evaluated by b - scan by the same senior technician .
axl was measured with an iol master ( carl zeiss ag , oberkochen , germany ) .
the lens opacity was assessed according to the lens opacities classification system iii ( locs iii developed by dr .
leo chylack , mass eye and ear ) under slit lamp examination by one investigator .
locs iii system has 4 categories : nc = nuclear color , no = nuclear opalescence , c = cortical cataract , and p = posterior subcapsular cataract . according to the previous study , nc and no were graded from 0.1 to 6.9 and
ss - oct anterior segment scan mode was used with 128 radial scans , with a depth of 6 mm and a length of 16 mm .
patients were instructed to fixate on the internal target and pull down the lower lid while the technician elevated the upper lid to expose the limbus .
a total of 64 b - scans taken from the anterior segment scan mode were analyzed for measurement of acv .
the instrument software automatically detected the boundaries of cornea , iris , and lens for each image , as shown in figure 1 .
manual adjustment was made if the software failed to detect the boundaries at the correct location .
angle width parameters derived from ss - oct included the angle opening distance 500 ( aod 500 ) , the trabecular iris space area 500 ( tisa 500 ) , the angle recess area 500 ( ara 500 ) , and the trabecular iris angle 500 ( tia 500 ) , which were determined as previously described for the superior , inferior , nasal , and temporal angles .
student 's t - test was used to compare differences in mean measurements between men and woman after normality tests and homogeneity of variance tests .
pearson and spearman 's correlation analyses were used to investigate the relationships between acv and demographic data ( pearson for continuous variables and spearman for categorical variables ) .
there were 29.35% ( 27/92 ) high myopic ( < 6.0 d ) patients and the average axl was 25.09 2.96 mm .
the average acv was 139.80 38.21 mm , range 59.41 to 254.09 mm .
we performed univariate analysis for influencing factors of acv and multivariable analysis to adjust for independent covariates .
the average acv was significantly larger in male patients than in female patients ( figure 2(a ) , p = 0.001 ) .
acv was negatively correlated with age and locs iii c grade of the lens ( figures 2(b ) and 2(c ) , pearson 's correlation analysis , r = 0.443 , p < 0.001 , and spearman 's correlation analysis , = 0.450 , p < 0.001 ) .
locs iii nc , no , and p grades were not correlated with acv narrowing ( spearman 's correlation analysis , = 0.127 , p = 0.246 , = 0.146 , p = 0.180 , = 0.024 , and p = 0.819 , resp . ) .
acv was also increased with increased axl ( figure 2(d ) , pearson 's correlation analysis , r = 0.552 , p < 0.001 ) .
table 3 presents the result from the multiple linear regression . with all of the covariates entered into the model ,
female gender ( p = 0.002 ) , greater age ( p = 0.015 ) , higher locs iii c grade ( p = 0.043 ) , and less myopia axl ( p = 0.001 ) were all correlated with decreased acv ( f = 10.252 p < 0.001 r = 0.498 ) .
objective , precise measurement of the anterior chamber volume has importantsignificance as a predictor of narrow angle glaucoma risk , assessment of pupil block , in addition to surgical planning in ac iol and phakic iol placement .
previous reports of anterior chamber volume measurements relied on the scheimpflug system , which requires protracted cooperation of patients during testing . by using scheimpflug system
, previous studies found that , with increasing age , acd and acv diminished and no correlation was found between acv and anterior chamber angle .
however , the scheimpflug system could only provide an estimation rather than a direct visualization of the anterior chamber , resulting in an inaccurate assessment of acv which also lacks reproducibility .
casia ss-1000 oct used a swept laser source , which could show high - resolution 3-dimensional images of anterior chamber by a very high - speed scanning system .
previous studies reported variable influencing factors on acv , including gender , acd , and age
. however , it remains unclear whether these factors remain significant after adjusting for independent covariates . in the study , we reported ss - oct data in healthy cataract patients and found that acv varied significantly among different subjects . influencing factors that contributed to reduced acv were female gender , increased age , locs iii c grade , and lower axl .
the female gender was related to decreased acv possibly because generally females have shorter axl , smaller body habitus , eyes , and therefore a narrower anterior chamber than males . however , there were almost twice as many females as males in our study , which might be a bias .
this was a limitation and we will verify the result in a population with similar percentages of men and women in the future . besides , the anterior chamber became shallower over time with thickening of the crystalline lens , which partly explained the negative correlation between increased age and acv .
consequently , locs iii c grade was also negatively correlated with acv . after adjusting for independent covariates , gender , age , locs iii c grade , and axl
these factors , along with ss - oct evaluation , are important for assessing risk in glaucoma patients and may impact the timing of therapeutic cataract surgery in patients with anatomic narrow angles or those with cataract - induced narrow angles .
moreover , patients needing ac iol or phakic iol placement can benefit from ss - oct assessment as a preoperative tool to assess risk and aid surgical planning .
further studies can examine if these risk factors are also found in patients before senile cataracts develop , in patients with open or narrow angle glaucoma , and how cataract surgery impacts the postoperative acv in patients at risk of narrow angles .
in addition , several anterior segment imaging modalities , such as scheimpflug system , anterior segment oct , and ultrasound biomicroscopy ( ubm ) , can be used to evaluate the volume of the anterior chamber .
however , compared to other instruments , the ss - oct used in the current study has several advantages : ( 1 ) it is a noncontact optical system compared to ubm , which avoids distortion of the eye anatomy and angle and reduces contagion .
( 2 ) it is faster than the scheimpflug system or ubm because of the swept laser source and captures data of acv in less than 1 second .
thus , patients could cooperate with this test easier than the scheimpflug system or ubm .
( 3 ) similarly to ubm , ss - oct provides direct angle visualization and generates an acv which is more precise and objective than the scheimpflug system of angle estimation . besides
, this ss - oct could automatically show accurate measurements of anterior chamber angle , which has advantages over ubm .
however , other methods still have their value in clinical ophthalmology . for instance , in cases of corneal scaring , ubm could still evaluate anterior chamber parameters while octs could not . to conclude , ss - oct could provide faster , objective , and more precise measurement of anterior segment than other methods in some conditions , which may have advantages in evaluation of acv but still need further confirmation in the future study . with ss - oct
, we found female gender , increased age , higher locs iii cortical grading , and decreased axl to be important predictors for smaller acv in normal cataract patients . | purpose . to evaluate the anterior chamber volume in cataract patients with swept - source optical coherence tomography ( ss - oct ) and its influencing factors
. methods .
anterior chamber volume of 92 cataract patients was evaluated with ss - oct in this cross - sectional study .
univariate analyses and multiple linear regression were used to investigate gender , age , operated eye , posterior vitreous detachment , lens opacity grading , and axial length ( axl ) related variables capable of influencing the acv . results .
the average acv was 139.80 38.21 mm3 ( range 59.41 to 254.09 mm3 ) .
the average acv was significantly larger in male patients than in female patients ( p = 0.001 ) .
acv was negatively correlated with age and locs iii cortical ( c ) grading of the lens ( pearson 's correlation analysis , r = 0.443 , p < 0.001 , and spearman 's correlation analysis , = 0.450 , p < 0.001 ) .
acv was also increased with axl ( pearson 's correlation analysis , r = 0.552 , p < 0.001 ) .
multiple linear regression showed that , with all of the covariates entered into the model , gender ( p = 0.002 ) , age ( p = 0.015 ) , locs iii c grade ( p = 0.043 ) , and axl ( p = 0.001 ) were still associated with acv ( f = 10.252 p < 0.001 r2 = 0.498 ) . conclusion .
with ss - oct , we found that , in healthy cataract patients , acv varied significantly among different subjects . influencing factors that contribute to reduced acv were female gender , increased age , locs iii c grade , and shorter axl . |
blue nevus is considered a benign lesion that only in rare cases may progress into melanoma .
the term malignant blue nevus includes both a melanoma that arose within a common or a cellular blue nevus or a de novo melanoma simulating a blue nevus .
a peculiar type of blue nevus is the so - called large plaque type blue nevus with subcutaneous cellular nodules ( lptbn - sn ) , a clinical variant characterized by large dimensions , plaque - like features , localization on the trunk , presence since birth or early childhood , and onset of discrete subcutaneous nodules many years later .
a 56-year - old caucasian man was hospitalized in our hospital in may 2012 because of a hemorrhagic cystitis with fever .
the patient was affected by severe cognitive impairment , mild blood hypertension , prostatic hypertrophy and had a permanent catheter . during the hospitalization ,
a dermatologic visit was required because of a large blue lesion on the trunk . on clinical examination ,
a large mammillated blue plaque involving more than half of the chest wall was observed ( figure 1 ) .
the anamnesis revealed that the patient was born with a large congenital blue nevus that had developed subcutaneous nodules in the last 15 years , slowly increasing in number and size .
the relatives of the patient reported on a previous large and deep biopsy , performed almost nine and a half years before in another hospital , which showed the presence of a melanoma on a congenital blue nevus . at that time , due to the dimensions of the lesion and the psychiatric illness of the patient , no treatment was performed .
unexpectedly , no metastases had developed and only a slow increase of the nodules was observed in the following years .
we decided to repeat a skin biopsy of a subcutaneous nodule , and the histopathology was consistent with a benign cellular blue nevus , while the revision of the previous biopsy confirmed the diagnosis of melanoma on a congenital blue nevus .
a strict follow - up of the patient every four months was undertaken , and after one year we observed the rapid increase of two of the subcutaneous nodules with ulceration ( figure 2 ) .
a new biopsy was performed , and in this case a histopathologic diagnosis of melanoma was made , confirming the final diagnosis of melanoma arising on an lptbn - sn ( figure 3 ) .
the patient was sent to the palliative care center of our hospital and died three months later .
the term large plaque type blue nevus with subcutaneous cellular nodules was coined in 1999 and refers to a huge blue nevus , usually located on the trunk , that develops subcutaneous nodules many years after the lesion has appeared [ 8 , 9 ] .
histopathologically , the nodules are characterized by areas resembling common blue nevi and areas resembling cellular blue nevi , with infiltration of subcutaneous and soft tissues , including the fascia and mammary tissues .
the potential malignancy of an lptbn - sn was discovered only in 2012 [ 36 ] .
melanoma arising on an lptbn - sn has a slow local growth and develops local recurrences after surgery or distant metastasis only after many years . in the case
reported by yeh and colleagues in 2012 , an interval of 22 years between the diagnosis of melanoma and death due to metastases occurred .
this is in contrast with the usually very quick course occurring when a nodular melanoma arises within a congenital nevus .
a possible explanation of this strange behavior is the slow accumulation of chromosomal mutations during the years , with a tumoral progression finally leading to an overt aggressive melanoma .
however , the absolute and relative risk for developing a melanoma in an lptbn - sn is still unknown .
there are three peculiar clinical entities that may have a biologic behavior similar to lptbn - sn : a variant of a nevus of ota showing a progressive evolution to melanoma with intermediate stages resembling a cellular blue nevus ; a giant congenital cellular blue nevus of the scalp ; and pigmented neurocristic hamartoma [ 1013 ] .
the nodules that develop in the context of such lesions can be regarded as true low - grade melanocytic malignancies that can progress to a higher grade of malignancy over many years .
initially , the lesions can have the trend to grow and infiltrate the surrounding tissues and , if untreated , metastasis and death can occur .
it belongs to the group of dermal melanocytic lesions and is characterized by hamartomatous lesions clinically and histologically similar to blue nevi , but with a neural crest - derived schwann cell component .
the case reported here highlights the malignant potential of an lptbn - sn , the very long course even without treatment and the possible coexistence of benign , borderline or malignant subcutaneous nodules in the same lptbn - sn .
patients with large congenital blue nevi should be advised on the potential oncologic transformation of these lesions , the importance of follow - ups should be emphasized and , whenever possible , a preventive complete surgical removal should be evaluated before subcutaneous nodules develop . | the term large plaque type blue nevus with subcutaneous cellular nodules ( lptbn - sn ) refers to a huge blue nevus , usually located on the trunk , that develops subcutaneous nodules many years after the lesion has appeared .
the potential malignancy of an lptbn - sn was only discovered in 2012.we report the case of a 56-year - old caucasian man that developed a cutaneous melanoma on an lptbn - sn of the trunk .
the first diagnosis was made more than 10 years before his death due to melanoma metastasis.the case reported here highlights the malignant potential of an lptbn - sn , the very long course even without treatment and the possible coexistence of benign , borderline or malignant subcutaneous nodules in the same lptbn-sn.patients with large congenital blue nevi should be advised on the potential oncologic transformation of these lesions , the importance of follow - ups should be emphasized and , whenever possible , a preventive complete surgical removal should be evaluated before subcutaneous nodules develop . |
the antibiotic resistance provides a great therapeutical and economic burden in the treatment of infectious diseases and it may threaten the success of antimicrobial chemotherapy .
the disproportion between the slow development of new drugs and the fast emergence of resistant strains necessitates the development and research of new antimicrobial agents or resistance modifiers .
one strategy employed to overcome resistance mechanisms is a combination therapy such as using clavulanic acid as inhibitor of -lactamase in drugs sulbactam and tazobactam .
however , the frequent use of clavulanate has led to the emergence of resistant bacterial strains .
the promising strategy is the use of synergistic effects of natural compounds , products of plant secondary metabolism .
traditionally , medicinal plants have been used throughout the world for centuries for a range of medicinal complications .
plant drugs are considered to be less toxic and free of side effects than synthetic ones .
some work demonstrated that plants either contain antimicrobials that can operate in synergy with antibiotics or possess compounds that have no intrinsic antibacterial activity but are able to sensitize the pathogen to a previously ineffective antibiotic [ 3 , 4 ] .
some plants can be sources of compounds that can potentiate the activity of antibiotics against resistant bacterial pathogens [ 59 ] .
these compounds are believed to play a role in the plant 's defense against infection by working in synergy with intrinsic antimicrobials .
it has been suggested recently that such compounds can potentially be used to improve the efficacy of antibiotics against bacterial pathogens .
salvia species are used as traditional medicines all around the world , possessing antibacterial , antioxidant , anti - inflammatory , and analgesic properties .
s. sclarea , popularly known as clary sage , is a biennial or perennial herb with diverse biological activities manifested by different components , mainly of eo .
salvia and other plants such as thyme , lavender , sage , basil , coleus , hyssop , and skullcap belong to the large plant family lamiaceae .
biological properties of eos and their antimicrobial activity have been attributed to their main compounds such as mono- , di- , and sesquiterpenes and a variety of low molecular weight aliphatic hydrocarbons , acids , alcohols , aldehydes , acyclic esters or lactones , coumarins , and homologues of phenylpropanoids .
these compounds have hydrophobic characteristics and interact with different sites of microbial cell , namely , with cytoplasmic membrane .
they affect the activities of membrane associated enzymes ; certain components of eos can act as uncouplers , which interfere with proton translocation over a membrane vesicle and subsequently interrupt adp phosphorylation .
specific terpenoids with functional groups , for example , phenolic alcohols or aldehydes , also interfere with membrane integrated or associated proteins , stopping their production , or activity eos are also able to inhibit the synthesis of dna , rna , proteins , and polysaccharides in fungal and bacterial cells [ 13 , 14 ] . the effect of eo may be associated with the cell envelope by interfering with the rigidity and integrity of the membrane , thereby changing the expression or function of cell wall - related genes such as penicillin - binding proteins ( pbps ) .
thymol , a p - cymene derived compound primarily found in some eo , suppresses the toxic shock syndrome toxin ( tsst-1 ) secretion in s. aureus and decreases the production of -hemolysin , sea , and seb enterotoxins in s. aureus .
diverse spectrum of eos ( s. officinalis , r. officinalis , a. alba , and e. caryophyllata ) as well as some of their major compounds ( limonene , eugenol , and eucalyptol ) inhibited qs gene expression in s. aureus .
extract from rhus javanica showed the inhibition of the genetic expression of virulence factors such as sea , agra , and sara in methicillin resistant s. aureus , as well as meca gene , which is responsible for staphylococcal resistance to -lactam antibiotics .
staphylococcus epidermidis was previously regarded as an innocuous commensal microorganism on the human skin and mucous membranes .
however , nowadays it is seen as an important opportunistic pathogen , one of the most prevalent causes of nosocomial infections associated with newborn , severely ill , and immunocompromised patients , and is also frequently isolated from postsurgical infections , especially in association with indwelling prosthetic devices .
it was found that approximately 70% of the s. epidermidis strains circulating in the hospital environment are resistant to methicillin and that the majority of them are also resistant to other antimicrobial classes .
resistance to methicillin is at 7590% among hospital isolates of s. epidermidis , which is even higher than the corresponding rate for s. aureus ( 4060% ) .
resistance to -lactam antibiotics in s. epidermidis , similarly as in s. aureus , is mediated by ( i ) production of -lactamase which hydrolytically destroys the -lactam antibiotics and ( ii ) the acquisition of meca gene that produces alternative pbp2 , which have low affinity to -lactam antibiotics .
gene meca is carried on a chromosomal genetic element designated as staphylococcal chromosomal cassette mec ( sccmec ) .
currently , there are several different types of sccmec elements , with several subtypes , characterized by a unique combination of the mec and the recombinase - encoding ccr gene complexes .
some reports suggest that , in coagulase negative staphylococci , namely , in s. epidermidis , sccmec structures are more diverse and include either mec - ccr combinations not yet described for s. aureus or more than one ccr allotype . in s. epidermidis
characterized 139 isolates of mrse and found that 41% of the isolates harbored sccmec type iv and 27% carried sccmec type iii , while sccmec types v , i , and ii were presented only in 6% , 4% , and 4% of the isolates , respectively .
of the 44 mrse isolates recovered from the blood of patients with prosthetic valve endocarditis , 2% harbored sccmec type i , 34% harbored type ii , 28% harbored type iii , and 36% harbored type iv .
expression of meca is inducible and can be controlled by either its cognate regulators meci ( dna binding repressor protein ) and mecr1 ( sensor / signal transducer ) or the structurally and functionally similar -lactamase regulators blai and blar1 , respectively .
the transcription of meca and blaz is corepressed by the regulators of the two regulons , meci and blai .
these regulators are almost identical and can replace each other and both meci and blai can bind as homodimers to the promoter / operator region of both meca and blaz . in the presence of antibiotics ,
the sensing is mediated by signal transduction via the two transmembrane inducers , mecr1 or blar1 , which will result in proteolytic autocleavage of the cytoplasmic domains of these proteins . to perform this autocleavage ,
autocleavage of the signal transducer is followed by cleavage of the cognate repressor , meci or blai , and by subsequent induction of the transcription of meca or blaz . in our previous work
we showed synergistic effects of oxacillin with plant extracts and eos from several plant species of lamiaceae family .
the purpose of the present study was to determine the effect of eo from s. sclarea on the expression of meca gene in strains s. epidermidis possessing different types of sccmec and determine synergistic effect of oxacillin and eo in these strains .
the aerial parts of s. sclarea were harvested at the optimal growing and development stage .
the plant material was air - dried and submitted to hydrodistillation for 4 h. isolated oil was diluted in n - hexane and dried over anhydrous sodium sulfate .
mrse strains were obtained from clinical samples of patients with positive hemocultures from the university teaching hospital old town , bratislava , slovak republic , and were kindly provided by dr .
slobodnkov from the institute of microbiology , faculty of medicine , comenius university , in bratislava , slovak republic .
the isolates were screened for their susceptibility towards oxacillin using disc diffusion method on mueller - hinton agar ( himedia , india ) in accordance with the clinical and laboratory standard institute guidelines ( clsi ) standards .
suspension of the tested bacteria ( 0.1 ml of 10 cells / ml ) was spread onto solid media plates .
antimicrobial susceptibility test discs ( himedia , india ) with ampicillin ( 10 g ) and oxacillin ( 1 g ) were placed on the incubated plates .
these plates , after 2 h of maintenance at 4c , were incubated for 24 h at 37c and the diameters of the resulting zones of inhibition were measured in millimeters .
extraction of genomic dna was performed by using dneasy blood and tissue kit ( qiagen , germany ) according to the manufacturer 's instructions .
all the isolates were tested for genes of the mec and bla operon using the polymerase chain reaction method .
each pcr sample contained 2.5 l of 10x star taq enzyme buffer ( gene craft , germany ) , 0.2 mm of each deoxynucleoside triphosphate ( gene craft , germany ) , 0.2 m of each of the forward and reverse primers , 1.5 u taq dna polymerase ( gene craft , germany ) , and 2 l template dna .
the pcr primers were designed by primer3 and primer - blast and synthesized by microsynth ( switzerland ) .
the cycling conditions were as follows : preheating for 5 minutes at 94c , followed by 30 cycles of denaturation at 94c/30 seconds , annealing at 55c/40 seconds , extension at 72c/30 seconds , and final extension for 5 minutes at 72c .
the sccmec type was determined using the protocol and primers proposed by zhang et al . .
multiplex pcr was performed in 25 l reactions with 2.5 l of 10x star taq enzyme buffer ( gene craft , germany ) , 0.2 mm of each deoxynucleoside triphosphate ( gene craft , germany ) , various concentrations of the respective primers , 1.0 u of taq dna polymerase ( gene craft , germany ) , and 2 l of template dna .
the amplification was performed using a biometra thermal cycler ( germany ) with an initial denaturation step at 94c/5 min followed by 30 cycles of denaturation at 94c/1 min , annealing at 55c/1 min , extension at 72c/2 min , and a final extension step at 72c/5 min .
minimal inhibitory concentrations ( mic ) for oxacillin ( sigma - aldrich ) and eo ( prepared as stated above ) were determined by standard broth microdilution method using 96-well microtiter plates in accordance with clsi in mueller - hinton broth .
the s. epidermidis suspension was adjusted to the 0.5 mcfarland standard and diluted to obtain a final turbidity in wells approximately 1 10 cfu / ml .
0.1 ml of bacterial suspension was mixed with an equal volume of each dilution of oxacillin or eo , and optical density ( od600 ) values were determined after 24 hours of growth at 37c using a 96-well plate reader varioskan flash ( thermo fisher scientific , finland ) .
the mic was defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism .
the study of the interaction between eo and oxacillin was done using the checkerboard method .
twofold serial dilutions of oxacillin prepared in horizontal rows of 96-well microtiter plate were subsequently cross - diluted vertically by twofold serial dilutions of eo .
microtiter plates were inoculated with test organism and incubated for 24 h at 37c .
mic values of the combinations were determined as the lowest concentration that completely inhibited bacterial growth recorded as the optical density at 600 nm using varioskan flash ( thermo fisher scientific , finland ) .
interaction between eo and oxacillin was then determined by calculating the fractional inhibitory concentration ( fic ) indices .
the fici is defined as follows : mic of substance a tested in combination / mic of substance a tested alone + mic of substance b tested in combination / mic of substance b tested alone .
the fici is interpreted as follows : fici < 0.5 , synergistic effect ; 0.5 < fic < 1 , additive effect ; 1 < fici < 4 , indifferent effect ; and fic > 4 , antagonistic effect .
the shape of the isobologram curve can be convex , linear , or concave , which is indicative of the synergistic , indifferent , and antagonistic interactions , respectively .
the time - kill assay was carried out in order to determine antibacterial and potential synergistic effects of eo when used singly and in combination with oxacillin .
bacteria ( 5 10 cfu / ml ) were exposed to oxacillin and eo alone or in combination in concentrations 1/2 mic and incubated at 37c .
aliquots ( 0.1 ml ) were taken at 0 , 6 , 10 , and 24 h and diluted in normal saline as needed to enumerate 30300 colonies .
the diluted cultures were spread thoroughly on plates containing mueller - hinton agar . after incubating at 37c for 24 h , the growing colonies were counted .
the experiment was performed in triplicate ; data are shown as mean standard deviation from three independent experiments .
mrse strains were treated with various concentrations of oxacillin and eo alone or with their combination for 30 minutes .
total rna was isolated by using rneasy mini kit ( qiagen , germany ) according to the manufacturer 's instructions .
dna contamination from the total rna preparations was removed with on - column rnase - free dnase treatment ( qiagen , germany ) .
to generate cdna , total rna was reverse transcribed using the gotaq 2-step rt - qpcr system ( promega ) with specific primers ( table 1 ) .
real - time pcr was performed at least three times for each examined gene with an applied biosystem 7500 fast real - time pcr system .
the ct values and the qpcr were normalized to the housekeeping gene for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) using the 2 method .
the experimental data are expressed as the mean standard deviation ( sd ) from three independent experiments .
the essential oil obtained from aerial parts of s. sclarea was analyzed by gas chromatography ( gc ) and 12 compounds representing 75.2% of the essential oil were identified .
the main components were linalyl acetate ( 38.67% ) , linalool ( 20.42% ) , germacrene ( 5.31% ) , geraniol ( 1.42% ) , and -caryophyllene ( 1.80% ) . in our previous work
we showed that addition of plant extracts and eos from some species of salvia plants increases the susceptibility of mrse to oxacillin .
eos and oxacillin in combination showed synergistic effect , inhibited growth , and even killed the bacteria . in this study we used 30 clinical isolates of mrse for study of potential synergistic effects of eo from s. sclarea and oxacillin , and by using checkerboard method we confirmed this effect in 23 isolates ( 76.7% ) ( fici from 0.125 to 0.381 ) .
the remaining 7 strains ( 23.3% ) showed additive effect ( fici from 0.531 to 0.793 ) .
based upon the phenotype analysis of methicillin resistance ( mic of oxacillin , table 2 ) , screening for presence or absence of meca , meci , mecr1 , blaz , blai , and blar1 gene using pcr ( figure 1 ) , and analysis of mec class ( figure 2 ) and on sccmec typization we selected four strains possessing different sccmec types i , ii , iii , and iva ( figure 3 ) , which cover all sccmec types and all variations of mec and bla operons present in collection of isolates .
we have attempted to assess if eo alone or in combination with oxacillin influences the expression of meca gene .
firstly , we characterize the meca / meci / mecr1 and blaz / blai / blar1 regions of our strains and determine the effect of presence or absence of mec and bla elements on the mic of oxacillin .
isolates r17 and r12 which possess sccmec types i and iva , respectively , and class b mec have genotypes + blai/+blar1 and meci / mutmecr1 ( absent meci and truncation of mecr1 by insertion of is1272 ) .
these strains showed high resistance , correlating with the fact that meci deletion can lead to increased resistance .
isolate r8 ( sccmec type ii ) has both complete repressor / sensor systems + meci/+mecr1 and + blai/+blar1 .
isolate r22 ( sccmec type iii ) has + meci/+mecr1 system but this isolate was the only one in which we were unable to confirm the presence of the bla operon .
we used real - time pcr to investigate the expression of genes from mec and bla operons and to determine the influence of eo on their expression .
quantification data for all genes were normalized to the reference gene for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) . in figure 4
regardless of constitutive or inducible expression of genes our data show that 30 min treatment of culture with subinhibitory concentrations of eo from s. sclarea leads to decreasing of expression of all genes from both operons .
it is known that eos influence membranes and proteins in cytoplasmic membrane ; therefore we can assume that they can influence conformation of mecr1 , or blar1 , which can have impact on transcription of both operons .
while eo showed similar and dose dependent effect on single genes from mec operon , in case of bla genes the effect of eo on blai and blar1 gene expression was weaker .
similar results , the inhibition of expression of resistant genes meca , meci , and mecr1 , against methicillin resistant s. aureus by hexane and chloroform fractions of salvia miltiorrhiza bunge , were described by lee et al . .
one isolate from each sccmec type was exposed to increasing amount of eo and as is shown in figure 5 , eo reduced the expression of meca gene in all strains .
regarding sccmec type and genetic background of mec and bla operons , strains r8 and r22 have mec a class with all three mec genes , while strains r17 and r12 have mec b class ( absent meci and truncation of mecr1 by insertion of is1272 ) . as is obvious from figure 5 , we did not find significant differences in expression of meca gene between strains with different mec class after treatment with eo at concentrations 1/4 and 1/2 of mic ; however in concentration 1/8 mic there was statistically significant ( p < 0.05 ) difference between strains of both classes of mec complex , when eo decreased the expression of meca gene more in strains r17 and r12 in the comparison with strains r8 and r22 .
we estimated the expression of meca gene in the presence of eo also in three random selected strains with iva type of sccmec and the results were similar ( data not shown ) .
finally we examined the expression of meca gene after 30 min treatment with oxacillin and eo .
the addition of eo not only inhibited the induction of expression , but also reduced the expression of meca gene in the comparison with untreated control ( figure 6 ) .
the combination of 1/4 mic of both compounds was more effective , probably due to lower concentration of oxacillin and lower induction of expression of meca gene .
we found similar trend as in the case of eo alone , that is , higher reduction of expression in strains r17 and r12 in the comparison with strains r8 and r22 .
the viable cell counts for four isolates of mrse differing in type of sccmec after exposure to 1/2 mic of oxacillin and eo alone and in combination at different times are shown in figure 7 .
the combination of 1/2 mic eo + 1/2 mic oxacillin completely inhibited the growth of all four strains after 24 h. the same combination caused an over 3 log10-fold reduction in the bacterial count yet after 10 h ( strains r17 and r8 ) in comparison with the most active compound alone . in strain
r12 the reduction was more than 5 log10-fold in 6 h and from 10 h the growth of this strain was completely inhibited . to get a better understanding of the modulatory effect of eo we examined the interaction between eo and oxacillin via the checkerboard method and described it in terms of fractional inhibitory concentration ( fic ) indices .
the fic indices of eo in combination with oxacillin were 0.381 for strain r17 , 0.156 for r8 , 0.125 for r22 , and 0.376 for r12 , which according to pillai et al .
the synergistic interaction can be read according to the curve indicating borderline synergy according to fic 0.5 .
isobolograms of all strains have convex shape beyond the borderline of synergisms and clearly show the potentiating effect of s. sclarea eo on oxacillin susceptibility in these strains .
our data reveal the potential of eo from s. sclarea to be candidate for combination therapy against mrse , because it has synergistic effect with oxacillin in all tested strains .
the killing effect of the combinatorial treatment is connected with reduction of expression of meca gene and other genes participating in staphylococcal resistance to -lactams antibiotics .
lower expression of these genes reduces the two major bacterial defense mechanisms against -lactam antibiotics .
although synergistic effect of eo may be caused by several mechanisms , observed different levels of the reduction of meca expression in strains with different types sccmec and different genetic background in mec and bla operons confirmed that reduction of meca gene expression can be one of major mechanisms . | the essential oil ( eo ) from salvia sclarea was shown to increase the susceptibility of methicillin resistant staphylococcus epidermidis ( mrse ) isolates to oxacillin .
the purpose of this study was to investigate the effect of eo from s. sclarea on expression of meca gene of mrse carrying different types of staphylococcal chromosomal cassette ( sccmec ) and to evaluate potential synergistic effect of eo with oxacillin . using real - time pcr we found that eo alone inhibited the expression of the resistant genes meca , mecr1 , and meci and blaz , blar1 , and blai .
the use of the combination of eo with oxacillin resulted in significantly inhibited expression of meca gene in all tested strains with different types of sccmec . using time - kill assay and checkerboard assay we confirmed synergistic effect of eo from s. sclarea and oxacillin in mrse . |
a clinical audit can identify activities that should or should not be performed in clinical settings ( 1 ) .
it not only improves care quality but also helps nurses to acquire and use the knowledge , skills , and attitudes necessary for creating a meaningful , productive , and satisfying working environment ( 2 ) . the first step in operationalizing the clinical audit is to identify specified indicators of the domain .
although using the nursing process improves nursing care quality , few studies have evaluated nursing performance in accordance with nursing process steps either nationally or internationally .
most studies on nursing audits evaluated only the quality of nursing records ( 3 - 5 ) to assess care quality ( 6 , 7 ) . in iran ,
khosravani et al . found that job satisfaction was significantly higher among nurses who used the nursing process in their practice ( 8) .
however , shoorideh and ashktorab showed that personal and management factors are among the barriers to implement the nursing process in clinical settings ( 9 ) .
given the importance of evaluation of nursing care , the need to develop appropriate criteria for clinical audits is obvious .
however , a review showed that few studies have used the nursing process as a framework to evaluate nursing care ( 3 - 5 , 7 ) .
this study aimed to audit nursing care on the basis of a nursing process model .
a cross - sectional study was carried out from february 2014 to april 2015 in 16 educational hospitals affiliated with tehran university of medical sciences ( tums ) .
healthcare providers in these hospitals include residents , fellows , interns , nurses , and nursing students . in this study , we specifically targeted nursing staff . first , a nursing audit checklist was designed based on a literature review and expert opinions obtained from in - depth interviews of 10 experts in nursing and accreditation .
interviews included the following question : what indicators should be considered if you want to evaluate clinical nursing services ? reflective and explorative questions were also used ( e.g. , why ?
a total of 18 interviews were conducted at this stage , with a mean duration of 44 minutes . to analyze the data ,
these codes were then reduced into 43 main codes in five main categories according to nursing process steps .
these secondary codes were used as the primary items to develop a checklist for auditing nursing practice . to assess face and content validity qualitatively , the checklist was sent to 15 experts in clinical nursing and accreditation of nursing practice and 10 experienced nurse educators .
some items were modified or deleted . at this stage , there were 25 items ( indicators ) in the checklist , covering the following domains : assessment ( 5 items ) , nursing diagnosis and identifying outcomes ( 5 items ) , planning ( 5 items ) , implementation ( 7 items ) , and evaluation ( 3 items ) .
most items were rated on a three - point scale ( never = 0 , sometimes = 50 , and always = 100 ) depending on the level of nurses compliance . however , some of the indicators employed a yes / no format ( yes = 100 ,
no = 0 ) according to the national hospital accreditation audit guide ( 10 ) .
mean scores were calculated for each indicator as well as for each domain and for the full checklist .
higher scores indicate better performance . the external consistency , internal consistency , and inter - rater reliability of the final version of the checklist were assessed .
external consistency was assessed through the test - retest method with a two - week interval ( r = 0.954 ) .
to assess inter - rater reliability , the checklist was simultaneously completed by two auditors ( first and corresponding authors ) for 10 care provider .
inclusion criteria were working full - time , having at least 2 years of clinical experience , and willingness to participate in the study . in a previous study ,
the prevalence of quality nursing care was 25% ( 12 ) . using cochran s sample size formula and the parameters p = 0.25 , q = 0.75 , d = 0.05 , and = 0.05
the number of nurses to be recruited each hospital was calculated by considering the total number of nurses in each .
the demographic questionnaire included questions on nurses age , gender , work experience , type of employment , setting , education level , and work shift .
observations , interviews , and document evaluation were used to audit nursing care ( using the audit guide ) .
the number of nurses to be audited in each hospital was calculated by considering the total number of nurses in each .
then , using a random number table , the required number of nurses was randomly selected from the list of nurses in each hospital .
two auditors , who were trained in auditing , performed the audit by using a guide designed before the audit process was started . to determine the rate of compliance with indicators , all of the nursing activities were observed during a single work shift and nursing documents
furthermore , in some cases , interviews were conducted with nurses as explained in detail in the audit guide .
some indicators needed to be assessed through a review of documents and nursing notes ( e.g. , recording and reporting ) .
some indicators had to be assessed through observation ( e.g. , collaborating with the client / family in decision making for clinical practice ) .
a few indicators were assessed through interviews of patients and their families ( e.g. , education about the home care plan ) .
this study was approved by the ethics committee of tehran university of medical sciences ( approval number : 92/s/130/1422 ) on september 28 , 2013 .
the research objectives were explained to all nurses participating in the study , and written informed consent was obtained .
all participants were aware that they would be observed for some time but they were not informed about the exact time of the audit .
all participants were also assured that their anonymity would be protected and their personal information would be kept confidential .
the kolmogorov - smirnov test was used to determine whether the quantitative variables were normally distributed .
descriptive statistics ( frequencies , percentages , means , and standard deviations ) were calculated for compliance with quality indicators .
pearson correlation analysis was conducted to examine the relationship between nurses compliance with auditing indicators and their age and work experience .
independent - samples t - tests were used to compare the mean compliance scores of nurses by dichotomous demographic variables .
first , a nursing audit checklist was designed based on a literature review and expert opinions obtained from in - depth interviews of 10 experts in nursing and accreditation .
interviews included the following question : what indicators should be considered if you want to evaluate clinical nursing services ? reflective and explorative questions were also used ( e.g. , why ?
a total of 18 interviews were conducted at this stage , with a mean duration of 44 minutes . to analyze the data ,
these codes were then reduced into 43 main codes in five main categories according to nursing process steps .
these secondary codes were used as the primary items to develop a checklist for auditing nursing practice . to assess face and content validity qualitatively , the checklist was sent to 15 experts in clinical nursing and accreditation of nursing practice and 10 experienced nurse educators .
some items were modified or deleted . at this stage , there were 25 items ( indicators ) in the checklist , covering the following domains : assessment ( 5 items ) , nursing diagnosis and identifying outcomes ( 5 items ) , planning ( 5 items ) , implementation ( 7 items ) , and evaluation ( 3 items ) .
most items were rated on a three - point scale ( never = 0 , sometimes = 50 , and always = 100 ) depending on the level of nurses compliance . however , some of the indicators employed a yes / no format ( yes = 100 ,
no = 0 ) according to the national hospital accreditation audit guide ( 10 ) .
mean scores were calculated for each indicator as well as for each domain and for the full checklist .
higher scores indicate better performance . the external consistency , internal consistency , and inter - rater reliability of the final version of the checklist were assessed .
external consistency was assessed through the test - retest method with a two - week interval ( r = 0.954 ) .
to assess inter - rater reliability , the checklist was simultaneously completed by two auditors ( first and corresponding authors ) for 10 care provider .
inclusion criteria were working full - time , having at least 2 years of clinical experience , and willingness to participate in the study . in a previous study ,
the prevalence of quality nursing care was 25% ( 12 ) . using cochran s sample size formula and the parameters p = 0.25 , q = 0.75 , d = 0.05 , and = 0.05
however , we recruited 300 nurses to compensate for possible attrition . the number of nurses to be recruited each hospital was calculated by considering the total number of nurses in each .
data were collected using a demographic questionnaire and the audit checklist . the demographic questionnaire included questions on nurses age , gender , work experience , type of employment , setting , education level , and work shift .
observations , interviews , and document evaluation were used to audit nursing care ( using the audit guide ) .
the number of nurses to be audited in each hospital was calculated by considering the total number of nurses in each .
then , using a random number table , the required number of nurses was randomly selected from the list of nurses in each hospital .
two auditors , who were trained in auditing , performed the audit by using a guide designed before the audit process was started . to determine the rate of compliance with indicators , all of the nursing activities were observed during a single work shift and nursing documents
furthermore , in some cases , interviews were conducted with nurses as explained in detail in the audit guide .
some indicators needed to be assessed through a review of documents and nursing notes ( e.g. , recording and reporting ) . some indicators had to be assessed through observation ( e.g. , collaborating with the client / family in decision making for clinical practice ) .
a few indicators were assessed through interviews of patients and their families ( e.g. , education about the home care plan ) .
this study was approved by the ethics committee of tehran university of medical sciences ( approval number : 92/s/130/1422 ) on september 28 , 2013 .
the research objectives were explained to all nurses participating in the study , and written informed consent was obtained .
all participants were aware that they would be observed for some time but they were not informed about the exact time of the audit .
all participants were also assured that their anonymity would be protected and their personal information would be kept confidential .
the kolmogorov - smirnov test was used to determine whether the quantitative variables were normally distributed .
descriptive statistics ( frequencies , percentages , means , and standard deviations ) were calculated for compliance with quality indicators .
pearson correlation analysis was conducted to examine the relationship between nurses compliance with auditing indicators and their age and work experience .
independent - samples t - tests were used to compare the mean compliance scores of nurses by dichotomous demographic variables .
further , 56% were non - permanently employed and almost 96% had a bsc in nursing .
the rate of compliance with nursing process indicators was 79.71 0.87 , confirming that the majority of nursing care was carried out based on the nursing process .
the overall compliance score did not significantly differ by gender ( t = -1.07 , p = 0.285 ) , nor did domain mean compliance scores , except that male nurses scored significantly higher on the diagnosis domain ( t = -2.25 , p = 0.030 ) .
however , mean overall compliance scores significantly differed between nurses with a bsc and those with an msc ( p = 0.04 ) .
this difference was also found in the mean compliance scores for three domains : diagnosis ( p = .001 ) , implementation ( p = 0.02 ) , and evaluation ( p = 0.01 ) ( table 2 ) .
in addition , the overall mean score did not significantly differ between nurses with permanent and non - permanent employment ( t = 0.123 , p = 0.42 ) . however , overall compliance scores were correlated with age ( r = 0.26 , p = 0.001 ) and work experience ( r = 0.273 , p = 0.001 ) .
table 3 presents the detailed data on the participants compliance with the audit items .
n / a ( not applicable ) : these indicators have two modes ( yes / no ) scored 0 or 100 according to the hospital accreditation audit guide .
in the present study , an audit tool was developed and used to evaluate nursing activities in accordance with nursing process indicators .
we also used a combination of interviews , observations , and document reviews to audit nurses performance .
also audited nurse performance but mostly used nursing documentation as the only data source ( 7 ) .
used a combination of methods to collect data on implementation of the nursing process ( 11 ) . in the present study , nurses obtained a relatively high score for compliance with the nursing process indicators , especially in the domains of assessment , diagnosis , and evaluation .
our findings are consistent with those of rivas et al . , who investigated the implementation of the nursing process in primary healthcare and reported that using the nursing process can improve the quality of care and people s health ( 12 ) .
training nurses on the nursing process and encouraging them to implement it can significantly improve the quality of nursing care ( 5 , 13 ) . in the present study ,
this is somewhat consistent with a previous study by sorensen , where assessment and care planning were introduced as the main indicators of healthcare system accreditation ( 3 ) . however , adib - hajbaghery and safa investigated nurses evaluation of their use and mastery of health assessment skills and reported that nurses use these skills below the desired level ( 14 ) .
although the nursing culture differs across hospitals , previous findings may have been influenced by managerial factors or the instruments used .
however , munroe et al . found that using a framework for assessment by nurses improved clinical practice and patient care outcomes ( 4 ) .
the present analysis showed that nurses experience and age had significant positive correlations with implementation of care indicators .
this suggests that nurses with more clinical experience are more likely to use the nursing process model to provide care and are more committed to it .
this differs from perez rivas et al.s findings that younger nurses had better adherence to the nursing process than older nurses did ( 12 ) .
in contrast , akbari kaji found that neither age nor experience was significantly associated with commitment to the nursing process ( 13 ) .
we did not find a significant association between level of education and compliance with the nursing process .
this is consistent with the findings of rivas et al . and akbari kaji ( 12 , 13 ) .
implementation would not only result in quality services but also would improve nurses professional prestige and both patient and nurse satisfaction .
thus , it seems not only necessary to retrain nurses through continuing education but also to insist that this model be implemented in practice . in the present study ,
our findings might have differed if we audit specialized units such as the icu or ccu . | background : although using the nursing process improves nursing care quality , few studies have evaluated nursing performance in accordance with nursing process steps either nationally or internationally.objectives:this study aimed to audit nursing care based on a nursing process model.patients and methods : this was a cross - sectional descriptive study in which a nursing audit checklist was designed and validated for assessing nurses compliance with nursing process .
a total of 300 nurses from various clinical settings of tehran university of medical sciences were selected .
data were analyzed using descriptive and inferential statistics , including frequencies , pearson correlation coefficient and independent samples t-tests.results:the compliance rate of nursing process indicators was 79.71 0.87 .
mean compliance scores did not significantly differ by education level and gender .
however , overall compliance scores were correlated with nurses age ( r = 0.26 , p = 0.001 ) and work experience ( r = 0.273 , p = 0.001).conclusions : nursing process indicators can be used to audit nursing care .
such audits can be used as quality assurance tools . |
endodontic surgery has advanced in recent years to increase the clinician 's ability to achieve more predictably successful clinical outcomes .
the properties of a root - end filling material are very critical in determining the success of periradicular surgery .
, to address the shortcomings of commonly used root - end filling materials . in a clinical study , saunders et al . , demonstrated that use of mta as a root - end filling material results in a high success rate but extended setting time and difficulties in handling mta resulted in limited use in many surgical situations .
also the set mta contains many voids in the form of air bubbles , pores , and capillary channels , which could affect the apical seal obtained with mta .
recently a new calcium alumino silicate cement has been developed by septodont called biodentine with excellent biocompatibility , increased physico - chemical properties like short setting time and high mechanical strength , which makes it clinically easy to handle for endodontic cases and dentin restorative procedures .
some investigations have focused on its removal while others have considered its effects on apical and coronal microleakage , bacterial penetration of the tubules , and the adaptation of root - end filling materials .
irrigation with doxycycline to remove the smear layer from dentinal surfaces has been shown to improve the apical seal of root - end filling materials , such as irm and amalgam .
mtad ( a mixture of tetracycline , acid and a detergent ) has been the focus of attention as an alternative root canal irrigant .
use of mtad has been reported to be more efficient in removing smear layer as compared with the use of edta and naocl , especially from the apical third without significantly changing the structure of the dentinal tubules .
, there has been no study investigating the use of mtad as an irrigant in root - end cavities .
hence the present study was designed to assess the apical seal , obtained after root - end cavity irrigation with mtad and retrograde filling with different restorative materials mta and biodentine .
sixty freshly extracted human maxillary central incisors with mature root apices and single canal were selected and stored in 10% formalin solution until further use . teeth with gross caries involving the root , cracks on the root surface , and exceptionally short and thin roots were excluded .
all the teeth were decoronated at the length of 14 mm from the apex with the help of diamond disc under copious water irrigation .
the working length was established and the root canals were instrumented with protaper rotary files ( dentsply tulsa dental , tulsa ) using x - smart rotary device ( dentsply maillefer ) .
instrumentation was done till f4 file with 2.5% naocl irrigation between each file instrumentation . ah plus sealer ( dentsply caulk , milford , de ) was mixed according to manufacturer 's instructions and was inserted into the canal with the help of lentulospirals .
protaper gutta - percha cone ( dentsply maillefer ) was used for obturation using lateral condensation technique and accessory cones used if required . the coronal opening for
the apical 3 mm of all the teeth were resected with a round diamond point in a water - cooled , high - speed hand - piece .
root - end preparations were made to a depth of 3 mm using proultra surgical tip no 2 ( dentsply maillefer ) .
thirty root - end preparations were irrigated with mtad ( biopure , dentsply , tulsa dental ) for 5 min ( group a ) , while 30 were irrigated with saline ( parenteral surgicals , india ) for 5 min ( group b ) prior to placement of root - end filling .
samples under each subgroup were then filled either with mta ( pro - root mta , dentsply , tulsa dental ) ( 1a and 2a ) or biodentine ( septodont , usa ) ( 1b and 2b ) and placed according to manufacturer 's instructions .
all teeth received a final 1 min rinse with saline after root - end filling .
the teeth were wrapped in moist gauze and stored in a sealed container at 37c for a minimum of 1 week .
samples from all the groups were coated with two coats of nail varnish except on the apical 3 mm .
after the application of nail varnish , the samples were dried and immersed in 5 ml of 2% methylene blue dye ( s d fine chemical ltd , india ) in 15 ml screw - capped bottle and stored at 37c 2c , at relative humidity in incubator for 72 hours .
after 72 hours , the samples were then washed under running tap water to remove the traces of the dye .
the teeth were then immersed and stored in freshly prepared 35% nitric acid ( s d fine chemical ltd , india ) for 72 hours in the centrifugal tube .
after 72 hours , the solutions were filtered using a fine grit filter paper in another centrifugal tube and the obtained sample solutions were centrifuged at 2000 rpm for one minute . the supernatant solutions thus collected were used to determine absorbency in a uv visible spectrophotometer at 670 nm .
the data was analyzed statistically by comparison of mean microleakage and t - test using spss 20 software .
all the teeth were decoronated at the length of 14 mm from the apex with the help of diamond disc under copious water irrigation .
the working length was established and the root canals were instrumented with protaper rotary files ( dentsply tulsa dental , tulsa ) using x - smart rotary device ( dentsply maillefer ) .
ah plus sealer ( dentsply caulk , milford , de ) was mixed according to manufacturer 's instructions and was inserted into the canal with the help of lentulospirals .
protaper gutta - percha cone ( dentsply maillefer ) was used for obturation using lateral condensation technique and accessory cones used if required .
the apical 3 mm of all the teeth were resected with a round diamond point in a water - cooled , high - speed hand - piece .
root - end preparations were made to a depth of 3 mm using proultra surgical tip no 2 ( dentsply maillefer ) .
thirty root - end preparations were irrigated with mtad ( biopure , dentsply , tulsa dental ) for 5 min ( group a ) , while 30 were irrigated with saline ( parenteral surgicals , india ) for 5 min ( group b ) prior to placement of root - end filling .
samples under each subgroup were then filled either with mta ( pro - root mta , dentsply , tulsa dental ) ( 1a and 2a ) or biodentine ( septodont , usa ) ( 1b and 2b ) and placed according to manufacturer 's instructions .
all teeth received a final 1 min rinse with saline after root - end filling .
the teeth were wrapped in moist gauze and stored in a sealed container at 37c for a minimum of 1 week .
samples from all the groups were coated with two coats of nail varnish except on the apical 3 mm . after the application of nail varnish , the samples were dried and immersed in 5 ml of 2% methylene blue dye ( s d fine chemical ltd , india ) in 15 ml screw - capped bottle and stored at 37c 2c , at relative humidity in incubator for 72 hours .
after 72 hours , the samples were then washed under running tap water to remove the traces of the dye .
the teeth were then immersed and stored in freshly prepared 35% nitric acid ( s d fine chemical ltd , india ) for 72 hours in the centrifugal tube .
after 72 hours , the solutions were filtered using a fine grit filter paper in another centrifugal tube and the obtained sample solutions were centrifuged at 2000 rpm for one minute . the supernatant solutions thus collected were used to determine absorbency in a uv visible spectrophotometer at 670 nm .
the data was analyzed statistically by comparison of mean microleakage and t - test using spss 20 software .
results suggest a significant difference ( p < 0.005 ) between group 1a ( mtad irrigation followed by mta placement ) and 2a ( saline irrigation followed by mta placement ) .
there was also a significant difference ( p < 0.1 ) between the groups 1b ( mtad irrigation followed by biodentine placement ) and 2b ( saline irrigation followed by biodentine placement ) .
there was a significant difference ( p < 0.005 ) between group 1a ( mta ) and 1b ( biodentine ) with mtad irrigation and also between group 2a ( mta ) and 2b ( biodentine ) with saline irrigation .
improved apical seal of root - end fillings has been pursued in an effort to improve the clinical results of apical surgery . surgical smear layer in endodontics
is defined as a smear layer , which contains micro - organisms and necrotic pulpal tissues which is formed on the dentinal surfaces , cut by the instruments during apicoectomy and retrograde cavity preparation .
citric acid , edta , 35% orthophosphoric acid and biopure mtad ( dentsply tulsa dental ) have been recommended for the removal of surgical smear layer .
smear layer removal from retrograde cavity preparation and dentin demineralization by the acidic agent of mtad i. e. , citric acid , has been shown to be associated with more rapid and complete cementum deposition on the root end , which produces rapid dentoalveolar healing .
doxycycline is a potent antibiotic and the long lasting substantivity of doxycycline on root surfaces supports the concept of using resected root surfaces as a substrate for the deposition and slow release for local doxycycline delivery .
mta has been considered as the material of choice as a root - end filling material .
nonetheless , mta does not provide a hermetic seal and it has been reported that fluid flow occurs at the material to the tooth interface .
recently , a novel biomaterial called biodentine has been introduced , and it has been shown that it can replace the traditional mta . in the present study ,
the results were recorded as a measure of absorbance of light . according to the beer - lamberts law ,
the absorbency of the solution is directly proportional to the concentration of absorbing species in the solution and path length .
thus for a fixed path length , uv visible spectroscopy can be used to determine the concentration of the absorber in the solution .
hence it can be interpreted that absorbance of the solution is directly related to the amount of microleakage .
the study utilizes a dye extraction method to quantify the amount of apical microleakage instead of dye penetration study as it has been shown that the depth of penetration of the dye is not uniform around the margins of a restoration and gives randomly chosen results , raising doubts about their reliability .
dye extraction method provides reliable results because it measures all of the dye taken up in the root and gives a quantitative reading .
the current investigation suggests that the removal of smear layer with mtad decreases the apical sealing ability of mta and improves the apical sealing ability of biodentine .
the results of the present study with regard to mta are in agreement with the findings of kubo et al . , who demonstrated demineralizing agents had a negative effect on the apical seal of mta when placed in a retrograde ( surgical ) model .
decreased leakage in smeared versus smear free mta obturated root end cavities might have been due to several factors , such as hydrophilic properties of mta and particle size of mta . the smear layer might act as a coupling agent enhancing the bond between the mta and root canal dentin due to hydraulic nature of mta which sets in the presence of moisture . the moist environment caused by the smear layer
might have a positive effect on the adaptation of mta to the root canal wall .
komabayashi and spngberg reported that the cumulative percentage of particles between 1.5 - 3 m was 70% for gray proroot mta .
garberoglio and brnnstrm showed that the tubule diameter was 0.9 - 2.5 m at different levels of the root canal in a scanning electron microscopy study .
consequently , the particle size of mta might not be suitable for penetration into the tubules .
the apical seal was reduced in root end cavity specimens filled with mta wherein the root end cavity irrigation was preceded with mtad which may be due to the ability of citric acid present in mtad to remove smear layer from dentinal surfaces .
the findings of the present study revealed that biodentine provided a better apical seal following mtad irrigation . in an earlier study by han and okiji , representative scanning electron microscope images and confocal images of biodentine samples showed tag - like structures within the dentinal tubules just beneath the interface which is due to the calcium and silica ion uptake into the dentine .
biodentine samples also showed an interfacial layer within the structure of dentin , just beneath the cement which is termed as
mineral infiltration zone ( miz ) and is associated with an altered intertubular micro - structure .
the dentinal tubules in the surfaces exposed to biodentine , appeared blocked and did not open to the surface , unlike the tubules in the clean , unexposed surfaces which explains its excellent sealing ability .
following hydration , the flowable consistency of the biodentine aids its penetration through opened dentinal tubules to crystallize over time within their structure , participating in the mechanical properties of the interface .
hence the removal of smear layer followed by biodentine placement facilitates the bonding to the exposed dentinal tubules .
the present study showed that biodentine produced less amount of microleakage compared to mta and the removal of smear layer improved the sealing ability of biodentine .
biodentine causes the uptake of calcium and silica in the adjacent root canal dentin in the presence of physiological solution .
biodentine has tricalcium silicate and zirconium particles of finer particle size , thus a higher value for specific surface area .
the rate of reaction of tricalcium silicate was higher for biodentine than mta owing to its optimized particle size distribution , the presence of calcium carbonate ( caco3 ) and the use of calcium chloride ( cacl2 ) . the interfacial layer formed between biodentine and dentine
it appears that the adhesion of biodentine cement to dentin may result from the physical process of crystal growth within the dentinal tubules leading to micromechanical bonding .
the removal of the loosely attached smear layer from the cavity surfaces with subsequent opening of dentinal tubules and demineralization of dentin creates a retentive surface for interlocking of restoratives .
within the limitations of the present study , it can be concluded that mta filled root - end cavities were more susceptible to microleakage after root - end irrigation with mtad , whereas the same mtad when used with biodentine significantly improved the apical seal of root - end cavities .
also the apical seal obtained with biodentine was superior to that obtained with mta irrespective of the presence of smear layer .
thus a regimen involving irrigation of the root - end cavity with mtad followed by retrofilling with biodentine can enhance the apical seal and augment osseous healing along with a sustained antibacterial effect , thus improving the success of periradicular surgery [ table 1 ] .
comparison between the mean micro leakage of mta and biodentine when used with mtad and saline | introduction : the present study is designed to assess the apical seal obtained after root end cavity irrigation with mtad and retrograde filling with mineral trioxide aggregate ( mta ) and biodentine.materials and methods : sixty extracted maxillary central incisors were instrumented and obturated .
apical 3 mm of all the roots were resected and retrograde preparations of 3 mm were made in all the teeth using ultrasonic tips .
thirty root end preparations were irrigated with mtad for 5 min ( group 1 ) , while 30 were irrigated with saline for 5 min ( group 2 ) .
each main group was subdivided into two subgroups containing 15 samples each .
samples under each subgroup were then filled either with mta ( 1a and 2a ) or biodentine ( 1b and 2b ) .
the amount of microleakage was assessed using a uv spectrophotometer.results:statistical analysis suggest a significant difference ( p < 0.005 ) between group 1a and 2a and between the groups 1b and 2b
( p < 0.1 )
. there was a significant difference ( p <
0.005 ) between group 1a and 1b and also between group 2a and 2b.conclusion:irrigation with mtad significantly improved the apical seal of biodentine , but it increased the microleakage in mta - filled root end cavities . also the apical seal obtained with biodentine was superior to that obtained with mta . |
during the last decades , we have witnessed a global epidemic of obesity . a sedentary lifestyle and an atherogenic diet , along with genetic predisposition , are probably the driving forces of this problem . according to the last national health and nutrition examination survey ( nhanes ) ,
obesity , particularly abdominal obesity , is associated with resistance to the effects of insulin on peripheral glucose and fatty acid utilization .
the resulting hiperinsulinemia and hyperglycemia , as the release of adipocyte cytokines , have been shown to induce vascular endothelial dysfunction , an abnormal lipid profile , hypertension , and vascular inflammation , all of which are atherogenic [ 36 ] .
the clustering of metabolic cardiovascular risk factors , under a common pathogenic process ( insulin resistance ) , was described for the first time in 1988 by reaven , leading to the concept of metabolic syndrome ( ms ) . since then , multiple definitions have been made , all of them considering some combination of insulin resistance , dyslipidemia ( hypertriglyceridemia , low hdl cholesterol ) , elevated fasting serum glucose , abdominal obesity , and hypertension ( table 1 ) [ 711 ] .
the ms affects over 20% of adults in western populations , and is becoming increasingly common [ 1315 ] , inclusively in adolescents .
however , the prevalence of ms varies widely according to geographic location , race , gender , and urbanization . since it was first described , an overwhelming body of evidence associated the ms with subsequent development of type ii diabetes mellitus and cardiovascular disease ( cvd ) [ 1725 ] .
recently , the evidence associating ms with cvd has been synthesized in three meta - analyses that showed an increased risk for incident cvd ( ranging from 53% to 118% ) and all - cause mortality ( ranging from 27% to 60% ) in subjects with ms [ 2325 ] . this increased risk appears related to the risk factor clustering , rather than to obesity alone , as normal weight people from the framingham population meeting the criteria for ms had a threefold increased risk for cvd , while obese individuals without ms did not have a significantly increased risk .
additionally , especially in the last decade , a number of other obesity - related disorders have been associated with the ms , including : fatty liver disease , polycystic ovary syndrome , obstructive sleep apnea , hyperuricemia , and , in particular , chronic kidney disease ( ckd ) .
chronic kidney disease is an important risk factor for end - stage renal disease ( esrd ) , cvd , and premature death [ 2731 ] and has become a global public health problem , with increasing social and economic impact due to its high prevalence [ 32 , 33 ] .
indeed , almost 5% of adult americans have ckd but only a minority reach esrd , suggesting that most of these patients die of other causes , such as cvd [ 26 , 33 , 34 ] .
the possible association between ms and ckd , in light of the growing epidemic of obesity , could represent a window of opportunity to identify individuals at risk of ckd ( and , evidently , cvd ) and to improve the burden of these diseases through early detection and prevention .
herein , we aim to critically review the existing clinical evidence that associates ms with renal disease and cvd , as well as the possible pathophysiologic mechanisms involved and actual treatment options .
chronic kidney disease is an independent risk factor for cvd and all cause - mortality and is considered a coronary heart disease risk equivalent [ 3537 ] . in patients with ckd stage 2 or higher , there is a direct relationship between the degree of renal function impairment and cardiovascular risk , independent of other known risk factors ( age , blood pressure , diabetes , and smoking ) , history of cvd , and presence of proteinuria .
even subtle reductions in renal function , in apparently healthy persons , increase the risk of cardiovascular morbidity and mortality [ 37 , 38 ] .
the main factors pointed out as responsible for this increased risk include volume overload and anemia leading to left ventricular hypertrophy , bone mineral disease causing valvular and vascular calcification , inflammation , oxidative stress , endothelial dysfunction , and sympathetic nervous system hyperactivity .
additionally , several epidemiologic studies point to a high prevalence of traditional and non - traditional risk factors for cvd in patients with ckd [ 35 , 4042 ] . moreover , the prevalence and magnitude of major risk factors for coronary heart disease increase as renal failure progresses ( e.g. , hypertension , insulin resistance , and hyperhomocysteinemia ) .
the prevalence of left ventricular hypertrophy and congestive heart failure is strikingly elevated in patients with ckd stages 2 through 5 [ 44 , 45 ] .
morbidity and mortality for congestive heart failure and coronary heart disease are also excessive in ckd [ 4548 ] .
for example , in the large study of left ventricular dysfunction ( solvd ) trial , moderate renal impairment was associated with a 41% increased risk for all - cause mortality , explained largely by heart failure , death and hospitalization . even in acute coronary syndromes , an estimated creatinine clearance less than 70
ml / min was associated with a 20% increase in mortality in both st and non - st elevation myocardial infarction . the close relationship between cvd and ckd
indeed , cardiovascular and renal diseases have many types of markers in common , including : clinical , pathophysiologic ( e.g. , increased angiotensin ii activity , upregulation of inflammatory and fibrosis producing cytokines ) , histopathologic ( e.g. , inflammatory infiltrates , vascular remodeling ) , biochemical ( e.g. , c - reactive protein , homocysteine , lipoprotein ( a ) and fibrinogen ) , acute and chronic inflammation and subclinical signs of atherosclerosis ( e.g. , increased common carotid artery intima - media thickness ) [ 5159 ] .
it is not surprising , then , that the ms , a constellation of cardiovascular risk factors with well established impact in the incidence of cvd and type ii diabetes mellitus , might be associated with the development and progression of ckd .
all the components of the ms have individually been associated with the incidence and progression of ckd . the mechanisms and impact of hypertensive and diabetic injuries , the two major etiologies of ckd in the world ,
obesity has also been associated with increased risk for esrd in several epidemiological studies [ 63 , 64 ] .
dyslipidemia , in particular atherogenic dyslipidemia ( low hdl cholesterol , high triglycerides ) , has also been shown to be an independent risk factor for the development and progression of ckd in observational studies and meta - analysis [ 42 , 6567 ] .
the clustering of all these risk factors should identify individuals at high risk for ckd . over the last decade a growing body of clinical evidence addressed this issue .
several observational studies found that individuals with the ms are at increased risk for presenting renal manifestations , namely , microalbuminuria and decreased glomerular filtration rate ( gfr ) .
in fact , epidemiologic studies have linked the ms with an increased risk for microalbuminuria , an early marker of glomerular injury and endothelial dysfunction [ 6873 ] . a cross - sectional survey of nondiabetic native americans that was conducted by hoehner et al . found that , after controlling for social , demographic , and comorbidity factors , the patients with one to two and those with three or more traits of the ms were 80% and 130% more likely to have microalbuminuria than those without ms , respectively .
chen et al . corroborated these findings in another cross - sectional analysis with data from 6125 individuals extracted from the national health and nutrition examination survey ( nhanes ) iii database - the multivariate - adjusted odds ratio for microalbuminuria in participants with the ms compared with participants without ms was 1.89 ( 1.342.67 ) , and the risk increased with the number of components of the ms .
similarly , a number of observational studies evaluated the association of ms and ckd ( table 2 ) [ 68 , 7381 ] .
almost all of them found a significant association between ms and ckd and consistently demonstrated an increased risk parallel to the number of ms traits [ 68 , 74 , 77 , 79 ] .
however , the majority of the studies were cross - sectional and , as so , unable to establish a cause - and - effect relationship , remaining unclear if the ms is a cause or a consequence of the reduced kidney function .
in fact , some reports suggest that renal dysfunction per se , even in early stages , is a cause of insulin resistance and ms which increases as the nephropathy progresses [ 82 , 83 ] .
additionally , experimental work indicates that uremia is a cause of insulin resistance rapidly reversible by peritoneal dialysis or hemodialysis , further suggesting the presence of a low - molecular - weight substance that functions as an insulin desensitizer in uremia .
on the other hand , most of these studies included patients with diabetes and hypertension , two known risk factors for the development and progression of ckd , further impairing the analysis . in order to address the issues of causality and impact of ms in the development of ckd ,
two relatively large longitudinal studies , that excluded diabetic patients at baseline , were published to date [ 74 , 76 ] .
showed an 88% ( or 1.88 ; 1.262.8 ) increased risk for ckd in patients with ms compared to those without , after a 3-year followup period .
however , when hypertensive patients at baseline were excluded from the analysis the increased risk was wiped out ( or 0.92 ; 0.4461.917 ) . probably , the most significant study to date was published by kurella et al .
the significant association between the ms and subsequent development of ckd was confirmed ( or 1.43 ; 1.181.73 ) .
moreover , this association remained true after excluding patients with hypertension at baseline ( or 1.46 ; 1.081.97 ) and patients with incident hypertension or diabetes during the followup ( or 1.24 ; 1.011.51 ) , although with marginal statistical significance .
however , even when hypertensive patients were excluded at baseline , the patients with ms had statistically significant higher blood pressure within the normal range ( at baseline and at the end of followup ) than the ones without ms , making it impossible to exclude the effect of hypertension .
additionally , in this and other studies [ 68 , 73 , 76 ] hypertension was the individual ms trait with the strongest association to ckd , even stronger than the ms itself .
the same consideration is valid for hyperglycemia in some studies [ 68 , 73 , 77 ] .
as bakker et al . elegantly demonstrated the problem with the concept of ms , as a way to predict renal ( and cvd ) risk , is the dichotomization of continuous variables and clustering correlated risk factors , resulting in a significant loss of predictive power .
a study of kitiyakara et al . has even suggested that the risk of ckd associated with the ms was different accordingly to the definition of ms used .
although the results of these studies suggest there is a close association between ms and renal disease , two questions still do persist does the ms add any relevant additional information in terms of renal risk ?
kurella et al . also found that there were graded relations among the number of ms traits , the homeostasis model assessment ( homa)-insulin resistance , fasting insulin levels and the risk for ckd , suggesting a pathophysiologic basis for their findings .
these findings indicate that that the ms directly contributes to the development of ckd and the claimed pathophysiologic link is obesity , a hallmark of the ms .
part of the explanation is , in fact , the link between obesity , diabetes and hypertension .
however , obesity ( in particular visceral obesity ) has independently been associated with the risk of ckd and esrd [ 63 , 64 , 68 , 76 , 87 ] , even when corrected for these two indirect mechanisms and proteinuria , suggesting the existence of additional direct pathomechanisms .
many studies evaluated the mechanisms by which the factors in ms mediate pathological and pathophysiological changes in the kidney .
the underlying mechanisms are still not completely understood but include insulin resistance itself , inflammation , renal endothelial dysfunction , oxidative stress , altered renal heamodynamics , activation of the reninangiotensin - aldosterone system ( raas ) and sympathetic nervous system ( sns ) , and dietary factors .
inflammation has been implicated in ms pathogenesis , especially as a mechanism of insulin resistance and endothelial dysfunction .
there is increased flux of free fatty acids into the liver leading to excessive hepatic production of triglycerides and resultant hypertriglyceridemia .
moreover , the adipocytes also secrete inflammatory cytokines ( e.g. , mcp-1 , tnf- , il-6 , leptin and c reactive protein ) , while there is relative deficiency of the anti - inflammatory and antiatherogenic cytokine adiponectin , resulting in a low - grade inflammatory state thought to be responsible for insulin resistance and endothelial dysfunction . at the molecular level ,
the link between inflammation and insulin resistance has already started to be elucidated [ 9092 ] , but is over the scope of this article .
the role of chronic inflammation in renal pathophysiological changes in the setting of ms , driven both by proinflammatory cytokines and adipocytokines , is not clear .
as stated , in obesity , the adipocyte - derived adiponectin , an insulin - sensitizing anti - inflammatory protein , is decreased along with an increase in the plasma levels of leptin .
increased adipokines have been shown to directly contribute to insulin resistance but their effects on ckd have not been examined .
however , adipokines such as leptin can cause renal vascular remodeling and disruption of renal function .
some evidence indicates that leptin may represent a link between obesity and increased sympathetic activity , through its action on the hypothalamus , to increase blood pressure .
it has also been shown that increased levels of tnf- promote generation of reactive oxygen species ( ros ) in glomerular cells and proximal tubular cells .
these ros contribute to renal injury in several ways like inducing renal endothelial dysfunction and microalbuminuria , matrix accumulation , mesangial expansion , and fibrosis [ 94 , 95 ] . within the kidney , insulin resistance and hyperinsulinemia associated with ms seem to induce local inflammation , an important pathophysiological pathway for ckd .
insulin may induce renal fibrosis through stimulation of mesangial cells and proximal tubule cells to produce tgf - b [ 96 , 97 ] .
moreover , insulin stimulates the production of igf-1 by vascular smooth muscle cells and other cell types , which has been implicated in the development of diabetic kidney disease .
igf-1 increases the activity of connective tissue growth factor , a cytokine that has profibrogenic actions on renal tubular cells and interstitial fibroblasts .
in addition , igf-1 decreases the activity of matrix metalloproteinase-2 , an enzyme responsible for extracellular matrix degradation , thereby promoting extracellular matrix expansion and renal fibrosis [ 99 , 100 ] .
additionally , insulin resistance promotes sodium and uric acid reabsorption , resulting in salt - sensitive hypertension and hyperuricemia . in the end , at the glomerular level , insulin resistance and the release of inflammatory cytokines induce mesangial expansion , basement membrane thickening , podocytopathy and loss of slit pore diaphragm integrity , leading to the so - called obesity - related glomerulopathy [ 89 , 101 ] .
this condition is characterized by a specific histopathologic pattern of glomerulomegaly ( 100% of cases ) , frequently accompanied by focal segmental glomerulosclerosis ( 80% of cases ) , and has been repeatedly described in obese patients without any other defined primary or secondary glomerular diseases ( including diabetic nephropathy , hypertensive nephrosclerosis , and secondary focal segmental glomerulosclerosis ) .
this glomerulopathy has pathologic features strikingly resembling the ones induced by diabetes and/or hypertension and , similarly , has a progressive clinical course .
of particular concern , the biopsy incidence of this condition had a 10-fold increase over a period of 15 years and has been observed in children as young as 3 years of age [ 101 , 102 ] .
the consistent observation of glomerulomegaly raised the hypothesis that glomerular hyperfiltration was a major mechanism in the pathogenesis of obesity - related glomerulopathy .
this was first elucidated in an animal model , the obese zucker rat , which has hyperphagia due to a defect in the brain leptin receptor , resulting in obesity and associated hyperglycemia , hyperinsulinaemia , insulin resistance , dyslipidaemia and hypertension , thus closely mimicking ms in humans .
this model has glomerular hyperfiltration and develops albuminuria , which progress to renal failure with histological characteristics of glomerulomegaly and focal and segmental glomerulosclerosis .
these authors demonstrated that obese patients , with clinical characteristics consistent with the diagnosis of ms , had a 50% and 30% increase in gfr and renal plasma flow ( rpf ) , respectively , as compared to lean controls , resulting in increased filtration fraction , an indirect indicator of glomerular hypertension .
moreover , when 17 morbidly obese patients who lost an average of 48 kg at 1 year after bariatric surgery , postprandial gfr and rpf were significantly decreased and approached normal levels , even though the patients were still obese . in a relatively recent study with young ( mean age 18 years ) healthy males , ms was associated with a 6.9-fold increase in the or of glomerular hyperfiltration .
the authors concluded that glomerular hyperfiltration has an early - onset in life , before any manifestations of cvd , and so may be a marker of metabolic risk .
it is well known that hyperfiltration ( even in nondiabetic patients ) leads to albuminuria ( a well established cardiovascular and renal risk factor ) , which is consistent with the aforementioned epidemiologic studies that showed a graded prevalence of microalbuminuria according to the number of ms traits [ 6873 ] .
besides the known effect of hyperglycemia , these renal haemodynamic changes have been attributed in experimental studies to a higher intake of dietary protein in these individuals [ 107109 ] .
the amino acid excess induces a profibrotic and proliferative mesangial injury response , mediated by increased formation of advanced glycation end - products , presumably the result of greater availability of free amino groups for glycation reactions [ 110 , 111 ] .
the presence of renal oxidative stress has been documented in animal models of insulin resistance , diabetic patients , and patients with ckd of various etiologies [ 112 , 113 ] . high glucose and free fatty acid increase mitochondrial ros in renal endothelial cells , which may contribute to tissue dysfunction by dysregulation of redox - sensitive signaling pathways or by oxidative damage to biological structures ( dna , proteins , lipids , etc . ) .
lipid peroxidation , a well - known consequence of excessive ros , is the first step in the generation of oxidized ldl , which accumulates in renal mesangial cells and form foam cells .
lipid peroxidation also induces endothelial damage and inflammatory response , impairs vasodilatation and activates macrophages [ 116 , 117 ] .
lipid peroxidation itself generates more free radicals and ros thereby increasing the potential for renal injury .
additionally , the aforementioned glomerular hyperfiltration may also be caused by mitochondrial ros , through the activation of cyclooxygenase-2 gene transcription followed by prostaglandin e2 overproduction and preglomerular vasodilation .
furthermore , excessive oxidative stress stimulates the synthesis of angiotensin ii ( via the nuclear factor - kb pathway ) , which further increases glomerular hypertension and hyperfiltration by causing efferent arteriole vasoconstriction .
angiotensin ii also stimulates the synthesis of tgf - b , thereby promoting glomerular fibrosis .
moreover , ros - dependent b - cell damage is thought to be involved in the progression from ms to type ii diabetes mellitus and ross enhance the formation of advanced glycation end products , which have a major pathophysiologic role in the development of diabetic nephropathy .
dysfunction of the vascular endothelium is an important factor in the pathogenesis of diabetic nephropathy and has its clinical counterpart in microalbuminuria [ 121 , 122 ] .
available evidence indicates that the insulin - receptor signaling pathway mediating glucose uptake in vascular endothelium requires stimulation of endothelial nitric oxide ( no ) synthase and subsequent no production , a potent vasodilatory and antithrombotic substance .
these actions mediate a hemodynamic component of energy distribution , enhancing tissue blood flow to optimize nutrient delivery . in insulin - resistant states , such as obesity , ms and type 2 diabetes mellitus ,
insulin- ( and adiponectin- ) induced no production and endothelium - dependent vasodilatation are impaired , representing one of several mechanisms linking abdominal obesity / insulin resistance and hypertension [ 125127 ] .
additionally , excessive oxidative stress further decreases no production and availability thereby contributing to endothelial dysfunction in diabetic and ms patients .
another product of endothelial cells , endothelin-1 , has been implicated in cvd , hypertension and several kidney diseases .
insulin stimulates the secretion of endothelin 1 from glomerular endothelial , mesangial and vascular smooth muscle cells through the insulin receptor .
endothelin-1 is associated with severe intrarenal vasoconstriction , reduced gfr , mesangial cell contraction and proliferation and sodium and water retention [ 128130 ] .
patients with the ms are hyperinsulinemic , suggesting that endothelin-1 may be involved in the development of nephropathy in these patients .
recently , another dietary factor - fructose - has been implicated in the pathophysiology of ms , and could establish the link between modern dietary patterns , systemic inflammation , ms and cardiovascular and renal disease progression . fructose is a simple sugar that is a component of table sugar as well as of high fructose corn syrup , which is a universally used sweetener .
fructose consumption has increased markedly worldwide during the last several decades , which correlates closely with the increasing incidence of obesity and ms .
the administration of fructose to both animals and humans has been showed to induce most of the features of ms [ 133 , 134 ] .
this is clinically relevant as some studies have reported that adolescents can easily ingest 15%20% of their energy intake as fructose . in recent years ,
experimental evidence associating fructose and systemic inflammation has accumulated , pointing out an increased expression of leukocyte adhesion proteins ( icam-1 ) and chemokines ( mcp-1 ) in various cell types ( endothelial , proximal tubular , and vascular smooth muscle cells ) .
the underlying mechanism appears to be related to increased uric acid generation and resulting hyperuricemia .
uric acid has been shown to induce oxidative stress , reduce nitric oxide generation , stimulate the local renin - angiotensin system and induce expression of proinflammatory chemokines , resulting in a significant increase in renal injury and inflammation [ 136139 ] .
these recent insights into the pathophysiology may represent new treatment opportunities ( e.g. , reduction of dietary fructose , uric acid lowering agents or , in the future , blocking the signaling pathways driving the response ) , as there is increasing evidence that systemic inflammation may increase the risk of cardiovascular outcomes and progression of renal disease [ 140142 ] . finally , the sympathetic nervous system ( sns ) and the renin - angiotensin - aldosterone system ( raas ) are considered important pathophysiologic factors in the development of ms , which can also contribute to the high cardiovascular risk associated with it .
some studies have associated plasma aldosterone levels with insulin resistance and ms , in both normotensive and hypertensive patients [ 143145 ] .
angiotensin ii is thought to have a role in the development of insulin resistance by inducing oxidative stress and also in the development of salt - sensitive hypertension by promoting sodium reabsorption , which could explain the benefits of blocking the raas on prevention of new cases of diabetes . in summary ,
apart from the effect of hypertension and diabetes , there are other contributing factors for development of kidney injury in ms .
these include oxidative stress and systemic inflammation ( probably induced by dietary factors like fructose and adipose tissue - derived cytokines ) , endothelial dysfunction , altered renal hemodynamics ( partially as the result of high dietary protein intake ) , excessive renal sodium reabsorption , activation of the raas and sns , atherogenic lipid profile , and even physical compression of the kidneys by adipose tissue .
complex interactions between all these factors ultimately lead to glomerular hyperfiltration , glomerular cell proliferation , matrix accumulation , and , finally , glomerulosclerosis and progressive loss of nephrons .
the association between ms and renal disease raises the question whether treating the components of the syndrome could prevent renal injury .
interventional randomized controlled trials addressing renal endpoints in the specific setting of ms have not been published to date . however
, each diagnostic criterion of ms identifies a risk factor for cvd that requires intervention independently of its contribution to ckd incidence or progression .
although the value of a multifactorial intervention for preventing cardiovascular events and ckd in the setting of ms has not been examined , it has already been proposed . according to this strategy , upon the diagnosis of ms
, clinicians should implement a multitargeted intervention with treatment goals based on global cvd risk assessment ( using , e.g. , the 10-year framingham risk score or the european risk score chart and the estimated gfr ) .
this multidimensional intervention should focus simultaneously on life - style changes , glycemic and blood pressure control , lipid lowering , and other proven beneficial treatments , like antiplatelet therapy ( figure 1 ) .
the value of simultaneous management of multiple risk factors has been tested in type ii diabetic patients ( a group for which ms is intended to be a high risk factor ) , resulting in a lower risk of cvd and nephropathy in the intensive treatment group ( stepwise implementation of behavior modification and pharmacologic therapy with strict treatment targets for hyperglycemia , hypertension , dyslipidemia , and microalbuminuria , plus secondary prevention of cardiovascular disease with aspirin ; treatment overseen by a project team ) compared to conventional treatment ( treatment for multiple risk factors from their general practitioner , according to current recommendations , with the possibility of being referred to specialists ) .
moreover , it has been shown in this group of patients that intensive blood pressure ( < 130/80 mm hg ) and blood glucose control ( aiming at glycated hemoglobin level 6.5% ) effectively prevent cardiovascular events , the development of microalbuminuria , and overt nephropathy [ 149 , 150 ] . despite that , the action to control cardiovascular risk in diabetes study group ( accord ) trials alert us that treating diabetic patients to normal values ( systolic blood pressure < 120 and glycated hemoglobin level 6.0% ) may not be beneficial and increase the risk of adverse events [ 151 , 152 ] .
these studies reinforce the notion that aggressive , although cautious , multitargeted management of the ms may confer protection for cvd and ckd .
in fact , several lines of evidence indicate that interventions directed at each component of the ms can protect the kidney
. lifestyle modifications , including physical activity and dietary interventions , with the objective of weight loss are a cornerstone of therapy .
physical activity and weight reduction improve insulin resistance even in people with renal impairment and esrd .
a weight loss of 5%10% of initial body weight has lowered the risk for diabetes and cvd , as well as significant ( p = .001 ) improvements in related modifiable risk factors , including hba1c , hdl cholesterol , triglycerides , and systolic and diastolic blood pressure .
beneficial renal effects , like reduction of glomerular hyperfiltration and albuminuria , have been demonstrated after weight loss ( either through diet or bariatric surgery ) [ 105 , 155 ] , although these results still need to be demonstrated by properly designed clinical trials .
additionally , the impact on renal outcomes resulting from improved insulin resistance through pharmacologic weight loss or the mediterranean diet has not been studied . moreover , in light of what is known about renal hemodynamics ( see above ) , the safety of the so popular high - protein low - carbohydrate weight reduction diet should also be analyzed .
finally , the potential effects of dietary fructose reduction in the incidence of ms and its renal and cardiovascular consequences represent an exciting field of research in the near future .
we strongly believe that a reasonable goal for obese patients with the ms would be a weight loss of 510% of body weight , through an effective ongoing program that includes an increase in physical activity to at least 150 min / week of moderate exercise , similar to the recommendations for prevention of type ii diabetes mellitus in patients with impaired glucose tolerance ( igt ) or impaired fasting glucose ( ifg ) .
as mentioned , intensive treatment of diabetes and hypertension has been shown to improve renal outcomes in diabetic patients . moreover , blocking the raas is extensively recommended as first - line anti - hypertensive therapy for renal and cardiovascular protection , especially in diabetic and/or proteinuric patients [ 159 , 160 ] . given the aforementioned role of raas activation in the pathophysiology of the ms and the importance of hypertension on ckd risk in the setting of ms , we should expect great impact of raas blockers on the development of kidney injury .
nevertheless , angiotensin - converting enzyme inhibitors and angiotensin receptor blockers should be the preferred antihypertensive drugs in patients with ms as in diabetics .
on the other hand , the use of thyazide diuretics and b - blockers should be avoided ( or restricted to treatment of resistant hypertension ) in patients with ms due to the risk of new - onset diabetes .
the therapeutic goal should be established according to the global cardiovascular risk , as recommended by the latest guidelines . in general , given that the ms is a constellation of cvd risk factors ( implying the presence of at least two traits ) and often with subclinical organ damage , a blood pressure lower than 130/80 mm hg should be attained in most patients .
probably , for renal protection , a lower target ( < 120/75 mm hg ) is beneficial in highly proteinuric patients ( > 1 gr / day ) .
however , as mentioned before , bringing systolic blood pressure to normal values ( < 120 mm hg ) may increase treatment sideeffects without additional benefit , especially in patients with advanced atherosclerotic disease .
given the known impact of diabetes in the development and progression of ckd , glycemic control in diabetic patients or preventing / delaying new - onset type ii diabetes mellitus is a main goal of therapy in patients with ms .
the use of agents that improve insulin sensitivity , like metformin or thiazolidenidiones , is of great interest but also raises some concerns .
metformin has been shown to reduce the incidence of both type ii diabetes mellitus and ms in subjects with igt . however , metformin was less effective than intensive lifestyle modification ( regular physical activity and low - fat diet ) [ 164 , 165 ] .
despite its beneficial effects , metformin is contraindicated in ckd patients with creatinine clearance lower than 60 ml / min due to the risk of severe lactic acidosis .
thiazolidenidiones ( peroxisome proliferator activator receptor- agonists ) , another class of antidiabetic agents with insulin sensitizing properties , have multiple pleotropic effects that , in theory , are of great interest in the setting of ms .
they reduce blood pressure and modulate dyslipidaemia , inflammation , oxidative stress , endothelial dysfunction , fibrosis and remodelling , and glomerular cell proliferation , and reduce urine albumin excretion .
moreover , thiazolidenidiones also reduce the incidence of type ii diabetes mellitus in prediabetic subjects .
however , their use causes weight gain and fluid retention , an undesirable effect especially for obese subjects and some drugs of this class have been associated with an increased incidence of myocardial infarction .
other classes of antidiabetic drugs have been shown to improve cardiovascular risk profile in diabetic and pre - diabetic patients , beyond glycemic control .
drugs that promote weight loss , improve control of other cardiovascular risk factors ( like blood pressure and lipid profile ) or that produce beneficial effects in surrogate markers of subclinical atherosclerosis , are very attractive in the setting of ms . for example , in a systematic review , the old alpha - glucosidase inhibitors have been shown to produce a modest weight reduction ( 1.2 kg ) as well as improvements in total cholesterol , ldl cholesterol , triglycerides , systolic and diastolic blood pressure in patients with igt and/or ifg . additionally , in the same group of patients , acarbose was associated with a significant reduction in the progression of carotid intima - media tickness ( imt ) compared to placebo - the annual progression of imt was reduced by 50% , with a mean followup of 3.9 years .
more importantly , in the study to prevent non - insulin - dependent mellitus ( stop - niddm ) , also in patients with igt , acarbose was found to reduce the risk of developing any cardiovascular event by 49% with an absolute risk reduction of 2.5% . on the opposite time - line end , also the new incretin mimetics
glucagon - like peptide-1 ( glp-1 ) receptor agonists and dipeptidyl peptidase-4 ( dpp-4 ) inhibitors - have beneficial pleotropic effects on various ms traits .
glp-1 receptor agonists are associated with progressive , dose - dependent weight loss in patients with type 2 diabetes while the dpp-4 inhibitors are generally considered weight neutral .
exenatide , a glp-1 receptor agonist , has been shown to produce significant reductions in total cholesterol , triglycerides , systolic and diastolic blood pressure in patients with type ii diabetes mellitus with the ms . the effect on blood pressure ,
a major determinant of renal and cardiovascular risk in patients with ms , is very significant and is greater in patients with systolic blood pressure > 130 mmhg ( 11.4 mm hg and 3.6 mm hg for systolic and diastolic blood pressure , resp . ;
similarly , dpp-4 inhibitors also produced favorable trends on lipid levels and systolic and diastolic blood pressure , even in patients without diabetes [ 173 , 176 ] .
moreover , glp-1 receptor agonists were recently associated with improvements in inflammatory state , insulin resistance and beta - cell function as compared to sulfonylureas in patients with type ii diabetes . at the present time , there are no data on glycemic control goals in patients with ms who are not diabetic . both for renal and cvd prevention
the obvious treatment goal is to prevent the onset of overt type ii diabetes in this group of patients .
current recommendations are to treat patients with ifg , igt or glycated hemoglobin of 5.7%6.4% though weight loss and physical activity . despite their beneficial effects ,
the use of antidiabetic drugs is actually not recommended in prediabetic subjects , maybe with the exception of metformin for patients at very highrisk of developing diabetes ( combined ifg and igt plus other risk factors such as glycated hemoglobin > 6% , hypertension , low hdl cholesterol , elevated triglycerides , or family history of diabetes in a first - degree relative ) who are obese and under 60 years of age . for patients with overt type ii diabetes mellitus , both for prevention of nephropathy and cvd risk reduction ,
the recommended goal is to achieve a glycated hemoglobin < 7% , although the available evidence is more compelling for microvascular disease prevention .
moreover , data from large diabetes trials suggest a small but incremental benefit in microvascular outcomes with glycated hemoglobin closer to normal [ 150 , 158 , 179 ] . therefore , despite the results of the aforementioned accord trial , for renal disease prevention a lower glycated hemoglobin might be pursued in selected patients ( those with short duration of diabetes , long life expectancy , and no significant cvd ) , with close monitoring for hypoglycemia and other adverse effects of treatment . in the setting of ms ,
the antidiabetic drugs with insulin sensitizing properties and/or with beneficial effects on other cardiovascular risk factors / markers should be considered for first - line therapy .
statins are the most widely used agents for dyslipidemia , but their role in the ms has not been well evaluated . nevertheless , for example , in the scandinavian simvastatin survival study ( 4s ) , among patients with elevated serum ldl - cholesterol and established coronary disease , those with characteristics of the ms had both the highest risk of major coronary events and the greatest benefit from statin therapy .
additionally , treatment of patients with known coronary disease and the ms with atorvastatin ( 80 mg / day ) , compared to atorvastatin ( 10 mg / day ) , decreased the rate of major cardiovascular events .
the effect of statins on kidney function has been addressed in several clinical studies and meta - analysis .
it was demonstrated that statins reduce kidney function loss in ckd patients and reduce albuminuria in microalbuminuric and macroalbuminuric patients [ 182 , 183 ] .
moreover , statins have been shown to slow down gfr decline in high cardiovascular risk patients with normal or near normal renal function at baseline and the patients with ms were the ones with the greatest benefit .
the role of statins for renal protection is attributed to their presumed pleiotropic effects including anti - inflammatory , antifibrotic , antihypertensive , and antioxidant properties .
fibrates are hypolipidaemic drugs very effective in reducing triglycerides , a major feature of ms .
some studies have suggested an improvement in proteinuria progression on diabetic patients treated with fibrates , but their role in preventing ckd in ms patients is still unknown . for cvd prevention ( and probably also for renal disease prevention and/or progression ) , the goal of therapy for ms patients , as for diabetic patients , should focus primarily in ldl cholesterol . in most cases ,
patients with ms represent a group with very high added risk for cvd ( framingham 10 year risk > 20% ) , even if they are not diabetic .
as so , most patients with ms and all patients with type ii diabetes mellitus ( a coronary heart disease equivalent ) should be treated to a primary treatment goal of ldl cholesterol < 100 mg / dl . in patients with overt cvd
, a lower target of < 70 mg / dl may be considered . for patients with lower cvd risk ( framingham 10 year risk 10%20% ) ,
secondary treatment goals , in this population are triglycerides < 150 mg / dl and hdl cholesterol > 40 mg / dl in men and > 50 mg / dl in women . to achieve these goals therapeutic lifestyle changes ( with emphasis on weight loss and physical activity ) should be implemented , but statin therapy will almost always be necessary and should be started simultaneously with lifestyle changes in high risk patients [ 9 , 158 ] .
the existence of ms as a unique pathophysiologic process and whether it confers risk beyond its individual components have been questioned and are still under debate . moreover , other clinical variables and risk scores have been proven superior to ms in indentifying individuals at risk for type ii diabetes mellitus or cvd [ 186 , 187 ] . despite that , in the last decade , a growing body of epidemiologic evidence has associated the ms with the risk for developing renal damage , clinically expressed in the form of microalbuminuria and/or ckd .
given the additional association between ms and cvd risk , which can be further aggravated if renal impairment is superimposed , ms represents , at least , a powerful awareness tool for the need to aggressively prevent and manage risk factors .
this need is underscored by the epidemic of obesity in the developed world , particularly affecting adolescents and young adults , which can pose in the future a huge burden on the health care system including dialysis facilities .
the association between ms and renal damage is , in part , explained by hypertension and impaired glucose metabolism .
however , experimental and epidemiologic data suggest that other aspects of the ms are associated with the development of renal abnormalities and should also deserve close attention as they are modifiable risk factors .
in particular , prevention of obesity and promotion of healthy lifestyles are likely to be the most effective approaches and should be a public health priority . an increasing body of evidence suggests that treating each component included in the ms definition can improve renal outcomes .
however , it has never been investigated whether treating patients with ms will prevent the development and progression of ckd .
clinical trials , in the setting of ms , to verify whether treating its components may effectively prevent renal impairment should be a priority .
these should include trials of weight loss and nutritional strategies ( e.g. , low protein and low fructose diets ) and multitargeted treatment of risk factors ( blood pressure and glycemic control , renin - angiotensin system inhibition , lipid lowering and insulin resistance ) .
finally , the new insights into the pathophysiology of systemic inflammation in the ms can provide , in the future , new treatment targets . | the metabolic syndrome ( ms ) and chronic kidney disease ( ckd ) have both become global public health problems , with increasing social and economic impact due to their high prevalence and remarkable impact on morbidity and mortality .
the causality between ms and ckd , and its clinical implications , still does remain not completely understood .
moreover , prophylactic and therapeutic interventions do need to be properly investigated in this field .
herein , we critically review the existing clinical evidence that associates ms with renal disease and cardiovascular disease , as well as the associated pathophysiologic mechanisms and actual treatment options . |
the theory of fetal origins of adult disease ( foad ) proposed by barker et al .
( 1986 ) claims that physiological changes during intrauterine development would promote the restriction of development , as well as ultrastructural and physiological changes that would predispose to the early development of cardiovascular and metabolic diseases in adulthood [ 13 ] . in a model of diabetes in pregnancy
, it was demonstrated that hyperglycemia promotes oxidative stress in the offspring , hence affecting the balance between oxidant and antioxidant factors , an increase in inflammatory markers , and a reduction in the number of glomeruli with aging .
the streptozotocin ( stz ) may cause mild or severe diabetes mellitus ( dm ) in the dam and lead to different effects in rat offspring .
early blood pressure , deficit in glomerular filtration and in renal plasma flow , and also glomerular hypertrophy were observed as the offspring aged .
acute renal failure ( arf ) is a kidney disorder which may result from reduced renal perfusion with no cell injury ; from ischemic , toxic , or obstructive injury of the renal tubules ; from tubulointerstitial inflammation and swelling ; or from reduced glomerular filtration rate associated with primary glomerular diseases .
the literature shows the relationship between pathological changes in adulthood and changes during intrauterine development , particularly dm .
thus , it is believed that renal injuries in a fetal programming model are caused by persistent renal inflammatory response due to an injury during intrauterine life . some authors demonstrated that gestational diabetes mellitus may lead to modifications in the placental transcriptome characterized by dominance of genes that regulate inflammatory responses .
cytokines play a crucial role in the establishment of inflammatory response and the systemic inflammation is associated with the development of endothelial dysfunction and hypertension which may result in progression of chronic disease . as inflammatory markers may predict later metabolic
[ 11 , 12 ] and vascular disease , it is extremely important to measure inflammatory markers in a fetal programming model .
therefore , based on the change of the current profile of patients with arf and on the association of this entity with several chronic diseases , the aim of this study was to evaluate the progression of folic acid - induced arf in a fetal programming model through the expression of proinflammatory markers .
this study aimed to demonstrate the influence of fetal programming on the development of arf , as well as the prognostic evolution of individuals that develop the disease .
this study was submitted to the ethics committee on animal use of the federal university of tringulo mineiro and approved under protocol number 168 .
male and female wistar rats , with initial weight ranging from 250 to 330 g , were maintained on a 12 h light - dark cycle at a constant temperature ( 25c ) .
dm was induced by stz at a dose of 50 mg / kg after twelve hours of fasting ; it was administered intraperitoneally at a single dose into female rats weighing 250330 g. control animals received the same dose of vehicle ( 0.1 m citrate buffer , ph 4.5 ) .
the diabetic state was confirmed after 48 hours by blood glucose measurement . only animals with blood glucose levels above 250 mg / dl were considered dams ( figure 1 ) . after the induction of diabetes , the estrous cycle of each rat
, the diabetic rats were mated at a ratio of one male to one female , and then a pregnancy test was performed through microscopic examination of vaginal secretions .
females that had spent a period of over seven days and/or two regular cycles with a male but that did not get pregnant were considered infertile and discarded in this study ( figure 1 ) .
approximately 21 days after birth , all the offspring from each dam were kept in lactation . in this stage ,
from the 28th day of life , the male rats were separated from their mothers and placed in collective cages .
male animals at two and five months of age were studied , comprising a total of 36 young males ( figure 1 ) .
the following experiments were performed when the animals reached the age of two and five months : the folic acid group received a single intraperitoneal injection of folic acid ( sigma , st .
louis , mo ) at a dose of 250 mg / kg in vehicle ( 0.2 ml of 300 mm nahco3 ) ; and the same volume of vehicle was administered to control animals ( figures 1 and 2 ) .
the animals were divided into four groups , as follows : ( 1 ) cc : control dam and control offspring ; ( 2 ) cfa : control dam and folic acid offspring ; ( 3 ) dc : diabetic dam and control offspring ; and ( 4 ) dfa : diabetic dam and folic acid offspring .
the animals were anaesthetized with a single injection of ketamine ( 60 mg / kg i m ) and xylazine ( 5 mg / kg i m ) , followed by exsanguination .
both kidneys of each animal were collected , weighed , and photographed , and then the renal capsule was sectioned and removed .
the right kidney was used for histological analysis and the left kidney was used for molecular biology analysis of gene transcripts .
half of the left kidney was divided into sections of approximately 50 mg , placed in a 1.5 ml polystyrene tube ( eppendorf , hamburg , germany ) , and immediately frozen in liquid nitrogen and transferred to a freezer at 80c ; the other half was fixed in karnovsky 's fixative for electron microscopy and in isopentane for further analysis .
gene expression of monocyte chemoattractant protein-1 ( mcp-1 ) , interleukin-1 ( il-1 ) , nos3 , transforming growth factor- ( tgf- ) , tumor necrosis factor- ( tnf- ) , and vascular endothelial growth factor ( vegf ) was measured by using taqman amplification system ( applied biosystems , branchburg , new jersey , usa ) , consisting of a commercial reagent mixture ( taqman universal pcr master mix , applied biosystems ) and of specific custom primers and probes ( assay - on - demand 20x , applied biosystems ) .
the reactions using taqman system consisted of the following volumes : mix taqman ( 5 l ) , primer / probe ( 0.5 l ) , cdna ( 1 l ) , and sterile water q.s
( 1 ) less than 100 mg of renal tissue was triturated in 1 ml of trizol ( invitrogen , carlsbad , california , usa ) using a polytron pt 1200 cl homogenizer ( kinematica ag , littau , switzerland ) ; ( 2 ) the homogenate was incubated for five minutes at room temperature , and 200 l of chloroform p.a .
was added ( merck , darmstadt , germany ) ; ( 3 ) the homogenate was incubated for three minutes and centrifuged at 12,000 g at 4c for 15 minutes ; the mixture formed three distinct phases : an organic phase containing proteins , a white interphase ( dna precipitate ) , and a colorless aqueous phase containing total rna ; ( 4 ) the aqueous phase was transferred to a new tube , to which 500 l isopropanol p.a .
( merck ) was added , thus promoting the precipitation of rna ; ( 5 ) the sample was incubated for ten minutes and then centrifuged for 10 minutes .
the precipitated rna formed a substance of gelatinous consistency ; the supernatant was removed and 1 ml of 75% p.a .
ethanol solution ( merck ) was added to remove the salts from the trizol reagent and then homogenized in a vortex mixer ; ( 6 ) this solution was centrifuged at 10,500 g in a refrigerated centrifuge for five minutes ; ( 7 ) the supernatant was removed , and the centrifuged solution was dried for ten minutes and was then rediluted in 50 l of rnase / dnase - free water ; ( 8) the rna was stored in a freezer at 80c .
it was synthesized from 2 g of total rna previously treated with dnase ( rq1 rnase - free dnase ) ( promega , madison , usa ) .
the following were added to the treated rna : 0.2 g of oligo(dt)12 - 18 ( ge healthcare , buckinghamshire , uk ) , 20 ng of bsa ( bovine serum albumin ) , 0.1 mol of dntps ( promega ) , and 400 u of m - mlv reverse transcriptase enzyme ( promega ) .
this mixture ( 50 ml ) was incubated at 37c for one hour for reverse transcription reactions .
all samples were run in triplicate containing a final reaction volume of 10 l , as follows : 5 l of taqman universal pcr master mix ; 1 l of cdna from each sample ; 0,5 l of primer and probe mix ; and , in the end , ultrapure water q.s .
the reaction was performed in a 7500 real - time pcr system thermal cycler ( applied biosystems , singapore ) .
amplification conditions for the taqman system are standardized and universal for any amplified pcr product . a comparative analysis of the cycle thresholds ( ct )
was used in order to determine the gene expression of mcp-1 ( rn00580555_m1 ) , il-1 ( rn00580432_m1 ) , nos3 ( rn02132634_s1 ) , tgf- ( rn00572010_m1 ) , tnf- ( rn99999017_m1 ) , and vegf ( rn01511601_m1 ) . for each sample
, the ct values of the target genes were normalized by their respective control gene , and the value used to demonstrate the relative expression of target genes was determined using the 2 expression .
n times regarding a control sample ; in this study , the same sample was always used at baseline .
for analysis between groups , one way anova ( f ) followed by tukey test was used .
rt - pcr analysis showed an increase in gene expression for mcp-1 in the groups that had received folic acid ( cfa2 and dfa2 ) in comparison with their respective control groups .
the cfa2 group presented mcp-1 significantly higher than the groups cc2 and dc2 ( figure 3(a ) ) .
mcp-1 expression was similar in animals aged five months that had been treated with folic acid .
this expression was higher in dc5 cases than in cc5 cases , however , without significant difference between groups ( figure 4(a ) ) .
there was a higher gene expression for il-1 in cfa2 , dfa2 , and dc2 cases in relation to cc2 cases , however , without significant difference between groups ( figure 3(b ) ) .
regarding the animals aged five months , il-1 expression was similar among the four groups analyzed ( figure 4(b ) ) .
gene expression for nos3 showed no significant difference in expression between the groups ( figures 3(c ) and 4(c ) ) . on the other hand ,
nos3 was found to be reduced in the dfa5 group in comparison with the cfa5 group , despite the absence of a significant difference ( figure 4(c ) ) .
nonetheless , a significant increase in gene expression of tgf- was observed between the groups that had received folic acid ( cfa2 and dfa2 ) when compared to their respective control groups .
the cfa2 group presented tgf- significantly higher than dc2 group ( figure 3(d ) ) .
there was no significant difference in the expression of tgf- between the groups analyzed at five months .
nevertheless , dc5 cases and the offspring of control and diabetic dams that had received folic acid had a higher expression of tgf- than cc5 cases ( figure 4(d ) ) .
the gene expression of tnf- was increased in the cases that had received folic acid ( cfa2 and dfa2 ) compared to their respective control groups , despite no significant difference ( figure 3(e ) ) .
there was also no significant difference in tnf- expression between the cases analyzed at the age of five months , even though a higher expression was observed in the dfa5 group in comparison with the dc5 and cfa5 groups .
the dc5 group had higher expression than the control group ( figure 4(e ) ) .
on the other hand , the gene expression of vegf was reduced in the cfa2 and dfa2 groups in relation to their respective control groups , however , without significant difference between groups ( figure 3(f ) ) .
furthermore , there was a significant reduction of vegf in the groups that had received folic acid ( cfa5 and dfa5 ) .
the dc5 group showed a higher expression of vegf than the cc5 group , despite no significant difference ( figure 4(f ) ) .
in this study , folic acid increased the expression of mcp-1 in the control offspring and in the diabetic offspring only in two - month group . in different experimental models ,
folic acid induces acute renal failure by tubular injury , which is characterized by apoptosis , proliferation of tubular cells , inflammatory infiltrate , mild fibrosis , podocyte edema , thinning of pedicel membrane associated with the presence of vesicles , reduced brush border , and tubular hypertrophy and obstruction due to the deposition of folic acid crystals .
therefore , mcp-1 seems to behave as an important biomarker of renal injury in the evaluated groups .
an experimental study on the offspring of diabetic rats correlated the expression of this proinflammatory cytokine with the chronic activation of the innate immune system in response to the increased insulin resistance .
moreover , because of fetal programming , it is believed that the dysfunction of fetal pancreatic cells caused by gestational dm results in permanent metabolic disturbance in postnatal and adult life [ 16 , 17 ] , severe insulin resistance [ 5 , 16 ] , a reduction in the number of nephrons and glomeruli , and impaired glomerular filtration rate .
therefore , these data may explain the higher expression of mcp-1 in dc5 cases compared to the cc5 cases .
the expression of il-1 was higher in the two - month groups , cfa2 , dfa2 , and dc2 .
this cytokine may mediate acute forms of renal injury , as well as being involved in the development of chronic kidney disease .
thus , it can be inferred that the injuries caused by folic acid and by fetal reprogramming increase the expression of il-1 in the two - month - old offspring .
an experimental study showed that intrauterine hyperglycemia causes the reduction of pancreatic islets , as well as a significant increase in glucose intolerance in the diabetic offspring from the 16th week of development .
these changes are correlated to the action of reactive oxygen species on pancreatic cells , which activate the transmembrane toll - like receptors responsible for the synthesis of il-1 and of other cytokines in response to the cytotoxic effect .
accordingly , fetal programming may led to a higher expression of il-1 in dc progeny than in cc progeny , as we observed in the two - month - old offspring . comparing the two groups that suffered renal injury by folic acid , a decreased nos3 in cases which suffered fetal reprogramming only in five - month groups
nos3 is expressed in the vascular endothelium of the afferent and efferent arterioles in the kidney .
however , in cases of renal injury , there is a severe uncoupling of nos3 via the nadph oxidase enzyme system and it is responsible for endothelial ros production .
thus , it can be inferred that oxidative stress interferes with its synthesis and activity .
dm compromises the activity of phosphatidylinositol 3-kinase and the activation of protein kinase b , which is responsible for phosphorylation and activation of nos3 . interference in this pathway inhibits the oxidation of l - arginine to no as well as the stability of nos3 mrna .
the reduced expression of dfa5 compared to cfa5 also indicates a greater impact of folic acid injury on the diabetic progeny , and it shows that fetal reprogramming interferes with the development of changes .
this data is in accordance with the literature , since folic acid is associated with mitochondrial dysfunction in renal failure .
an experimental study showed that tgf- levels increase up to four times six days after induction of renal injury .
therefore , the higher mrna synthesis of this cytokine in podocytes contributes to mesangial matrix expansion , gbm thickening , and proliferation , hypertrophy , and apoptosis [ 2325 ] of podocytes and proximal tubular cells .
thus , these findings seem to explain the higher expression of this cytokine in the cfa2 and dfa2 groups .
tgf- gene expression also showed the influence of age on the analyzed progeny , since the dc5 group was older .
the hyperglycemic state promotes glycation of proteins , diacylglycerol synthesis , and activation of protein kinase c that favor its expression [ 23 , 26 ] .
moreover , the higher expression in the dc5 group compared to cc5 group indicates a more evident late renal failure .
tnf- expression was higher in both the cfa2 and dfa2 groups and also in the dfa5 group when compared to the dc5 and cfa5 groups .
studies have showed that the levels of this inflammatory cytokine are increased by 25 times in the renal tissue of rats 48 hours after administration of folic acid , which was associated not only with a higher prevalence of epithelial apoptosis and necrosis [ 27 , 28 ] , but also with glomerular hypercellularity and mesangial expansion .
the literature shows that the development of maternal insulin resistance appears to be responsible for the increase in tnf- levels from the first stage of prenatal development to the 28th week .
the increased expression of tnf- in dfa5 cases in relation to cfa5 cases also indicates a greater impact of folic acid injury in diabetic offspring , hence indicating that fetal reprogramming interferes with the progressive changes that appear in late adulthood .
vegf gene expression was lower in both the cfa2 and dfa2 groups , as well as in the cfa5 and dfa5 groups .
folic acid appears to interfere with vegf expression , since there is a reduction in the expression of this growth factor 14 days after administration .
the progression of nephrotoxicity is believed to be responsible for both attenuating receptor expression and for interfering with vegf synthesis , which occurs via intracellular signaling pathways . on the other hand ,
similarly , hyperglycemia caused by activation of extracellular signal - regulated kinase in podocytes and by advanced glycation end products ( ages ) is responsible for the expression of this factor .
dm causes early ultrastructural changes , such as mesangial and endothelial cell proliferation , gbm thickening , and erasure of pedicels , as well as late ultrastructural changes , such as apoptosis of renal cells through increased expression of proapoptotic proteins bim and bax , and consequent increase in the activation of caspase-3 .
therefore , the progression of this disease may have promoted gene expression of vegf in diabetic offspring aged two and five months in this study .
studies on fetal reprogramming in humans are scarce due to the difficulty of selecting adults who have suffered some type of aggression during their fetal development , which may be confirmed through maternal medical records and , therefore , be able to assess the late consequences of these aggressions . in the literature are found some longitudinal and cohort studies on human fetal reprogramming .
there are also some studies with holocaust survivors born during world war ii proving the late effects of nutritional deficiency during fetal development on adults and their offspring due to maternal malnutrition and hunger .
the results of these studies indicate that these individuals were more likely to present dyslipidemia , hypertension , vascular disease , diabetes mellitus , metabolic syndrome , and premature osteopenia / osteoporosis [ 3739 ] .
however , acquiring human samples of biopsies and associating the data with intrauterine aggression history may be very difficult or even infeasible . because of this , experimental studies on fetal reprogramming are extremely important to simulate something that may occur in humans and , thus , be able to understand the molecular mechanisms involved in this process .
this experimental study suggests that metabolic dysfunctions triggered by dm during pregnancy and by folic acid during intrauterine life culminate in fetal programming of descendant offspring in postnatal life .
the decrease in nos3 and increase in tnf- indicate a greater impact of folic acid injury in diabetic offspring , and they also show that fetal reprogramming interferes with the development of changes that appear in late adulthood . | objective . to evaluate the expression of inflammatory markers in experimental renal failure after fetal programming .
methods .
the offspring aged two and five months were divided into four groups : cc ( control dams , control offspring ) ; dc ( diabetic dams , control offspring ) ; cfa ( control dams , folic acid offspring , 250 mg / kg ) ; and dfa ( diabetic dams , folic acid offspring ) .
gene expression of inflammatory markers mcp-1 , il-1 , nos3 , tgf- , tnf- , and vegf was evaluated by rt - pcr . results .
mcp-1 was increased in the cfa and dfa groups at two and five months of age , as well as in dc5 when compared to cc5 .
there was a higher expression of il-1 in the cfa2 , dfa2 , and dc2 groups .
there was a decrease in nos3 and an increase in tnf- in dfa5 in relation to cfa5 .
the gene expression of tgf- increased in cases that had received folic acid at two and five months , and vegf decreased in the cfa5 and dfa5 groups .
dc5 showed increased vegf expression in comparison with cc5 .
conclusions .
gestational diabetes mellitus and folic acid both change the expression of inflammatory markers , thus demonstrating that the exposure to harmful agents in adulthood has a more severe impact in cases which underwent fetal reprogramming . |
calpain ( ec 3.4.22.17 ) is a ca - dependent cysteine protease first isolated in 1978 , with a ph optimum between 7.0 and 8.0 .
there are at least 15 distinct calpain genes present in the human genome and several have a number of isoforms ( up to 10 ) . along with the atp - dependent proteasome , calpain appears to be responsible for the majority of non - lysosomal targeted proteolysis .
it is a member of the papain superfamily a group of proteases that includes papain , calpain , streptopain , ubiquitin - specific peptidases and many families of viral cysteine endopeptidases .
calpain is a protein of ancient origin with homologues found in vertebrates , insects , crustaceans , nematodes , fungi , higher plants , dictyostelium , kinetoplastid protozoa , and bacteria and evolved from a gene fusion event between an n - terminal cysteine protease and a c - terminal calmodulin - like protein , an event predating the eukaryote / prokaryote divergence .
the enzyme cleaves preferentially on the c - terminal side of tyrosine , methionine or arginine , preceded by leucine or valine ( i.e. p1 = y , m , or r ; p2 = l or v according to the established nomenclature ) .
calpain occurs either as a heterodimer with a small regulatory subunit and a large catalytic subunit or as the catalytic subunit alone .
the catalytic domain is divided into two subdomains ( 2a and 2b ) with the cysteine residue lying in domain 2a and the histidine and asparagine in 2b .
, calpain undergoes a conformational change , dissociates from or cleaves the associated calpastatin and finally cleaves its own first domain to become fully active .
substrates of this enzyme appear to be recognised principally by the presence of pest sequence(s ) within the protein although exceptions are known .
pest sequences were first described in 1986 and are short subsequences ( usually 10 60 residues ) within proteins that are bounded by but do not contain basic residues ( h , k or r ) , and are enriched in proline ( p ) , glutamate ( e ) , serine ( s ) , threonine ( t ) and aspartate ( d ) residues .
an algorithm ( the pest - find score ) has been described for assessing the significance of such subsequences : a score of 5 or greater is regarded as significant .
pest sequences are found in ~10% of all cellular proteins in the organisms analysed to date and are typically found in highly regulated proteins .
pest + ve ( pest sequence containing ) proteins typically have short half lives ( 0.5 to 2 hours ) in intact cells compared with most other proteins ( > 24 hours ) . in pest + ve proteins ,
removal or disruption of the pest sequence increases the protein 's half life to more " normal " values while insertion or creation of a new pest sequence within a pest -ve ( pest sequence free ) protein decreases that protein 's half life to a value typical of a pest + ve protein .
the authors found the inhibitors used were ~100 times as potent ( ic50 ~10 m ) than the other protease inhibitors ( chymostatin , leupeptin , pepstatin a and bestatin ) examined .
erythrocytes normally contain only calpain 2 and it was not clear at the time if the effect of these inhibitors was as a result of inhibition of the parasite 's and/or of the erythrocyte 's calpain .
this has been clarified recently by hanspal et al . who reinvestigated this effect in calpain 2 knock - out mice .
the mouse erythrocytes were shown to have no detectable calpain activity but still supported the invasion and growth of p. falciparum in culture .
a third paper has shown that removal of cafrom the growth medium results in growth arrest in the late trophozoite stage and failure to invade erythrocytes
findings consistent with a role for calpain in the parasite life cycle . with the recent publication of the entire genome [ 16 - 18 ] a search for the calpain and calpastatin genes or their homologs and pest + ve proteins was undertaken to investigate this further .
the flat files ( version 2 ) were downloaded from the plasmodb web site .
multiple errors were found in the annotation including an anomalous start ( aaa , cac , gta , tag ) and termination codons ( aat , ata , aag , ctt , gaa , ggg , ttc , ttt ) , introns of length 2 and 5 bases , unusual intronic splice sites ( ta , tt ) , introns with exceptionally high gc content ( up to 44% ) and the absence of a number experimentally known genes . in view of these findings , the alternative annotation of chromosome 2 , as revised in september 2002 , was used .
sequences of 10 + residues bounded by , but not containing basic residues ( h , k or r ) , are first identified . the mole percent ( mp ) of this subsequence
is then determined after subtracting one mole equivalent of p , e / d and s / t .
the normalised hydrophobicity value is the value of the kyte - doolittle index for that residue multiplied by 10 plus 45 , giving values between 0 and 90 .
stellwagen has suggested that a value of 58 for tyrosine rather than 32 as originally given gives a more reliable pest score : the former value was used here .
the average hydrophobicity ( ho ) of a subsequence is determined by summing the mp of each residue and its normalised hydrophobicity value .
the pest - find score is 0.55 ( mp ) - 0.5 ( ho ) .
( the original paper has a misprint with pest - find = 0.5 ( mp ) - 0.5(ho ) )
forty - four pest sequences with scores > 5 were found with lengths varying from 12 to 94 ( 30.3 /-19.9 ) amino acids in 27 ( 13.1% ) proteins .
the proteins fall into four groups ( a ) hypothetical proteins 4 ( b ) dna binding proteins 2 ( origin recognition complex subunit 5 and chromatin - binding protein ) ( c ) metabolic proteins 3 ( a phosphatase , ribosome releasing factor , atp - dependent acyl - coa synthetase ) and ( d ) cell surface associated proteins 18 ( erythrocyte membrane proteins ( emp ) 1 and 3 , rifins , merozoite surface proteins ( msp ) 2 and 5 , serine - repeat antigens , transmission blocking target antigen pfs230 and two predicted secreted proteins ) .
the pest sequences occur throughout proteins with some bias towards the n - terminal end ( 60% are found in the first half ) .
the pest + ve proteins had significantly lower predicted pis ( 6.63 and 8.13 respectively : t = 3.86 , p < 0.0005 ) and were significantly longer ( 1181 and 731 amino acids respectively : t = 2.91 , p < 0.004 ) than the average .
18 ( 66.7% ) of the pest + ve proteins have introns , a figure slightly higher than the mean ( 57.5% ) .
there was no significant difference in the number of introns per protein ( 1.12 and 1.32 , t = -0.16 , p > 0.8 ) . on chromosome 13 , a putative calpain gene ( mal13p1.310 ) containing a single intron
the gene is unusually large ( 2047 residues ) and has a biologically implausible " intron " that appears to have been created to avoid an unrecognised frameshift [ see additional file : 1 ] .
the 5 ' end of the gene contains a low - complexity region and is more than twice the size of other known calpains ( generally 600 800 residues ) .
the catalytic domain is the only part of the enzyme with homology with the vertebrate enzymes and in the p. falciparum gene this domain is unusually distant from the n - terminus ( residues 10021470 ) : in other organisms the active site lies within 150 residues of the n - terminus . it seems probable that a fusion event has occurred at the original 5 ' end of the calpain gene with a second , to date , unidentified gene . if activation of the p. falciparum calpain is similar to that in other organisms , the 5 ' domain would be removed during activation and this new element may be responsible for the selective toxicity of the inhibitors or may play some regulatory role .
a re - annotation of the genome will shortly be completed and it is intended that it will be possible to compare the two annotations ( huestis r. , personal communication ) .
pest sequences have not been previously described in p. falciparum and the sequences found here appear to be very similar to those known in other organisms .
the presence of pest sequences in hypothetical proteins , dna - binding proteins and proteins involved in intermediary metabolism was expected , while the finding that the majority of pest + ve sequences were surface exposed proteins was not . the greater length and
the lower predicted pi of the pest + ve proteins and locational bias of the pest sequences towards the n - terminus are consistent with earlier findings .
several families of surface exposed proteins are present in chromosome 2 : pest sequences were found in all pfemps , msp 2 and 5 , a subset of sera antigens ( 6 of 8) and rifins ( 2 of 7 ) .
cytoadherence - linked asexual genes ( clags ) and sub - telomeric variable open - reading frames ( stevors ) were all pest -ve .
the presence of pest sequences in a subset of sera and rifins is suggestive of differential processing or cellular turnover and may shed some light on the reason for the large number of these genes in the genome .
the presence of pest sequences in surface exposed proteins prompted a search for these sequences in other proteins known to be involved in merozoite invasion .
msp-1 and -2 , the seras , and erythrocyte binding antigen ( eba)-175 are pest + ve while apical membrane antigen ( ama)-1 , rhoptry associated protein ( rap)-1 , -2 and -3 , maebl , merozoite capping protein-1 and acidic / basic repeat antigen ( abra ) are pest -ve .
spectrin , band 4.1 and ankyrin are pest + ve erythrocyte proteins known to be bound by p. faciparum merozoites during the invasion process .
band 3 , another pest + ve erythrocyte protein , is eliminated from the site of contact with the merozoite .
the involvement of calpain in other cell fusion reactions and the presence of pest + ve membrane proteins suggest that this system may be involved in cell adhesion processes in p. falciparum . as the parasite progresses from the trophozoite to the schizont stage
, there is a thirty - fold increase in the level of transcription of calpain .
the erythrocyte normally maintains a submicromolar intracellular caconcentration which rises thirty - fold as the parasite matures .
given the presence of a pest sequence in dna origin recognition complex protein and the effects on caremoval on trophozoite - to - schizont progression , it is tempting to speculate that the lack of camay inhibit calpain activation and that this is responsible for the effects seen here [ 28 - 32 ] . bearing in mind a report for a central role for falcipain 1 in merozoite biology , the increase in transcriptional levels seen in the schizont and the effect of inhibitors on erythrocyte invasion suggest that calpain too may play a role here .
the p. falciparum calpain gene differs significantly from those found to date in vertebrates and this may partly explain the selective toxicity of the inhibitors .
there are many calpain inhibitors presently available and the majority of these are small peptides that can be freeze - dried and stored at room temperature .
several have been used in phase 2 human trials for treatment of myocardial infarction , stroke and cancer . given the need for novel drugs to treat malaria , the selective toxicity of these inhibitors , several known crystal structures and the possibility of recognising probable target proteins and gene sequence , these agents may be worth exploring further .
dm conceived of the study , downloaded the data used , wrote the software , analysed the results and wrote the drafts of the manuscript .
we would like to thank robert huestis for his help with the calpain gene annotation and two anonymous referees for their suggestions on an earlier version of this manuscript . | backgroundinhibitors of the protease calpain are known to have selectively toxic effects on plasmodium falciparum .
the enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids ( pest sequences ) .
the genome of p. falciparum was searched for this protease , its natural inhibitor and putative substrates.methodsthe publicly available p. falciparum genome was found to have too many errors to permit reliable analysis .
an earlier annotation of chromosome 2 was instead examined .
pest scores were determined for all annotated proteins .
the published genome was searched for calpain and calpastatin homologs.resultstypical pest sequences were found in 13% of the proteins on chromosome 2 , including a surprising number of cell - surface proteins .
the annotated calpain gene has a non - biological " intron " that appears to have been created to avoid an unrecognized frameshift .
only the catalytic domain has significant similarity with the vertebrate calpains .
no calpastatin homologs were found in the published annotation.conclusiona calpain gene is present in the genome and many putative substrates of this enzyme have been found .
calpastatin homologs may be found once the re - annotation is completed . given the selective toxicity of calpain inhibitors , this enzyme may be worth exploring further as a potential drug target . |
the strong association between abdominal fat and increased risk of cardio - metabolic risk suggests that measures of central fat distribution such as waist circumference ( wc ) and whr may be better than measures of general obesity , such as bmi , in predicting type 2 diabetes
. this may be particularly important in the elderly population in whom bmi is known to be a weaker indicator of obesity because visceral fat increases and muscle mass declines with age .
the national heart lung and blood institute recommends the use of wc as an additional means of assessing the risks associated with excessive adiposity .
although several prospective studies have shown wc to be a better predictor of diabetes than bmi [ 48 ] , the results are inconclusive [ 913 ] .
indeed , in a recent meta - analysis based on 32 studies , bmi , wc and whr were all shown to have similar associations with risk of incident type 2 diabetes , suggesting that all measures are important in predicting the risk of diabetes . however , this meta - analysis did not stratify analyses by sex , which may be a potentially important omission .
furthermore , few studies have compared the use of these anthropometric markers in older individuals and determined whether the use of bmi combined with measures of body fat distribution ( wc or whr ) improves the ability to predict diabetes .
this is an important clinical question as the greatest relative increase in type 2 diabetes prevalence in developed countries in the next 25 years will be in those aged over 65 years .
uncertainties also remain about the best combination of anthropometric measures to use in men and women and the optimal cut - off points , particularly in older people .
we have examined the predictive power of bmi , wc and whr in predicting incident diabetes and evaluated wc cut - off points to assess diabetes risk in a prospective study of men and women aged 6079 years .
we have also examined the combined use of bmi and measures of fat distribution ( wc and whr ) in predicting type 2 diabetes .
data from the british regional heart study and the british women s heart and health study were used .
the british regional heart study is a prospective study of cardiovascular disease involving 7,735 british men drawn from general practices in 24 british towns and cities who were followed up from 1978 to 1980 [ 16 , 17 ] . in 19982000
, all surviving men , now aged 6079 years , were invited for a 20th year follow - up examination ; 4,252 men ( 77% of survivors ) attended for examination . in 19992001 , a parallel study of 4,286 women ( 60% of those invited ) of the same age and drawn from 22 of the same 24 towns and cities was established , with the addition of one more study city ( bristol ) .
the study population was predominantly ( > 95% ) described as white by examining nurses .
similar protocols for data collection were used in both studies . in both studies , nurses administered questionnaires , made physical measurements and collected fasting venous blood samples , from which serum was stored at 70c for subsequent analysis . men and women completed detailed questionnaires on : medical history ; medication ; and lifestyle , including cigarette smoking , alcohol consumption and physical activity .
details of measurement and classification methods for smoking status , physical activity , social class , blood pressure and blood lipids in the two cohorts have been described elsewhere [ 1821 ] .
all participants provided written informed consent to the investigation and ethical approval was provided by all relevant local research ethics committees .
fasting glucose measurements were available in 3829 women ( 89% ) and 4032 men ( 95% ) .
we excluded all men and women with prevalent diabetes ( defined as the presence of a diagnosis by a doctor of diabetes and/or a fasting glucose of 7 mmol / l ) ( n = 481 men , n = 377 women ) and four women whose diabetes status could not be determined .
we further excluded those with missing bmi , waist and hip measurements ( n = 32 men ; n = 44 women ) .
after these exclusions , a total of 3519 non - diabetic men and 3404 non - diabetic women were eligible for the current study .
height was measured with a holtain ( crosswell , uk ) stadiometer to the last complete 0.1 cm and weight was measured with a soehnle ( waagen , murrhardt , germany ) digital electronic scale to the last complete 0.1 kg .
body mass index ( weight / height in kg / m ) was calculated for each individual .
waist and hip circumferences were measured in duplicate with an insertion tape ( cms , london , uk ) ; hip circumference was measured at the point of maximum circumference over the buttocks .
the waist measurement was taken from the midpoint between the iliac crest and the lower ribs measured at the sides .
follow - up all men and women were followed up for all - cause mortality , cardiovascular morbidity and development of type 2 diabetes .
losses to follow - up remained exceptionally low ( < 3% ) in both cohorts .
the analysis is based on follow - up from re - screening ( 19982000 ) to june 2006 in men and from 19992001 to september 2007 in women , a mean follow - up period of 7 years ( 68 years ) .
information on the incidence of new events has been obtained from the combination of regular two yearly reviews of patients notes ( including hospital and clinic correspondence ) through to the end of the study period and from personal questionnaires to surviving participants , repeated since the initial examination , asking whether a doctor had ever told them that they had diabetes , the year of diagnosis and reported use of medications specifically for diabetes .
self - report of doctor - diagnosed diabetes has shown to be a valid measure of recording diabetes in the present study .
the medical - record review was compared with the patient 's recall of doctor - diagnosed diabetes . in 97% of men who reported a doctor diagnosis of diabetes on the questionnaire ,
statistical analysis the study population was stratified into sex - specific quartiles of bmi , wc and whr .
a cox proportional hazards model was used to assess age - adjusted and multivariate - adjusted hazard ratio ( hr ) .
age , systolic blood pressure , log triacylglycerol and log fasting glucose were fitted as continuous variables in the multivariate analysis .
physical activity , smoking , parental history of diabetes , manual social class , pre - existing chd and alcohol intake were fitted as categorical variables .
finally , receiving - operating characteristic ( roc ) curve analyses and the respective auc were used to compare the predictive power of baseline wc , whr , and bmi on risk of type 2 diabetes in men and women .
during follow - up , there were 165 incident cases of type 2 diabetes in the 3,519 men ( 7.15/1,000 person - years ) and 128 cases in the 3,404 women ( 5.40/1,000 person - years ) .
bmi correlated most strongly with wc in both men and women ( r = 0.87 and r = 0.83 , respectively ) and correlated more with whr in men than in women ( r = 0.53 and r = 0.35 ) .
sex - specific baseline characteristics of the study sample are shown in table 1 .
compared with the women , men were more physically active and had higher rates of smoking , consumption of alcohol and pre - existing chd , but lower rates of family history of diabetes .
table 1baseline characteristics in men and women with no prevalent diabetescharacteristicmen ( n = 3,519)women ( n = 3,404)p valueage ( years)68.6 ( 5.5)69.3 ( 5.5)<0.0001bmi ( kg / m)26.7 ( 3.5)27.3 ( 4.8)<0.0001wc ( cm)96.5 ( 10.0)85.4 ( 11.7)<0.0001hip circumference ( cm)102.1 ( 6.9)104.6 ( 10.0)<0.0001whr0.94 ( 0.06)0.81 ( 0.07)<0.0001inactive , % ( n)32.6 ( 1109)63.0 ( 2065)<0.0001current smokers , % ( n)13.0 ( 455)11.05 ( 376)0.0127parental history of diabetes , % ( n)5.5 ( 195)9.3 ( 318)<0.0001manual social class , % ( n)50.7 ( 1784)48.9 ( 1518)0.1439systolic blood pressure ( mmhg)148.5 ( 23.9)146.2 ( 24.9)0.0001hdl - cholesterol ( mmol / l)1.34 ( 0.34)1.67 ( 0.45)<0.0001triacylglycerol ( mmol / l)1.58 ( 1.13 , 2.15)1.62 ( 1.2 , 2.16)0.0549glucose ( mmol / l)5.55 ( 5.52 , 5.91)5.68 ( 5.4 , 6)<0.0001pre - existing chd , % ( n)10.7 ( 375)15.3 ( 515)<0.0001daily and weekend drinking , % ( n)63.6 ( 2192)38.89 ( 1216)<0.0001data are mean values ( sd ) unless otherwise statedincident cases of diabetes : 165incident cases of diabetes : 128geometric mean ( interquartile range ) baseline characteristics in men and women with no prevalent diabetes data are mean values ( sd ) unless otherwise stated incident cases of diabetes : 165 incident cases of diabetes : 128 geometric mean ( interquartile range ) the relationships between wc , whr and bmi and risk of type 2 diabetes in men and women are shown in table 2 .
all indicators were strongly and significantly associated with risk of type 2 diabetes in both men and women after adjustment for potential confounders including age , social class , alcohol intake , smoking status , physical activity , parental history of diabetes and pre - existing chd .
further adjustment for systolic blood pressure , hdl - cholesterol and log plasma glucose reduced the associations , but they remained significant . in men bmi and wc showed similar associations ; whr was the weakest predictor .
table 2age - adjusted and multivariate - adjusted hazard ratios and 95% ci for type 2 diabetes according to quartiles of anthropometric indicators in menvariablecasesrate/1,000 person - yearsage - adjustedadjustedadjustedadjustedadjustedall men1657.15quartiles bmi ( kg / m ) < 24.38111.941.001.001.001.001.00 24.38223.761.94 ( 0.944.00)1.85 ( 0.893.81)1.72 ( 0.833.57)1.40 ( 0.672.92)1.43 ( 0.682.99 ) 26.42386.403.29 ( 1.686.43)3.04 ( 1.555.96)2.21 ( 1.114.39)1.88 ( 0.923.83)1.57 ( 0.773.23 ) 28.719416.828.90 ( 4.7616.6)7.35 ( 3.9013.84)4.71 ( 2.459.03)3.36 ( 1.567.21)2.48 ( 1.145.42 ) p trend quartiles<0.001<0.001<0.001<0.0010.014 wc ( cm ) < 90.1132.231.001.001.001.001.00 90.1274.602.08 ( 1.074.02)1.95 ( 1.003.78)1.52 ( 0.782.97)1.48 ( 0.762.89)1.27 ( 0.652.48 ) 96.5406.863.1 ( 1.665.8)2.84 ( 1.525.33)1.93 ( 1.023.65)1.69 ( 0.893.24)1.31 ( 0.682.54 ) 102.58515.357.06 ( 3.9412.65)5.89 ( 3.2610.65)3.53 ( 1.926.48)2.17 ( 1.084.37)1.59 ( 0.773.26 ) p trend quartiles<0.001<0.001<0.0010.0260.214 whr < 0.907162.71.001.001.001.001.00 0.907356.002.25 ( 1.254.07)2.03 ( 1.123.69)1.64 ( 0.92.97)1.53 ( 0.842.79)1.26 ( 0.682.3 ) 0.95427.232.74 ( 1.544.88)2.41 ( 1.354.32)1.75 ( 0.983.15)1.46 ( 0.811.27)1.20 ( 0.662.19 ) 0.987213.135.10 ( 2.978.78)4.38 ( 2.537.59)2.76 ( 1.584.82)2.25 ( 1.273.98)1.65 ( 0.922.95 ) p trend quartiles<0.001<0.001<0.0010.030.069adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes and pre - existing chdadjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c and log blood glucoseadjusted for age and bmi / wcadjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c , log blood glucose and bmi / wc age - adjusted and multivariate - adjusted hazard ratios and 95% ci for type 2 diabetes according to quartiles of anthropometric indicators in men adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes and pre - existing chd adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c and log blood glucose adjusted for age and bmi / wc adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c , log blood glucose and bmi / wc the positive association between bmi and the risk of type 2 diabetes remained strong even after adjustment for whr in both men and women ( p for trend < 0.0001 for both sexes ; tables 2 , 3 ) . in men
bmi was a significant predictor of diabetes after adjustment for age and wc and remained significant even after additional adjustment for other potential confounders and possible mediators ( p = 0.01 ) .
however , in women bmi was not a predictor of diabetes when adjustments were made for age and wc alone .
by contrast wc remained significantly associated with diabetes in women in the fully adjusted model that included adjustment for bmi . in men
adjustment for bmi attenuated the association between wc and diabetes in men and this association was not significant after adjustment for potential confounders and mediators .
adjustment for bmi attenuated the association between whr and diabetes in men but made little difference in women .
table 3age - adjusted and multivariate - adjusted hazard ratio and 95% ci for type 2 diabetes according to quartiles of anthropometric indicators in womenvariablecasesrate/1,000 person - yearsage - adjustedadjustedadjustedadjustedadjustedall women1285.40quartiles bmi ( kg / m ) < 24.07122.031.001.001.001.001.00 24.07122.010.98 ( 0.442.17)0.99 ( 0.442.20)0.86 ( 0.381.92)0.64 ( 0.291.44)0.64 ( 0.281.44 ) 26.65264.352.14 ( 1.084.23)2.01 ( 1.014.00)1.64 ( 0.823.28)0.94 ( 0.461.94)0.92 ( 0.441.91 ) 29.787813.36.46 ( 3.5211.88)5.80 ( 3.1110.81)4.10 ( 2.167.79)1.34 ( 0.612.92)1.27 ( 0.572.82 ) p trend quartiles<0.001<0.001<0.0010.1620.231 wc ( cm ) < 77.150.841.001.001.001.001.00 77.1132.162.60 ( 0.937.28)2.54 ( 0.907.12)2.30 ( 0.816.46)2.40 ( 0.856.76)2.22 ( 0.796.29 ) 84.05233.864.68 ( 1.7812.3)4.34 ( 1.6411.44)3.83 ( 1.4410.16)4.00 ( 1.4910.77)3.60 ( 1.339.73 ) 92.38715.1218.34 ( 7.4445.16)16.64 ( 6.7041.35)12.18 ( 4.8330.74)13.68 ( 5.1136.66)10.82 ( 3.9829.37 ) p trend quartiles<0.001<0.001<0.001<0.001<0.001 whr < 0.77101.641.001.001.001.001.00 0.77121.991.25 ( 0.542.9)1.24 ( 0.542.88)1.08 ( 0.462.54)1.05 ( 0.452.43)0.95 ( 0.42.24 ) 0.81335.613.61 ( 1.787.32)3.29 ( 1.626.69)2.85 ( 1.45.82)2.56 ( 1.255.23)2.3 ( 1.124.74 ) 0.867312.838.39 ( 4.3316.27)7.36 ( 3.7814.36)5.61 ( 2.8411.09)5.38 ( 2.7510.52)4.25 ( 2.138.47 ) p trend quartiles<0.001<0.001<0.001<0.001<0.001adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes and pre - existing chdadjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c and log blood glucoseadjusted for age and bmi / wcadjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c , log blood glucose and bmi / wc age - adjusted and multivariate - adjusted hazard ratio and 95% ci for type 2 diabetes according to quartiles of anthropometric indicators in women adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes and pre - existing chd adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c and log blood glucose adjusted for age and bmi / wc adjusted for age , smoking , physical activity , alcohol intake , parental history of diabetes , pre - existing chd , systolic blood pressure , hdl - c , log blood glucose and bmi / wc there was no significant interaction between age or parental history and adiposity measures with risk of diabetes and no significant interaction was seen between wc and bmi and diabetes in men or women ( p = 0.49 and p = 0.77 for men and women , respectively ) .
unadjusted roc analyses indicate that bmi and wc were similar in predicting type 2 diabetes in men ; whr was the weakest ( fig . 1 ) .
in men , the auc was 0.726 for bmi , 0.713 for wc ( p = 0.20 for differences in auc for wc vs bmi ) and auc = 0.656 for whr .
addition of wc to bmi did not improve prediction in men ( difference in auc = 0.001 p = 0.73 ) .
when stratified by bmi levels ( < 25 kg / m , 2529.9 kg / m and 30 + kg / m ) , wc did not improve prediction of diabetes in any of the bmi groups . in women ,
wc was the best predictor of type 2 diabetes ( auc = 0.780 ) and was significantly better than bmi ( auc = 0.733 , p = 0.002 ) and whr ( auc = 0.728 , p = 0.005 ) .
wc improved prediction within all bmi groups , particularly those who were not obese ( bmi < 30 kg / m ) .
the addition of bmi or whr to wc did not improve prediction in women ( difference in auc = 0.0007 , p = 0.73 for adding bmi ; difference in auc = 0.008 , p = 0.63 for adding whr ) .
1the roc curves ( unadjusted ) for bmi , wc and whr for ( a ) men and ( b ) women .
roc area in women : bmi 0.733 ; whr 0.728 ; wc 0.780 the roc curves ( unadjusted ) for bmi , wc and whr for ( a ) men and ( b ) women .
roc area in women : bmi 0.733 ; whr 0.728 ; wc 0.780 we also assessed the improvement of prediction of the anthropometric measures when age and parental history were included in the model . in both men and women bmi and wc
this increased significantly to 0.73 with the addition of bmi and 0.71 with wc ( for all differences in auc p < 0.0001 ) .
for women the auc for age and parental history was 0.60 and this improved significantly with the addition of bmi ( auc = 0.75 ) or wc ( auc = 0.79 ; for all differences in auc p < 0.0001 ) .
the addition of wc to the model with age , parental history and bmi improved the prediction in women ( auc = 0.79 , p = 0.01 ) .
we also assessed the optimal cut - off points for bmi and wc for identifying diabetes in men and women .
table 4 shows the sensitivity , specificity and positive predictive value for the various cut - off points for bmi and wc in men and women . for women ,
92 cm appeared to be the optimal wc cut - off point in terms of the sum of the sensitivity and specificity ; for men , the optimal cut - off point for wc was 100 cm . for bmi
the optimal cut - off point for men was between 28 and 29 kg / m and for women the optimal cut - off point was between 29 and 30 kg / m .
table 4sensitivity , specificity and positive predictive value ( ppv ) and percentage of cases identified using sex - specific cut - off points for bmi and wc to predict diabetes over 7 yearsvariablemen ( n = 3,519)women ( n = 3,404)ncases%sensitivityspecificityppv ( % ) ncases%sensitivityspecificityppv ( % ) bmi ( kg / m)252,38214767.789.133.46.2227011466.789.134.25.0261,96813955.984.245.57.1191610756.383.644.85.6271,50612342.874.558.88.215839946.177.354.76.3281,1079931.560.069.98.912909237.971.963.47.1297998922.753.978.811.110248430.165.671.38.2305197114.743.086.613.78127823.960.977.69.6waist ( cm)802,18912064.393.836.85.5812,07111860.892.240.45.7821,94911657.390.644.06.0831,83311253.987.547.56.1841,71211050.385.951.16.4851,59710946.985.254.66.8861,49410443.981.357.67.0871,38910140.878.960.77.3881,2839837.776.663.87.6891,1749634.575.067.18.2901,0719331.572.770.18.7919759228.671.973.09.4928908926.169.575.510.0937998523.566.478.210.6942,11013860.083.641.26.57237621.259.480.310.5951,96913355.980.645.36.86607419.457.882.111.2961,82612851.977.649.47.0971,67612347.674.553.67.3981,49911942.672.158.87.9991,33511237.967.963.58.41001,20210634.264.267.38.81011,0719930.460.071.09.21029319126.555.275.09.8 sensitivity , specificity and positive predictive value ( ppv ) and percentage of cases identified using sex - specific cut - off points for bmi and wc to predict diabetes over 7 years
in this study of older men and women , aged 6079 years , both general adiposity ( bmi ) and the common measures of regional adiposity ( wc and whr ) strongly and independently predicted incident diabetes in both men and women after adjusting for potential confounders and risk factors , including plasma lipids , plasma glucose and blood pressure .
our study confirms previous studies showing that both overall and abdominal adiposity play an important role in diabetes [ 414 ] and extends these findings to older adults .
there has been much discussion of which anthropometric measure best predicts diabetes and the usefulness of wc or whr over bmi .
our study also provides new information regarding the possible optimal range of clinical utility for wc in predicting future diabetes in older adults . by analysis of roc curves
we have shown that in men , wc and bmi showed similar associations with incident diabetes ; whr was the weakest predictor . however , in women the wc was a significantly better indicator of diabetes risk than bmi or whr , and enhanced prediction of diabetes beyond bmi .
previous prospective studies comparing wc , bmi and whr for predicting diabetes risk have been inconsistent [ 413 ] . indeed , in a recent meta - analysis of 32 studies worldwide it was concluded that these three obesity indicators have similar associations with incident diabetes .
most of the studies included were carried out in populations with wide age - ranges and analyses but , critically , the analyses were not stratified by sex or age group .
our finding that wc is a better predictor of diabetes than bmi or whr in older women but not in older men is consistent with and extends data from several other prospective studies .
the iowa women s health study of postmenopausal women ( aged 5569 years ) showed those in the top quintile of the wc distribution to be associated with the largest relative risk compared with the top quintile of bmi and whr . in the monica / kora augsburg cohort study , wc emerged as the stronger predictor of type 2 diabetes in women but in men bmi appeared to be the stronger predictor , as was seen in our study . in the nurses health study of women aged 3055 years , wc was the strongest predictor of diabetes when compared with bmi and whr . in the health professionals follow - up study of men aged 4075 years , bmi and wc showed similar associations ; whr was the weakest predictor . in the epic - potsdam study of men and women aged 3565 years
wc was a better predictor of incident diabetes than bmi among women but not among men . in the atherosclerosis risk in communities ( aric ) study of adults aged 4064 years
, bmi and wc showed similar associations in men , with whr being the weakest predictor , in close agreement with our findings .
however , in female aric participants all anthropometric measures showed similar associations with diabetes risk , in contrast to our findings . taken together , the results of these studies and our data suggest that the measure of obesity that best predicts diabetes may be dependent on age and sex , and that waist circumference is a better predictor of diabetes in women . the measurement of wc is recommended in the us national institutes of health clinical guidelines for the assessment or management of obesity .
however , its utility in clinical practice has been questioned because of the training required to ensure that reliable wc data are collected ; other weaknesses , including lack of consensus regarding the best thresholds for defining obesity in different populations , have been discussed .
of the few prospective studies which have assessed whether the combined use of general obesity with measures of body fat distribution can improve prediction of diabetes , some have suggested that combined use does not provide additional information beyond any single measure [ 9 , 13 ] , as was observed in this study . in older men , there is no evidence that measurement of wc provides additional clinical information beyond that provided by bmi , and vice versa in women .
the finding that wc is a far better predictor of diabetes than bmi in older women lends support to the use of wc in older women ( and perhaps in women in general ) to identify those at high risk of developing diabetes .
further study would be required , however , before this could be done reliably in clinical practice .
the cut - off point for wc recommended for use in clinical practice remains controversial .
the atp iii criteria define abdominally obese as wc > 88 cm for women and > 102 cm for men .
however , in the present study , roc curve analysis showed that wc thresholds of about 92 and 100 cm in women and men , respectively , provided optimal discrimination for diabetes , at least as defined in terms of the sum of sensitivity and specificity .
however , for women sensitivity would be substantially lowered if this method for defining optimal threshold was applied and a higher threshold of 92 cm was used .
the threshold of 88 cm yielded 76% sensitivity ( specificity 63% ) whilst a threshold of 92 cm would yield a sensitivity of under 70% but a specificity of 75% .
by contrast in men , the results suggest that for diabetes prediction the current recommended wc guidelines of 102 cm could be lowered to 100 cm for identifying diabetes risk , supporting the findings of wang et al . .
this method of calculating optimal cut - points gives equal weighting to the notion of sensitivity and specificity and may not be ideal for the assessment of screening thresholds for diabetes in clinical practice .
it is likely that optimal thresholds will depend on weighing the issues of cost as well as maximising the identification of those at high risk .
we appreciate the need for replication of our findings in independent studies before changes to thresholds are made .
it was carried out in an older , predominantly white , european population ; we can not generalise our findings to younger populations or to other ethnic groups , in which the relations between anthropometric measurements and diabetes risk are clearly different .
the determination of diabetes incidence in this study relied on documented doctor - diagnosed cases of diabetes , which inevitably results in under ascertainment of cases .
this may also influence the performance of anthropometric measurements as bmi is the more routine marker measured in clinical practice .
however , if anything , wc emerges as the far stronger predictor in women and this argument would bias against wc being a better predictor . conversely it might be argued that since markers of central obesity ( wc or whr ) have been shown to be more strongly associated with chd risk than bmi in women in several studies [ 7 , 26 ] , women with higher wc are more likely to have health problems and more contact with their doctors and thus are more likely to be diagnosed . to address this possible bias we examined cross - sectionally the relationship of wc , bmi and hyperglycaemia ( fasting glucose concentration
wc was a significantly stronger predictor of hyperglycaemia than bmi in women , but in men wc and bmi showed similar predictive power .
these findings suggest ascertainment bias is unlikely to explain wc being a better predictor of clinically diagnosed diabetes in women .
the stronger association between wc and fasting blood glucose ( a strong determinant of diabetes ) compared with bmi in women may contribute to explaining why wc is a better predictor of diabetes than bmi .
however , wc remained a significantly better predictor than bmi after adjustment for fasting blood glucose .
finally , our study can not determine why wc should be a better predictor of diabetes in elderly women compared with elderly men , but suggests this as an area for future investigation .
both overall and central adiposity indices are strong predictors of type 2 diabetes in older adults .
bmi is as strong as wc in predicting type 2 diabetes in men . in women , however , wc was a significantly better measure for the identification of diabetes risk
. the use of bmi combined with wc does not improve identification of type 2 diabetes risk beyond single measures of bmi ( in men ) or wc ( in women ) .
current recommended guidelines for wc in men of 102 cm could be lowered to identify diabetes risk , whereas those for older women could be increased .
finally , future simple diabetes - prediction algorithms could usefully include wc in those applicable to women , though for changes to clinical practice one would need to ensure that better prediction in research settings is not negated by poorer reliability of the measurement in routine practice . | aims / hypothesisthe aim of this study was to examine whether waist circumference ( wc ) or whr improve diabetes prediction beyond body mass index in older men and women , and to define optimal cut - off points.methodsin this prospective study , non - diabetic men ( n = 3,519 ) and women ( n = 3,404 ) aged 6079 years were followed up for 7 years .
there were 169 and 128 incident cases of type 2 diabetes in men and women , respectively.resultsbmi , wc and whr all showed strong associations with incident type 2 diabetes independent of potential confounders . in men ,
the adjusted relative risks ( top vs lowest quartile ) were 4.71 ( 95% ci 2.459.03 ) for bmi , 3.53 ( 95% ci 1.926.48 ) for wc and 2.76 ( 95% ci 1.584.82 ) for whr . for women , the corresponding relative risks were 4.10 ( 95% ci 2.167.79 ) , 12.18 ( 95% ci 4.8330.74 ) and 5.61 ( 95% ci 2.8411.09 ) for bmi , wc and whr , respectively .
receiver - operating characteristic curve analysis revealed similar associations for bmi and wc in predicting diabetes in men ( auc = 0.726 and 0.713 , respectively ) ; whr was the weakest predictor ( auc = 0.656 ) . in women ,
wc was a significantly stronger predictor ( auc = 0.780 ) than either bmi ( auc = 0.733 ) or whr ( auc = 0.728 ; p < 0.01 for both )
. inclusion of both wc and bmi did not improve prediction beyond bmi alone in men or wc alone in women .
optimal sensitivity and specificity for the prediction of type 2 diabetes was observed at a wc of 100 cm in men and 92 cm in women.conclusions/interpretationin older men , bmi and wc yielded similar prediction of risk of type 2 diabetes , whereas wc was clearly a superior predictor in older women . |
the current application of physiologically based pharmacokinetic ( pbpk ) modeling in drug development was summarized by three industry representatives ( links to all presentations are provided in supplementary table 1 and a comprehensive meeting report is available online1 ) .
jan snoeys ( janssen ) provided an overview of pbpk in drug development and two additional presenters gave specific examples of the use of pbpk in industry . in the first presentation two approaches to pbpk modeling were described : models using only in vitro / in silico data ( bottom up ) and those combining in vitro / in silico data with observed pk data .
the importance of validated in vitro assays was stressed , ideally with adoption of common assay methodology across different laboratories .
also highlighted was that the process of model optimization using sensitivity analysis and parameter optimization is integral to development of robust pbpk models and must be conducted recognizing information gaps and consequences on model utilization .
often optimization of several independent factors may lead to improved simulated pk profiles . in such circumstances
it is of value to test such hypotheses with experimental data . in system model verification ,
drug - independent system components and virtual populations may be verified with compounds representing a range of physicochemical properties .
differences in ugt - mediated clearance and ugt2b15 polymorphism between japanese and caucasian subjects were considered in the model of an investigational drug .
in the second example , a pbpk model included drug disposition mediated by organic anion - transporting polypeptides ( oatp ) .
literature data of seven compounds and a scaling method were employed to develop the model .
the model was valuable in prospectively predicting human pk for four investigational drugs tested to be oatp substrates in vitro .
patrice larger ( novartis ) described pk predictions using two pbpk pregnancy models : one with limited pregnancy factors and a second expanded with additional factors such as fetal
specific pregnancy factors in the second model did not significantly impact the predicted pharmacokinetic profiles for the reference compounds .
risk evaluations , providing a mechanistic basis for extrapolation beyond the clinical trial population , reducing uncertainty , and enabling better labeling around drug drug interactions ( ddis ) and in special populations ( e.g. , elderly , pediatric , etc ) .
pbpk is increasingly submitted as part of marketing authorization applications ( maas ) . to date
these have comprised mainly ddi applications and have been included in a number of european summaries of product characteristics ( smpc ) and/or european public assessment reports ( e.g. , halavan,2 jakavi,3 and olysio4 ) .
so far , few submissions with application to pediatric dose selection have been received . pbpk - thinking in drug development
is encouraged , as it leads to a mechanistic understanding of the processes involved in drug disposition .
the modeling approach helps to identify gaps in understanding of absorption , distribution , metabolism , and excretion ( adme ) , informs improved study designs , complements other modeling and simulation ( m&s ) approaches and builds confidence for extrapolation .
when systematically applied over many drugs , this will support continued development and validation of system models .
the continued evolution of pbpk is key to facilitating greater confidence in extrapolation ( e.g. , pediatric , elderly , ddi , renal impairment , etc . ) , thereby reducing experimental data requirements .
this meeting was viewed as the beginning of an important dialog towards defining the standards to facilitate a greater role of pbpk in european regulatory decision making .
there is now sufficient experience within the european regulatory system to support development of regulatory standards , guidelines , and practice .
a pbpk concept paper has been published5 which will lead to a specific european guideline on qualification and reporting of pbpk modeling and analysis .
the guideline will be informed by comments received during the consultation period and discussions reported here and the earlier us food and drug administration ( fda ) workshop.6
the steps involved in pbpk model development relevant for regulatory applications within clinical pharmacology are illustrated in figure
1a , which formed the basis for selection of the four discussion topics :
drug - specific input parametersclinical data for verification of drug - specific input parametersqualification of system modelsreporting drug - specific input parameters clinical data for verification of drug - specific input parameters qualification of system models ( a ) schematic illustrating overlay of discussion topics implicit in best practice during pbpk model development and reporting .
key points and recommendations from discussion sessions a consensus should be developed on the important input parameters for specific applications , e.g. , a list of important input parameters for each category ( ddi , specific populations , such as pediatrics , biopharmaceutics ) .
more consideration is required on the incorporation of uncertainty in input parameters in models ; consideration of covariance of parameters is also important .
agreement and adoption of common reference standards to be utilized across companies should be encouraged .
if companies develop their own scaling factors then they must fully and transparently justify these .
all companies should be encouraged to present quantitative drug disposition diagrams as part of their clinical pharmacology documentation .
a statement should be developed , supported by appropriate rationale that explains the expectation of iv data as a key element in the quantitative mechanistic understanding of drug disposition .
general guidance should be developed around the choice of parameters and range of values included in sensitivity analysis based on the physico chemical properties of a molecule , and the experimental system .
companies should systematically document the relationship between in vitro ki , ki , and in vivo ddi results to inform the range for sensitivity analysis for perpetrators .
companies should consider whether it is better to resolve uncertainty experimentally where this is possible , rather than addressing this issue solely through sensitivity analysis .
a follow - up meeting should be convened to address pbpk system model validation and explore solutions ( such as open source validation sets ) , while considering the needs of software companies .
a working group should be established to compare the use of system model evaluation and assessment terminology in other related fields such as statistics , mathematics , and modeling and simulation with the aim of reaching agreement on the terminology and definitions for pbpk .
a fit for purpose model development story should be included in the pbpk report . a clear statement of the assumptions underlying the modeling , the input parameters and the relationship of the parameters and the appropriateness of these assumptions , as well as the impact on the predictions ,
should also be included . a clear account of the purpose of the modeling effort should be included in the report .
relevant targets for successful prediction vs. actual clinical data should be set with reference to the therapeutic index of the drug .
the objective of session 1 was to help establish best practice for defining drug - specific input parameters .
key parameters were identified prior to the meeting ( see link in supplementary table 1 ) .
questions discussed were : ( 1 ) model improvement ( optimization of parameters and use of scaling factors ) , ( 2 ) sensitivity testing of parameters , and ( 3 ) methodology to define inputs .
it was agreed that optimization is routinely performed but should be well justified and that scaling factors vary in plausibility ; some being physiologically based but others more empirical , and may vary in size between analyses .
sensitivity testing of model predictions to changes in model parameters was considered to be essential , with the suggestion that the importance of a parameter and the range of sensitivity analysis performed be defined depending on the physiochemical properties of the drug and/or the experimental systems under consideration .
it was noted that in vitro methodology to determine input values often varies across and sometimes within companies .
important points identified for follow up were the need for a full understanding and description of methodology to measure input parameters , preferably with adoption of common reference standards across companies , and a need for greater clarification and justification of empirical scaling factors . in session 2 ,
verification of pbpk models was discussed and best practice in the use of adme data ( in vitro and in vivo ) in the verification of drug - specific input parameters was explored .
an example of a useful quantitative mass balance diagram for a compound administered both orally and intravenously is illustrated in figure 1b .
based on the feedback , the following points were identified as important to support a quantitative mass balance diagram : ( 1 ) what are the clearance pathways and their quantitative contributions ? ( 2 ) what is the extent of absorption of the drug and is parent drug in feces a result of lack of absorption ( fa ) or biliary / intestinal excretion ? ( 3 ) when oral bioavailability is low , what are the contributions of intestinal and hepatic first pass loss ? ( 4 ) what is the rate - limiting step in hepatic drug clearance ( basolateral uptake or metabolism / biliary secretion ) ? in general , attendees agreed that intravenous data can be important in pbpk model development ( depending on the pathways ) . sensitivity analysis for inhibition constant ( ki ) ,
fraction unbound in plasma , microsomes and gut ( fup , fumic , fugut , depending on the gastrointestinal model utilized ) , and permeability is considered crucial when simulating the impact of a possible cyp3a perpetrator on the pk of a victim drug .
further simulations for sensitivity evaluation should address confidence in liver or intrahepatic concentration and intraenterocyte concentration when inhibition occurs intracellularly . pbpk modeling of enzyme induction was also discussed : it was highlighted that currently there is insufficient confidence within the european regulatory system to support waiver of studies based on pbpk .
an important point identified for follow up was development of a statement , with a supporting rationale , which explains the expectation of intravenous data ( unless its absence can be justified ) as a key element in the quantitative mechanistic understanding of drug disposition . the system qualification discussion ( session 3 ) focused on commercially available software , which is utilized in the vast majority of regulatory applications of pbpk .
the three main companies providing pbpk software used in clinical pharmacology regulatory submissions to date ( gastroplus , pk - sim , and simcyp ) provided short summaries of their approach to system model qualification .
the system validation consists of tracking and documenting ( user manual , publication ) changes and ( re)validating the software using former or new test cases .
the libraries ' qualification consists of continuously updating the databases with emerging knowledge from the literature or provided by ( pharmaceutical ) companies .
additional elements presented by the companies included : version stamp for model output , training datasets with tutorials available to users , validation datasets that remain with the software to allow verification of continued system model reliability , and transparency regarding source of data and assumptions within the system .
overall it was concluded that each software provider has developed internal systems to evaluate and track the reliability of their system models and associated libraries .
further discussion focused on the framework proposed for assessment of systems pharmacology models7 and referred to the us national academies framework on validation , verification , and uncertainty quantitation.8 in summary , the framework recommends that the software should be open access , bug - free , with no copying errors .
in addition , two types of data should be clearly differentiated : training data published with the model and novel and varied validation data .
it was acknowledged that in the field of pbpk , open source software published with training datasets presents a challenge for software providers wishing to protect their intellectual property .
other solutions , such as open source validation datasets ( for a specific condition of use ) , could potentially serve the same purpose .
importantly , there is some mismatch between the terminology used within pbpk and computational science communities : qualification or verification vs.
also acknowledged was that system model qualification is an area that has not been extensively discussed within the pbpk community and that standards are not currently agreed , and that progress to establish best practice is needed .
session 4 discussed best practice in pbpk reporting focusing on what information is needed in reports for regulatory review .
it was considered that reports should clearly address the regulatory question , the clinical context , and whether the simulations will lead to changes in dose recommendations .
there was general agreement that including background information ( clinical pharmacology , purpose of the modeling effort , history of pbpk model development , etc . )
it was also pointed out that background information on clinical pharmacology is of limited value without integration across studies .
uncertainty , sensitivity analysis , and plausibility of the assumptions should always be presented and discussed .
reports of simulations used in support of a waiver of an in vivo study ( such as a ddi study ) require particular care to integrate the simulations with the rest of the clinical data , including a discussion of the implications for drug dosing , taking into account the pkpd profile of the drug .
additional supporting information may be found in the online version of this article . supporting information | under the remit of the ministerial industry strategy group ( misg ) , the association of the british pharmaceutical industry ( abpi ) and medicines and healthcare products regulatory agency ( mhra ) hosted a meeting to explore physiologically based pharmacokinetic modeling and simulation , focusing on the clinical component of regulatory applications .
the meeting took place on 30 june 2014 with international representatives from industry , academia , and regulatory agencies .
discussion topics were selected to be complementary to those discussed at an earlier us food and drug administration ( fda ) meeting .
this report summarizes the meeting outcomes , focusing on the european regulatory perspective . |
to extract sij from the experimentally accessible gij , we formally link ( 1 ) and ( 2 ) via ( 3){dxidxi=1dxidxj=k=1nxixkdxkdxjij}.
equation ( 3 ) is exact and the sum accounts for all network paths connecting i and j ( supplementary note s.i.1 - 2 ) .
it is of limited use , however , as it requires us to solve n coupled algebraic equations . in supplementary note s.i.1 we show that ( 3 ) can be reformulated as ( 4)s=(gi+d(sg))g1 , where i is the identity matrix and d(m ) sets the off - diagonal terms of m to zero . to obtain an approximate solution for s we use that fact that typically , perturbations decay rapidly as they propagate through the network , so that the response observed between two nodes is dominated by the shortest path between them .
this allows us to approximate d(sg ) with d((gi)g ) ( supplementary note s.i.3 ) , obtaining ( 5)s=(gi+d((gi)g))g1 .
equation ( 5 ) , our main result , provides sij from the experimentally accessible gij .
it achieves this through a ' silencing effect , in which direct response terms are preserved , while indirect responses are silenced . to understand this
consider a specific term in gij , documenting the response of node i to j 's perturbation .
( 3 ) , this response is a consequence of all direct and indirect paths leading from j to i. as we document below , the transformation ( 5 ) detects the indirect paths and silences them , maintaining only the contribution of the direct paths ( fig .
1d - f ) . to demonstrate the predictive power of ( 5 ) , we implemented michaelis - menten dynamics on a model network ( supplementary note s.iii ) , as commonly used to model generegulation .
we obtained gij by perturbing the activity of each node and then calculated sij using ( 5 ) .
figure 2a shows the gij and sij terms associated with interacting ( green ) and non - interacting ( orange ) node pairs .
although gij is higher for direct interactions , the overlap between the orange and the green symbols indicates a lack of a clear threshold q that separates direct and indirect interactions .
in contrast , sij displays a clear separation between direct and indirect interactions , accurately predicting each direct link .
2b , red ) has an area of auroc = 0.91 , reflecting inherent limitations in separating direct from indirect interactions based on gij only .
in contrast for sij we obtain auroc = 0.997 ( blue ) , where the true positive rate ( tpr ) reaches 100% with a false positive rate ( fpr ) of less than 10 . also , although for gij precision increases gradually with the threshold q ( fig .
hence , in our well controlled model system effectively any non - zero sij corresponds to a direct link .
the performance of ( 5 ) is due to the silencing effect : it leaves gij unchanged if i and j are linked , while it systematically lowers all gij not rooted in a direct interaction . to quantify this effect we measured the discrimination ratio g = gijdir/gijindir ( s = sijdir/sijindir ) which captures the ratio between gij ( sij ) terms associated with direct links and those associated with indirect links ( fig .
we find that sij is much more discriminative than gij owing to its silencing of indirect responses . to quantify this effect we measure the silencing ( 6)=sg , which captures the increased power of sij to discriminate between direct and indirect links compared to gij . in our model system we find that = 15 , a silencing of more than an order of magnitude ( fig .
furthermore , the longer is the distance dij between two nodes , the larger is the silencing ( fig .
as an illustration , consider a linear cascade in which changes in any node result in a finite response gij by all other nodes ( fig .
equation ( 5 ) silences all indirect responses , while leaving the response of direct links effectively unchanged , offering a discriminative measure that enables a perfect reconstruction of the original network . to test the predictive power of ( 5 ) on real data we used the e. coli datasets distributed by the dream5 network inference challenge .
the input data include a compendium of microarray experiments measuring the expression levels of 4,511 e. coli genes ( 141 of which are known transcription factors ) under 805 different experimental conditions ( supplementary note s.iv.1 ) .
we constructed three separate global response matrices gij between the 141 transcription factors and their 4,511 potential target genes , based on ( i ) pearson correlations ; ( ii ) spearman rank correlations ; and ( iii ) mutual information , which are three commonly used methods for link detection ( supplementary note s.iv.3 ) . from each of the three gij matrices we obtained sij via ( 5 ) , and compared the performance of gij with the pertinent sij . to validate our predictions we relied on the gold standard used in the dream5 challenge , consisting of 2,066 established gene regulatory interactions .
measuring auroc from gij and sij , we find an improvement of 56% for pearson correlations ( fig .
we further tested the discrimination ratio , , and the silencing , , for each of these methods , finding that indirect correlations are subject to an average of two - fold silencing in the transition from gij to sij ( fig .
3d ) . silencing is especially crucial in the presence of the cascade and co - regulation motifs shown in figures 3e - f , where most inference methods indicate a spurious link between x and y owing to the indirect correlation mediated by node i. indeed , the transformation ( 5 ) silences these indirect correlations by a factor of three or more for pearson and spearman correlations and by a smaller factor for mutual information , overcoming one of the most common hurdles of inference methods , which tend to over - represent triadic motifs .
as all experimental data is subject to noise , the global response matrix , gij , is characterized by some degree of uncertainty . to test the performance of our methodology in the presence of noise
, we repeated the numerical experiment of figure 2 , this time adding gaussian noise to gij , which allows us to calculate silencing as a function of increasing the signal to noise ratio ( fig .
4 ) . as expected , silencing is unaffected by small values of , so that features a plateau below 0.1 . for large , silencing decays as ~ , demonstrating that the performance of the method decreases slowly with increasing the signal to noise ratio . indeed ,
as opposed to a rapid exponential decay , the observed slower power - law dependence indicates that the method is rather tolerant against noise .
silencing is lost only when the noise reaches the critical level c 0.75 , when the signal is almost completely overridden by noise , leading to = 1 ( supplementary note s.v.1 ) .
they represent the fact that in most cases we are unable to read the states of all nodes in the system . to illustrate the effect of the hidden nodes on the performance of the silencing method
, we consider the case of a simple cascade i k j , where the intermediate node k is hidden . in this scenario ,
( 5 ) will not be able to silence the indirect i j link because in the observable system the gij term can not be attributed to any indirect path .
hence , absent any other information about the system , it is mathematically impossible to infer the indirectness of gij , as the removal of k isolated i from j. this touches upon the fundamental mechanism of silencing : as illustrated in figure 1 ( and supplementary note s.i.2 ) the silencing transformation ( 5 ) exploits the flow of information through indirect paths . consequently , if as a result of hidden nodes the network fragments into several components such that the node pair i and j become isolated from each other , then all indirect paths between them became hidden and the pertinent gij term will not be silenced ( fig .
is expected to fail only when the network breaks into many isolated components so that most node pairs become isolated .
fortunately , a fundamental property of complex networks is that with average degree k > > 1 , one needs to remove a large fraction of the nodes to fragment the underlying giant connected component .
therefore we can build on percolation theory , which allows us to analytically predict how the size of the largest connected component changes with the random removal of a certain fraction of nodes .
the calculation shows that silencing is maintained as long as the fraction of hidden nodes is smaller than ( 7)c1k , where =2ln(2 + 2)1.7 ( supplementary note s.v.2 ) .
this equation indicates that for large k the method will be reliable even if a large fraction of the nodes are hidden . to test this prediction
, we revisited the numerically obtained gij analyzed in figure 2 and measured the degree of silencing after randomly removing an increasing fraction of nodes . in each case
we find that , as predicted , the degree of silencing is driven mainly by , approaching 1 ( no silencing ) when 1 , namely when the isolated pairs begin to dominate the network ( fig .
( 7 ) predicts c 0.57 , i.e. the method will fail only when almost 60% of the nodes are hidden .
note that for biological networks k is expected to be in the range of k 10 , predicting c 0.8 .
namely , one needs to lose access to 80% of the nodes for silencing to lose its effectiveness .
to extract sij from the experimentally accessible gij , we formally link ( 1 ) and ( 2 ) via ( 3){dxidxi=1dxidxj=k=1nxixkdxkdxjij}.
equation ( 3 ) is exact and the sum accounts for all network paths connecting i and j ( supplementary note s.i.1 - 2 ) .
it is of limited use , however , as it requires us to solve n coupled algebraic equations . in supplementary note s.i.1 we show that ( 3 ) can be reformulated as ( 4)s=(gi+d(sg))g1 , where i is the identity matrix and d(m ) sets the off - diagonal terms of m to zero . to obtain an approximate solution for s we use that fact that typically , perturbations decay rapidly as they propagate through the network , so that the response observed between two nodes is dominated by the shortest path between them .
this allows us to approximate d(sg ) with d((gi)g ) ( supplementary note s.i.3 ) , obtaining ( 5)s=(gi+d((gi)g))g1 .
equation ( 5 ) , our main result , provides sij from the experimentally accessible gij .
it achieves this through a ' silencing effect , in which direct response terms are preserved , while indirect responses are silenced . to understand this
consider a specific term in gij , documenting the response of node i to j 's perturbation .
( 3 ) , this response is a consequence of all direct and indirect paths leading from j to i. as we document below , the transformation ( 5 ) detects the indirect paths and silences them , maintaining only the contribution of the direct paths ( fig .
to demonstrate the predictive power of ( 5 ) , we implemented michaelis - menten dynamics on a model network ( supplementary note s.iii ) , as commonly used to model generegulation .
we obtained gij by perturbing the activity of each node and then calculated sij using ( 5 ) .
figure 2a shows the gij and sij terms associated with interacting ( green ) and non - interacting ( orange ) node pairs .
although gij is higher for direct interactions , the overlap between the orange and the green symbols indicates a lack of a clear threshold q that separates direct and indirect interactions .
in contrast , sij displays a clear separation between direct and indirect interactions , accurately predicting each direct link .
2b , red ) has an area of auroc = 0.91 , reflecting inherent limitations in separating direct from indirect interactions based on gij only .
in contrast for sij we obtain auroc = 0.997 ( blue ) , where the true positive rate ( tpr ) reaches 100% with a false positive rate ( fpr ) of less than 10 . also , although for gij precision increases gradually with the threshold q ( fig .
hence , in our well controlled model system effectively any non - zero sij corresponds to a direct link .
the performance of ( 5 ) is due to the silencing effect : it leaves gij unchanged if i and j are linked , while it systematically lowers all gij not rooted in a direct interaction . to quantify this effect we measured the discrimination ratio g = gijdir/gijindir ( s = sijdir/sijindir ) which captures the ratio between gij ( sij ) terms associated with direct links and those associated with indirect links ( fig .
we find that sij is much more discriminative than gij owing to its silencing of indirect responses . to quantify this effect we measure the silencing ( 6)=sg , which captures the increased power of sij to discriminate between direct and indirect links compared to gij . in our model system we find that = 15 , a silencing of more than an order of magnitude ( fig .
furthermore , the longer is the distance dij between two nodes , the larger is the silencing ( fig .
consider a linear cascade in which changes in any node result in a finite response gij by all other nodes ( fig .
equation ( 5 ) silences all indirect responses , while leaving the response of direct links effectively unchanged , offering a discriminative measure that enables a perfect reconstruction of the original network .
to test the predictive power of ( 5 ) on real data we used the e. coli datasets distributed by the dream5 network inference challenge .
the input data include a compendium of microarray experiments measuring the expression levels of 4,511 e. coli genes ( 141 of which are known transcription factors ) under 805 different experimental conditions ( supplementary note s.iv.1 ) .
we constructed three separate global response matrices gij between the 141 transcription factors and their 4,511 potential target genes , based on ( i ) pearson correlations ; ( ii ) spearman rank correlations ; and ( iii ) mutual information , which are three commonly used methods for link detection ( supplementary note s.iv.3 ) . from each of the three gij matrices we obtained sij via ( 5 ) , and compared the performance of gij with the pertinent sij . to validate our predictions we relied on the gold standard used in the dream5 challenge , consisting of 2,066 established gene regulatory interactions .
measuring auroc from gij and sij , we find an improvement of 56% for pearson correlations ( fig .
we further tested the discrimination ratio , , and the silencing , , for each of these methods , finding that indirect correlations are subject to an average of two - fold silencing in the transition from gij to sij ( fig .
3d ) . silencing is especially crucial in the presence of the cascade and co - regulation motifs shown in figures 3e - f , where most inference methods indicate a spurious link between x and y owing to the indirect correlation mediated by node i. indeed , the transformation ( 5 ) silences these indirect correlations by a factor of three or more for pearson and spearman correlations and by a smaller factor for mutual information , overcoming one of the most common hurdles of inference methods , which tend to over - represent triadic motifs .
as all experimental data is subject to noise , the global response matrix , gij , is characterized by some degree of uncertainty . to test the performance of our methodology in the presence of noise
, we repeated the numerical experiment of figure 2 , this time adding gaussian noise to gij , which allows us to calculate silencing as a function of increasing the signal to noise ratio ( fig .
4 ) . as expected , silencing is unaffected by small values of , so that features a plateau below 0.1 . for large , silencing decays as ~ , demonstrating that the performance of the method decreases slowly with increasing the signal to noise ratio . indeed ,
as opposed to a rapid exponential decay , the observed slower power - law dependence indicates that the method is rather tolerant against noise .
silencing is lost only when the noise reaches the critical level c 0.75 , when the signal is almost completely overridden by noise , leading to = 1 ( supplementary note s.v.1 ) .
they represent the fact that in most cases we are unable to read the states of all nodes in the system .
to illustrate the effect of the hidden nodes on the performance of the silencing method , we consider the case of a simple cascade i k j , where the intermediate node k is hidden . in this scenario ,
( 5 ) will not be able to silence the indirect i j link because in the observable system the gij term can not be attributed to any indirect path .
hence , absent any other information about the system , it is mathematically impossible to infer the indirectness of gij , as the removal of k isolated i from j. this touches upon the fundamental mechanism of silencing : as illustrated in figure 1 ( and supplementary note s.i.2 ) the silencing transformation ( 5 ) exploits the flow of information through indirect paths .
consequently , if as a result of hidden nodes the network fragments into several components such that the node pair i and j become isolated from each other , then all indirect paths between them became hidden and the pertinent gij term will not be silenced ( fig .
is expected to fail only when the network breaks into many isolated components so that most node pairs become isolated .
> > 1 , one needs to remove a large fraction of the nodes to fragment the underlying giant connected component .
therefore we can build on percolation theory , which allows us to analytically predict how the size of the largest connected component changes with the random removal of a certain fraction of nodes .
the calculation shows that silencing is maintained as long as the fraction of hidden nodes is smaller than ( 7)c1k , where =2ln(2 + 2)1.7 ( supplementary note s.v.2 ) .
this equation indicates that for large k the method will be reliable even if a large fraction of the nodes are hidden . to test this prediction
, we revisited the numerically obtained gij analyzed in figure 2 and measured the degree of silencing after randomly removing an increasing fraction of nodes . in each case
we find that , as predicted , the degree of silencing is driven mainly by , approaching 1 ( no silencing ) when 1 , namely when the isolated pairs begin to dominate the network ( fig .
( 7 ) predicts c 0.57 , i.e. the method will fail only when almost 60% of the nodes are hidden .
note that for biological networks k is expected to be in the range of k 10 , predicting c 0.8 .
namely , one needs to lose access to 80% of the nodes for silencing to lose its effectiveness .
( 5 ) is scalable and requires no assumptions about the network topology . by silencing indirect effects , it turns the raw correlation data into a predictive sij matrix , dominated by direct interactions .
it is especially suited to treat perturbation data , such as genetic perturbation experiments , in which case gij describes the response of all genes ( dxi ) as a consequence of the perturbation of the source gene ( dxj ) . in practice , however , gij could be the result of a broader set of experimental realizations where other measures are used to evaluate the association between nodes , typically statistical measures such as pearson or spearman correlation coefficients . still , our empirical results ( fig .
3 ) clearly show that the transformation ( 5 ) successfully applies to these empirically accessible measures as well .
the method 's broad applicability is rooted in the fact that it does not depend on the value of each specific term in gij , but rather on the global relationships between them . indeed , the global structure of gij reflects the patterns of propagation of the perturbations along the network .
equation ( 5 ) helps uncover these paths from the raw data , disentangling the direct from the indirect effects .
these patterns of information flow are inherent to the underlying network structure , and should not depend on the specific experimental realization of ( 1 ) . for instance , a cascade i j k will be characterized by a decreasing correlation propagating along the arrows , a large correlation between i and j and a weaker one between i and k. although the magnitude of these correlations might depend on the size or the form of i 's perturbation as well as on the statistical measure we used to evaluate them , the decay pattern required to infer the structure of the cascade is an inherent property of the network flow and can be successfully detected by the silencing method ( supplementary note s.i.4 ) .
hence it is expected to apply under rather general conditions . however , as equation ( 5 ) indicates it requires that the input matrix , gij , is invertible .
for instance in the empirical results of figure 3a we constructed gij from pearson correlations , using the states of 4,511 nodes measured under 805 experimental conditions .
in general , if the number of experimental conditions is smaller than the number of nodes the resulting pearson correlation matrix may be singular . in this case
additional processing will be required before ( 5 ) could be applied . here , following the dream5 protocol , we only focused on the correlations between the 141 known transcription factors and the rest of the nodes , which lead to an invertible gij ( supplementary note s.iv ) . other means to ensure gij 's invertibility are discussed in supplementary note s.iv.4 . isolating indirect effects in correlation data , a fundamental challenge of network inference ,
in contrast , the success of the silencing method is rooted in its exploitation the global network topology : it relies on the fundamental principles of network structure and dynamics to identify and silence the effects of indirect paths .
the ability to extract sij from gij could also have implications for our understanding of network dynamics .
indeed , gij is a global network measure , as its magnitude is determined by the numerous indirect paths connecting i and j. hence , for a given dynamics , the gij matrix will take a different form depending on the network topology , making it a poor predictor of the system 's dynamics . by eliminating indirect effects , sij measures the effect gene i would have on gene j
it thus helps us quantify the dynamical mechanism that governs individual pairwise interactions , avoiding the convolution of dynamical and topological effects present in experimental data .
the structure of gij reflects the microscopic mechanisms that govern the pairwise interactions , e.g. genetic regulation and biochemical processes .
it is difficult , however to extract this information from gij since its terms are a convolution of many interactions , reflecting the many paths leading from i to j. the transition to sij , via ( 5 ) , allows us to treat each isolated interaction on its own , providing a direct observation into the microscopic interaction mechanism .
direct application of this fact could be the derivation of a rate equation that governs the system 's dynamics from gij , as well as predicting the universality class and the scaling laws governing the system 's response to perturbations . hence ( 5 ) helps translate the ever - growing amount of data on global correlations into valuable local information .
| predicting physical and functional links between cellular components is a fundamental challenge of biology and network science .
yet , correlations , a ubiquitous input for biological link prediction , are affected by both direct and indirect effects , confounding our ability to identify true pairwise interactions . here
we exploit the fundamental properties of dynamical correlations in networks to develop a method to silence indirect effects .
the method receives as input the observed correlations between node pairs and uses a matrix transformation to turn the correlation matrix into a highly discriminative silenced matrix , which enhances only the terms associated with direct causal links . achieving perfect accuracy in model systems , we test the method against empirical data collected for the escherichia coli regulatory interaction network , showing that it improves on the best preforming link prediction methods .
overall the silencing methodology helps translate the abundant correlation data into valuable local information , with applications ranging from link prediction to inferring the dynamical mechanisms governing biological networks . |
basal cell carcinoma ( bcc ) is the most common cutaneous malignancy , the most common human malignancy overall , and its incidence is increasing . for more than 80 years , convincing epidemiological evidence has linked sun exposure with skin cancer .
bcc is more frequent in patients with higher cumulative sun exposure , with more sun - sensitive skin types , from areas of high ambient solar irradiance , and on sun - exposed body sites [ 1 , 2 ] .
both the quality and quantity of such epidemiological evidence have improved over those 80 years , and more recent data have given us direct evidence of ultraviolet- ( uv- ) induced mutations in genes important to bcc development .
uv - induced mutations in the p53 tumor - suppressor gene have been found in more than half of bcc cases .
mutations that activate the hedgehog intercellular signaling pathway genes , including ptch , sonic hedgehog , and smoothened , play a significant role in the development of bcc , and these mutations have also been shown to be uv induced .
uvb exposure seems to be the most important risk factor in developing bcc [ 2 , 47 ] .
uva has also been implicated as a risk factor for bcc , including exposure during the use of tanning beds [ 9 , 10 ] .
with the propensity of both epidemiologic and direct evidence indicating the chief role of uv in the development of bcc , one may expect a similar propensity of evidence indicating that uv blockade by sunscreen protects against bcc . to date
, there has only been one randomized controlled trial examining sunscreen 's role in the prevention of skin cancer , and it showed no significant protective benefit of sunscreen with relation to bcc .
there was both a daily spf 16 sunscreen group and a no - sunscreen group that included no placebo lotion . at the end of this trial , 75% of
the daily sunscreen group patients were applying sunscreen to their head , neck , arms , and hands 3 or 4 days per week , and this correlated well with the measured weight of sunscreen at scheduled study - clinic visits . in the no - sunscreen group , 74% of
patients were not using sunscreen at all or no more than 1 - 2 days per week .
the no - sunscreen group was in fact allowed to wear sunscreen , and 26% of that group was using sunscreen more than 2 days per week .
skin cancer diagnoses were obtained by scheduled study - clinic visits 2 and 4 years after the initiation of the study , utilizing blinded dermatologists to make clinical diagnoses , which were also confirmed histologically .
diagnoses of skin cancers made by study patients ' local doctors at other times were obtained and confirmed with review of medical records .
the use of the spf 16 sunscreen to the head , neck , arms , and hands had no effect on the incidence of bcc tumors or the total number of bcc tumors occurring at these sites .
the incidence of squamous cell carcinoma was significantly reduced in the daily sunscreen group . to assess latency of the protective effects of sunscreen , an 8-year follow - up study of all patients included in the original study
again , a trend , but not a significant reduction in the total number or incidence of bcc , was observed with daily sunscreen use . when isolated to the late follow - up period alone
, there was a non - significant reduction in bcc incidence with daily sunscreen users ( rr 0.75 , 95% ci 0.491.14 ) , indicating a possible trend towards decreased bcc incidence over longer time periods .
green et al . 's data did not support what years of prior epidemiological and direct evidence would have predicted : a protective benefit against bcc by using sunscreen . however , there are four important real - world concepts that were not addressed by this study : sunscreen abuse , sunscreen misuse , sunscreen formulation , and cumulative uv exposure
. the idea of sunscreen abuse has been elucidated by three randomized trials in europe that examined the behavior of subjects on vacation using sunscreen with known spf values .
these studies elucidated the idea of intentional sun exposure ( ise ) and nonintentional sun exposure ( nise ) .
ise is sun exposure with an intention to stay in the sun with large areas of uncovered skin or to acquire a tan .
these three trials found that the subjects with ise had differing patterns of behavior with different spf sunscreens assigned for use .
higher known spf values were associated with a dramatic increase in time spent in the sun , specifically sunbathing [ 1416 ] .
higher spf values were also associated with dramatic decreases in the amount of sunscreen used , the amount of clothing used to cover up while in the sun , and the time of day when sun exposure was obtained , with the high spf group sunbathing closer to the noon hours .
the abuse of sunscreen occurs because sunburn is delayed , and the users are able to practice sun exposure behavior that would not be possible if they were burning .
the users ' perceived endpoint , sunburn , is thus delayed but still obtained . in green 's trial ,
the daily sunscreen group was aware that they were using sunscreen , and the nonsunscreen group had no placebo , and thus they knew that they were not being protected . according to the three aforementioned studies , this knowledge alone would have significantly altered the sun exposure behavior practices of the two groups .
sunscreen misuse is nearly ubiquitous when respect is given to the fact that 2 mg / cm is the amount of sunscreen used when spf is measured .
studies examining the actual amount applied by most users have revealed amounts closer to 0.5 mg / cm , or one quarter of the recommended amount , which yields an effective spf of about 1/3 the labeled spf .
theoretical calculations suggest that there is an exponential relationship between spf and the amount of sunscreen used , and that exponential growth has been suggested by studies in human skin .
green 's study did not mention the actual amounts of sunscreen applied , but with a spf 16 sunscreen and the realistic amounts used by the subjects , the actual spf obtained was likely exponentially lower than 16 .
the other form of blatant sunscreen misuse in green 's study was the fact that in the daily sunscreen group , 75% of patients were applying sunscreen to parts of their body 3 - 4 days per week , and this was supported by measured sunscreen weights . in the daily group , 75% of the subjects were using sunscreen roughly 50% of the time , and one may consider that more consistent use of the daily assignment may have yielded different results .
in addition , the control group was allowed to wear sunscreen , and 26% of that no - sunscreen group was in fact wearing sunscreen more than 2 times per week .
thus , the actual protective benefit is likely far greater than that stated in green 's study . in the 1990s ,
suspicion of uva 's role in cutaneous carcinogenesis fueled an explosion of new broad spectrum
one may be hard pressed to find a sunscreen resembling the antiquated formulation used in green 's study on a shelf for sale today .
green 's study formulation did utilize avobenzone but had no concurrent uva stabilizer . with today 's standards , both the low spf and lack of stable uva coverage provided by this sunscreen may cause providers and consumers alike to shy away . with the new us food and drug administration ( fda ) labeling standards , this sunscreen from green 's study would have to live up to its purported spf exactly to claim that it prevents skin cancer ; if it is tested under spf 15 , it could only claim to prevent sunburn . with issues over the stability of its uva coverage , it may not meet the standards that today 's sunscreen formulations are required to reach to be labeled broad spectrum [ 18 , 19 ] .
unfortunately , no randomized trials exist to date examining what effect a newer sunscreen formulation may have with relation to basal cell carcinoma and other skin cancer development .
the likelihood of developing bcc increases with age , having a median age at diagnosis of 67 years and a mean age of 64.4 years .
the most common age group affected is 5080-year - old individuals , with sun damage starting to accumulate at an early age and likely not manifesting as bcc for upwards of 20 years , thus there is a very significant lag time between sun exposure and development of bcc .
with such an apparent contribution of cumulative uv exposure , it brings to question how applicable a 4.5-year and even an 8-year extended follow - up period is with respect to actual bcc risk , when patients have likely been accruing uv - induced damage for triple that amount of time .
a nonsignificant downward trend was noted in bcc when isolated in the extended 8-year endpoint of green 's study , and a longer period of sunscreen use followed over a more substantial time frame may very well have shown a statistically significant benefit .
dermatologists recommend to their patients the daily use of high spf and broad spectrum sunscreens applied in liberal amounts as part of a photoprotection plan , but no studies have examined the effects of sunscreen on bcc with parameters comparable to these recommendations .
published literature finding no effect of sunscreen on bcc did not utilize true daily use of high spf and broad spectrum sunscreens with consistent application and likely involved inadequate amounts applied .
the fda has addressed some of these issues in its new rules for sunscreens . by the summer of 2012
, sunscreens will have to provide a minimum spf of 15 and pass the new broad spectrum test procedure to claim to protect against cancer when used with other methods of photoprotection ; if they only achieve one of these two standards , they can only claim to prevent sunburn .
sunscreens will also not be able to claim to provide more than two hours of protection or provide protection immediately after application unless they submit data to prove these claims and are approved by the fda .
all sunscreens will require directions on when to reapply , and sunscreens that are not water resistant will have to instruct consumers to seek water resistant options if swimming or sweating .
when viewed as a whole , the fda 's new rules have addressed many of the real - world concepts that have been problematic with sunscreen use in the past .
sunscreen abuse , sunscreen misuse , and cumulative uv exposure have been addressed with mandatory and deliberate directions on the label about reapplying , not providing more than two hours of protection , and sunscreen only being part of a photoprotection plan .
the improved capabilities of sunscreen formulations have been respected by requiring both a mandatory spf minimum and a tested uva minimum to claim broad spectrum coverage and prevent skin cancer . while the conclusions of the studies performed to date can not be extrapolated to real - world physician - patient practices that have proper patient education and compliance , the frontier for research with new sunscreen formulations under new standards and practices is exciting .
the field of dermatology can be hopeful that the true role of sunscreen in the prevention of uv - induced skin cancer may soon be reliably understood . | basal cell carcinoma ( bcc ) is the most common human malignancy .
both epidemiological and direct evidence have established ultraviolet ( uv ) exposure from the sun as the most important risk factor for bcc development
. there has only been one randomized and controlled study to examine sunscreen 's role in the prevention of bcc , and no significant protective benefit was found .
this study did not address four important concepts : sunscreen abuse , sunscreen misuse , sunscreen formulation , and cumulative uv exposure .
thus , the results of this study are difficult to interpret and extrapolate with real - world sunscreen practices . |
alzheimer 's disease ( ad ) is the most common neurodegenerative disease , accounting for more than 50% of all dementia types .
its twice more common in females which could be due to increased longevity and sex difference in brain size .
women with alzheimer have lower endogenous estrogen level which lead to the hypothesis that estrogen could be neuroprotective .
hormone replacement therapy ( hrt ) has been extensively studied in the past , but results were inconclusive .
recent studies suggest that 17 -estradiol based therapies may provide the most beneficial neuroprotective effect .
early introduction and prolonged therapy preferably for < 5 years with 17 -estradiol prevents ad .
observational studies have examined both hrt and estrogen replacement therapy ( ert ) , in relation to ad .
an inverse relationship was seen for the duration of ert and risk for alzheimer . increased risk of alzheimer is seen with younger age at oophorectomy ( bilateral or unilateral ) .
these findings suggest that earlier age of surgical menopause increases the risk of cognitive impairment . in women 's health initiative memory study ( whims )
a total of 4532 women with natural menopause were randomized into a trial comparing conjugated equine estrogen ( cee ) with medroxyprogesterone ( mpa ) versus placebo .
study revealed that women who received cee with mpa demonstrated greater cognitive decline compared with the placebo group .
additional analysis revealed that risk for dementia was doubled for women who received cee with mpa compared with the placebo group . taken together , data from the whims demonstrated a higher incidence of dementia and greater cognitive decline among hormone user .
predominant estrogen in premenopausal women is estradiol and its decline is more than estrone in post - menopausal age .
cee contains predominantly estrone rather than estradiol ; it also contains other hormones which are not desired .
in contrast 17 -estradiol , administration achieved a hormone state close to that observed prior to menopause .
studies suggest that 17 -estradiol significantly improved the verbal memory by enhancing verbal information processing and decreased forgetfulness .
17 -estradiol increased the metabolism in receptive language and auditory association area . for getting the desired result , a minimum of 3 months of therapy with no upper cut off is needed.-amyloid and tau proteins are involved in the structural changes that lead to ad pathology , particularly in the hippocampus , medial temporal , parietal and frontal cortical regions .
evidence has shown that estrogen particularly 17 -estradiol provides protection against -amyloid induced damage and tau - related changes .
neuroimaging outcomes have also been supportive of the benefits of 17 -estradiol , particularly in the brain regions that show preclinical abnormalities in individuals who are at risk for ad .
17 -estradiol , increases the blood flow to the hippocampus and superior temporal gyrus and it also activates left middle , superior frontal cortex and inferior parietal cortex during verbal memory encoding task on functional magnetic resonant imaging.17 -estradiol also reduces the level of amyloid precursor protein ( app ) through enhanced alpha secretase processing resulting in marked reduction of app - c terminal fragment , amyloid beta and plaque burden .
it also enhances the level of transthyretin in the brain , which inhibits the aggregation of amyloid eta into plaque.17 -estradiol also promotes the growth and survival of cholinergic neurons , increases the density of hippocampal neurons and increases the synaptic plasticity in the hippocampus which enhance the short and long term memory .
it selectively benefits healthy neurons ( healthy cell bias ) or when neuronal stress has just started . in degenerating neurons particularly in the presence of apolipoprotein e4 ,
critical period for post - menopausal women during which 17 -estradiol selectively provides a beneficial effect .
younger post - menopausal women have higher density of muscarinic receptor than an older one .
17 -estradiol in early post - menopausal women significantly decreased the anticholinergic induced verbal memory task
. in cache county study investigators enrolled 1768 women between 1995 and 2006 at the age of menopause who were given hrt particularly17 -estradiol without progesterone . when started within a critical period of 5 years post - menopausal
ideally 17 -estradiol should be started within a critical period of 5 years post - menopause in a dose of 50 g / day transdermally .
transdermal route byepasses the hepatic metabolism and hence results in steady state plasma concentration of estradiol .
it also maintains 1:1 estrone to estradiol ratio which is normally observed in selected phases of the menstrual cycle .
favorable results are noted from both short and long term therapy . food and drug administration recommends hrt at lowest possible dose for the shortest duration but in 2013 british and international menopause society put no arbitrary limit on duration of hrt .
if symptoms persist , the benefits of hormone therapy outweigh the risk . the major concern with estrogen therapy
is increased risk of venous thrombosis , coronary artery disease , breast and endometrial carcinoma , dysmenorrhea , abnormal vaginal bleeding and hypersensitivity .
screening of patients who are at risk to develop above complications and mammography should be done in every case .
regular annual follow - up for early detection of complication is advisable just like any other hrt .
recent studies suggest that estrogen particularly 17 -estradiol has a positive effect in increasing blood flow , stimulating dendrites , protecting against oxidative stress and modulating neurotransmitters .
there appears to be a critical window ( window of opportunity ) between 50 and 60 years of age , ideally within first 5 years of menopause during which estrogens have this positive effect but thereafter the cells deteriorate and estrogen may accelerate cell damage , the so called healthy cell bias .
so it is advised to start 17 -estradiol in early menopause for longer duration as an inverse relation exists between hrt and risk for ad .
additional research is needed to optimize hormone therapy to delay , prevent and treat ad and to prevent estrogen related complications such as carcinoma and venous thrombosis . | alzheimer disease ( ad ) is a crippling neurodegenerative disorder .
it is more common in females after menopause .
estrogen probably has a protective role in cognitive decline .
large amount of research has been carried out to see the benefits of hormone replacement therapy with regards to alzheimer still its neuroprotective effect is not established .
recent studies suggest a reduced risk of ad and improved cognitive functioning of post - menopausal women who used 17 -estradiol in the critical period .
use of 17 -estradiol in young and healthy post - menopausal women yields the maximum benefit when the neurons are intact or neuronal stress has just started .
hence intervention in the critical period is key in the prevention or delay of ad in post - menopausal women . |
changes in olfactory functions have been found in many neurodegenerative and psychiatric disorders , including parkinson s disease , alzheimer - type dementia , schizophrenia , obsessive compulsive disorder , and attention deficit / hyperactivity disorder ( adhd ) , as well as autism spectrum disorders ( asds).1,2 in a recent systematic review of olfaction in child and adolescent psychiatric disorders , schecklmann et al2 emphasized that specific changes in olfactory function were found mainly in disorders with a dopaminergic pathology ( eg , adhd , autism , schizophrenia , and 22q11-deletion syndrome ) .
unequivocal findings in early onset schizophrenia showed diminished discrimination and identification with unaltered sensitivity , and thus olfactory identification was seen as a highly promising biomarker candidate for schizophrenia .
patients with 22q11-deletion syndrome featured severe olfactory impairments in all olfactory domains ; however , when the authors statistically controlled the results for craniofacial abnormalities , which might contribute to diminished olfactory findings , no significant olfactory abnormalities were found .
studies investigating adhd and autism relative to olfaction have produced inconsistent results.2 there have been few studies to deal with olfactory abnormalities in asd .
suzuki et al3 measured odor - detection threshold and odor identification in twelve adult males with asperger s syndrome ( as ) and twelve matched control subjects using the cain two - bottle test and the university of pennsylvania smell identification test ( upsit ) . as subjects did not have impaired odor detection , but did have significantly impaired olfactory identification .
bennetto et al4 focused on olfactory identification and compared 21 participants with autism ( age 1018 years ) with 27 well - matched controls . using the sniffin sticks test
tavassoli and baron - cohen5 examined 38 adult participants with asd and 42 controls , and found no difference in olfactory detection thresholds between the groups .
brewer et al6 investigated olfactory identification in 15 children with high - functioning autism ( hfa ; mean age 77.8 months ) and 15 controls ( matched for age , sex , and intelligence quotient ) utilizing a modified visual analog of the upsit .
however , smell identification was negatively correlated with age in the hfa group , a finding that was opposite to the relationship found in controls .
may et al7 reported results of a 5-year follow - up of children with hfa that followed a previous study .
additionally , unirhinal olfactory identification was compared between hfa , as , and controls ( twelve participants per group ) .
unirhinal olfactory identification was reduced in hfa compared to as and controls , suggesting an orbitofrontal compromise in hfa , but orbitofrontal integrity in as . in our previous reports , we found children and adolescents with asd , in comparison to healthy controls , being significantly impaired relative to odor - detection thresholds ( 6.3 vs 7.9 , p=0.025 ) .
there was no significant difference between autistic and control subjects on the total score of olfactory identification ( 10.7 vs 10.6 , p=0.799).8 autistic subjects assessed all odors as overall less pleasant than control subjects assessment ; however , the difference was not significant ( 45.1 vs 42.0 , p=0.123).9 the aim of the present study was to analyze further the data of the autistic group from our previous research in order to determine if and to what extent a relationship between olfactory functions ( odor - detection thresholds , odor identification , and odor preference ) and autism severity and sensory - related psychopathology exists in children with asd .
subjects with asd were recruited from the department of child psychiatry , university hospital motol ( prague ) .
all participants and their legal guardians agreed to participate in the study ; legal guardians signed informed consents for subjects under 18 years of age , as per czech law .
inclusion criteria for autistic subjects were : 1 ) diagnosis of as , high - functioning autism , or other high - functioning asd , confirmed using the autism diagnostic interview revised ( adi - r)10 and/or the childhood autism rating scale ( cars);11 2 ) age 618 years ; and 3 ) an intelligence quotient 70 .
our sample consisted of 35 autistic patients with a mean age of 10.83.6 years ( range 6.118.5 years , 31 boys ) . diagnoses made according to the international classification of diseases ( 10th revision)12 included 27 patients with as , five patients with childhood autism , and three patients with unspecified pervasive developmental disorders .
olfactory threshold and identification were examined using sniffin sticks , a commercially available three - part olfaction test ( burghardt medical technology , wedel , germany ) : threshold , discrimination , and identification .
the test has been reliably standardized , is appropriate for both adults and children,13,14 and has been previously used in the czech population.15 two parts of the test ( threshold and identification ) were used in this study . we also developed a simple method to assess odor pleasantness.9 odor pleasantness was assessed using the identification part of the sniffin sticks test on a 5-point scale , with 1 being most pleasant , 3 being neutral , and 5 being most unpleasant .
we supplemented the scale with emoticons , which was thought to be useful in autistic children , in order to facilitate decisions . during testing
the cap was removed by the investigator and the pen tip was placed centrally , approximately 2 cm in front of the nose .
the tests were administered in sequence starting with testing of odor thresholds , followed by testing of odor identification ( this sequence matched the manufacturer s guidelines ) . finally , odor pleasantness was examined . for more details on testing procedures
, please see the detailed description in dudova et al.8 the severity of autistic psychopathology was measured using the cars.11 for calculations , we used the total score and the score of item 9 ( taste , smell , and touch response and use ) , which was directly connected to olfactory behavioral abnormalities .
statistical analysis was performed using spss version 21.0 ( ibm , armonk , ny , usa ) .
the spearman correlation analysis was used to explore relationships between autistic psychopathology and odor - detection threshold , odor identification total score , and odor pleasantness total score .
subjects with asd were recruited from the department of child psychiatry , university hospital motol ( prague ) .
all participants and their legal guardians agreed to participate in the study ; legal guardians signed informed consents for subjects under 18 years of age , as per czech law .
inclusion criteria for autistic subjects were : 1 ) diagnosis of as , high - functioning autism , or other high - functioning asd , confirmed using the autism diagnostic interview revised ( adi - r)10 and/or the childhood autism rating scale ( cars);11 2 ) age 618 years ; and 3 ) an intelligence quotient 70 .
our sample consisted of 35 autistic patients with a mean age of 10.83.6 years ( range 6.118.5 years , 31 boys ) . diagnoses made according to the international classification of diseases ( 10th revision)12 included 27 patients with as , five patients with childhood autism , and three patients with unspecified pervasive developmental disorders .
olfactory threshold and identification were examined using sniffin sticks , a commercially available three - part olfaction test ( burghardt medical technology , wedel , germany ) : threshold , discrimination , and identification .
the test has been reliably standardized , is appropriate for both adults and children,13,14 and has been previously used in the czech population.15 two parts of the test ( threshold and identification ) were used in this study .
we also developed a simple method to assess odor pleasantness.9 odor pleasantness was assessed using the identification part of the sniffin sticks test on a 5-point scale , with 1 being most pleasant , 3 being neutral , and 5 being most unpleasant .
we supplemented the scale with emoticons , which was thought to be useful in autistic children , in order to facilitate decisions . during testing
, odorants were presented using modified felt - tip pens . for odor presentation , the cap was removed by the investigator and the pen tip was placed centrally , approximately 2 cm in front of the nose .
the tests were administered in sequence starting with testing of odor thresholds , followed by testing of odor identification ( this sequence matched the manufacturer s guidelines ) .
please see the detailed description in dudova et al.8 the severity of autistic psychopathology was measured using the cars.11 for calculations , we used the total score and the score of item 9 ( taste , smell , and touch response and use ) , which was directly connected to olfactory behavioral abnormalities .
statistical analysis was performed using spss version 21.0 ( ibm , armonk , ny , usa ) .
the spearman correlation analysis was used to explore relationships between autistic psychopathology and odor - detection threshold , odor identification total score , and odor pleasantness total score .
as we published previously , the mean odor - detection threshold was found to be 6.33.1 , mean total score of olfactory identification was 10.72.6,8 and mean total score of odor pleasantness was 45.18.2.9 we found no significant correlations between autism severity ( as expressed by the total cars score ) and odor - detection thresholds , odor identification , or odor pleasantness .
there was no significant relationship either between the cars item 9 score ( taste , smell , and touch response and use ) and odor - detection thresholds , odor identification , or odor pleasantness . for details , see table 1 .
as we demonstrated in the introductory part of our article , pediatric results68 differed from results obtained from studies involving adolescents4 and adults.3,5 brewer et al6 suggested that the absence of an olfactory identification deficit in hfa was caused by immature olfactory identification at younger ages .
they formulated the hypothesis that those with hfa may appear to grow into the deficit as they mature and enter puberty ; in other words , olfactory identification abilities in hfa children will appear to deteriorate with age , but only when compared to controls .
this was confirmed , in some hfa participants , in a 5-year follow - up of children with hfa by may et al.7 they found that olfactory identification improved in both groups ( hfa and controls ) during the follow - up , but development was more heterogeneous in children with hfa .
the term heterogeneous is at best rather vague , and our interpretation of it is that most if not all controls had improved odor identification over time , while those with hfa had mixed results , ie , with some showing improvement with age , and others not . based on this interpretation of the data , may et al s results agree with our previous results , which were obtained from a group of participants of comparable age .
we found odor - identification ability to be positively correlated with age in the control group , but not in the autism group.8 it is reasonable to conclude that chronologic aging contributes to some of the problems encountered during olfactory research associated with autism . to our knowledge , there has been only one study that explored the relationship between autistic psychopathology and olfactory functions .
however , the methodology was different , and thus the results are not directly comparable .
bennetto et al4 examined olfactory and taste functioning in individuals with autism to determine if there was a connection between olfaction and social impairment in autism .
they examined the relationship between olfactory identification and comparable scores taken from the adi - r , the standardized parent interview for autism . in participants with autism , olfactory identification
was marginally related to their ability to engage in social verbalization or chatting , and significantly related to their skill at maintaining a reciprocal conversation .
children with a poor performance on the olfactory identification test were more likely to have greater social impairment .
generally , it can be said that studies attempting to correlate autism symptoms and/or severity with various neurophysiological abnormalities have not been entirely successful.16 this is not only with regard to olfactory functions .
for example , inconsistent results have also been obtained in research looking at electroencephalogram ( eeg ) abnormalities and skin - conductance reactions .
some studies found a positive association between eeg abnormalities and autism severity,17 while some did not.18,19 similarly , some studies found abnormal skin - conductance reactions in autism,20,21 while others found no relationship.22 the probable interpretation of our negative results is that olfactory abnormalities in autism represent a trait marker for the disorder rather than a state marker .
our results are limited by sample size and the age diversity of the study group , which included children as well as adolescents .
most of our patients were diagnosed with as ( 27 of 35 patients ) , which is atypical and does not represent the most common diagnosis in the asd group.23 another limitation was the use of the cars , which is a quite simple rating scale , and the behavior related to sensory input was concentrated into just one item .
however , similar methodology using the cars has already been successfully used in other autism studies.18,24 in future , the possible relationship between sensory - related behavior and olfactory functions could be better examined by using more sophisticated scales , such as the infant / toddler sensory profile.25
we did not find any significant relationship between the severity of autistic psychopathology and odor - detection thresholds , odor identification , or odor pleasantness . | backgroundchanges in olfactory functions have been found in many neurodegenerative and psychiatric disorders , including autism spectrum disorders ( asds ) .
the aim of the present study was to evaluate the relationship between olfactory functions ( odor - detection thresholds , odor identification , and odor preference ) and autism severity and sensory - related behavior in children and adolescents with asd.subjects and methodsour sample consisted of 35 high - functioning patients with asd ( mean age 10.83.6 years , 31 boys ) .
olfactory testing ( threshold and identification ) used the sniffin sticks test .
odor pleasantness was assessed on a 5-point scale using the identification part of the sniffin sticks test .
the severity of autistic psychopathology was measured using the childhood autism rating scale ( cars).resultsusing spearman s correlation , we found no significant correlations between autism severity ( as expressed by total cars score ) and odor - detection thresholds ( r=0.144 , p=0.409 ) , odor identification ( r=0.07 , p=0.966 ) , or odor pleasantness ( r=0.046 , p=0.794 ) .
there was also no significant relationship between cars item 9 ( taste , smell , and touch response and use ) and odor - detection thresholds ( r=0.170 , p=0.330 ) , odor identification ( r=0.282 , p=0.100 ) , or odor pleasantness ( r=0.017 , p=0.923).conclusionwe did not find any significant relationship between the severity of autistic psychopathology and olfactory functions . |
multi - wavelength observations of coronal mass ejections ( cmes ) can be successfully used to investigate the main physical plasma parameters of the solar corona . for the most energetic cme events , a forward fast - mode magnetohydrodynamic ( mhd ) shock
coronal shocks are thought to be driven by coronal mass ejections and/or by flare ejecta .
alternatively , their origin can be attributed to flare - ignited blast waves . however , even after several decades , the origin of coronal shock waves is not completely understood as yet ( see , e.g. , and references therein ) . generally , cme - driven shocks are too faint to be detected by white - light coronagraphs in the visible range , but they can be easily identified in radio dynamic spectra as type ii radio bursts , that is , narrow bands of enhanced radio emission generated near the local electron plasma frequency fpe and/or its harmonics . however , due the lack of information about the location of the type ii emission in the corona , it is important to integrate the data with ancillary observations from instruments having also spatial resolution .
in the past decade , spectroscopic observations from the soho s ultraviolet coronagraph spectrometer ( uvcs ) have been effectively used for coronal shock detection [ 38 ] .
uv emission lines can be very important sources of diagnostic information about the physical properties of the solar corona .
in fact , line intensities and profiles critically depend on several parameters , such as the electron density , the electron temperature , the kinetic temperature of the emitting ions and the outflow velocity . in the present paper
, we analyze multi - wavelength observations of a cme / shock event observed on november 1st 2003 at 1.7r beyond the southwest limb of the sun . the main goal of this work is to retrieve information on the local alfvn mach number and magnetic field strength of the pre - shock plasma in the middle corona .
a candidate cme / shock event observed on 2003 november 1st included in the online cdaw lasco cme catalog ( http://cdaw.gsfc.nasa.gov/cme_list/index.html ) was selected by using a uvcs catalog of cmes composed by over 1000 events crossing the uvcs slit during the cme detection .
in particular , we searched for high speed events ( > 700 km / s ) with a metric type ii radio burst detected in the time window from the cme onset to the end of uvcs observations . in order to have the best spatial coverage for events observed at different wavelengths , we searched for a cme also detected in the low corona by the mlso / mk4 ground - based coronagraph .
the dynamics of the cme was inferred through lasco coronagraph white - light data ( f.o.v .
2.330r ) , fig . 1 , and mlso / mk4 coronagraph data ( f.o.v .
1.12.8r ) , while the shock dynamics was investigated using uv spectrographic observations of uvcs , fig . 2 , and ground - based radio spectral observations of culgoora ( australia ) , fig . 3 , and holloman ( new mexico ) .
the cme was first detected around 22:30 ut in the o vi 1031.931037.62 doublet spectral lines by uvcs that at the time of the cme passage was performing a sit - and - stare observation at 1.7r at a polar angle of 245. later , it was also detected by the lasco instrument at 23:06 ut , propagating beyond the southwest limb of the sun .
the associated type ii radio emission was finally detected by ground - based radio spectrographs from about 22:35 to 23:10 ut .
from the analysis of the spectral emission of the oxygen doublet o vi 1032/o vi 1037 observed by uvcs , we detected a significant increase of the o vi line intensities and a line broadening of about 30% in a portion of the uvcs slit .
these observational features are attributed to plasma compression and ion heating , as expected at the transit of a coronal shock propagating ahead of the front of a fast cme . in general , the observed o vi intensity i is given by the sum of a collisional component icolllne2dl and a radiative scattering component iradidisklnefd()dl , where idisk is the exciting disk intensity , ne the electron density and fd the doppler dimming function which depends on the ion outflow velocity , v. the observed lines intensities ratio , r = iovi1032/iovi1037 , is expected to range between the value of 4 in the case of totally radiative contribution and the value of 2 in the case of totally collisional contribution . from the o vi doublet intensity diagnostics , as shown in fig .
4 , we verified that at the shock passage ( at 22:40 ut ) r tends to the value of 2 due to the rapid acceleration and compression of the plasma . yet
, r still remains > 2 because of the line - of - sight ( l.o.s . ) contribution of fore / back - ground material , as expected . at the cme transit ( at 23:10 ut ) r 2 due to the high density of the expanding cme plasma that fills a much greater portion of the l.o.s . in order to estimate the shock speed , vshock , from the type ii frequency drift observed in the radio dynamic spectra
, we inferred the local coronal density profile ne(r ) through inversion of the polarized brightness ( pb ) as derived from the white - light coronagraph observations , thus obtaining a shock speed of 800 km / s .
for the determination of the plasma density we have used the on - line available pb calibrated data from mlso / mk4 instrument ( specifically the one at 22:30 ut of 11.01.2003 , just before the cme detection ) . with the van de hulst s inversion method ( assuming spherical symmetry , hypothesis generally accepted during solar maximum ) , we derived a radial density profile in the region of the shock propagation .
this profile , that was fitted with a third degree polynomial , was found to be quite similar to a typical newkirk 1967 profile ( ne(r ) = 4.2 10 10 ) that , by the way , is generally the one used in literature in order to determine shock velocities above streamer from radio dynamical spectra . on the basis of the lasco and mk4 observations
, we further estimated the kinematics of the cme front , finding a cme speed of 750 km / s ( i.e. the shock propagates faster by about 6% than the cme itself ) .
a band - splitting of the emission lines is often observed in the radio dynamic spectra and it is attributed to emission behind and ahead of the shock front . from the squared ratio of the frequencies of the two split bands , as observed in the metric radio dynamic spectra , we were able to estimate the compression ratio x = fh2fl2=nhnl=1.39 . assuming perpendicular propagation of the shock with respect to the ambient magnetic field
the sound speed is much less than the alfvn speed va , so that vms va .
hugoniot relationships for 1 ( that is , the ratio of the plasma pressure to magnetic pressure ) , yielding ma = x(x+5)2(4-x)=1.30 , we estimated vavshockma610km / s . with the above information , by using the electron density obtained from the inversion of the pb , we were able to compute the coronal magnetic field strength , decreasing from about 4.4 to 1.5 g in the height range from 1.2 to 1.5r , as shown in fig .
we have analyzed a cme / shock event observed by uvcs / soho in the ultraviolet on november 1st 2003 .
the data analysis , performed also with the aid of radio and visible observations from ground- and space - based instruments , allowed us to obtain estimates of important properties of the coronal plasma crossed by the shock ( basically the alfvn speed va = 610 km / s and the magnetic field strength b = 4.41.5 g ) that were found to be consistent with estimates that were previously inferred with different techniques at comparable heights in the corona . | the analysis of the spectral properties and dynamic evolution of a cme / shock event observed on november 1st 2003 in white - light by the lasco coronagraph and in the ultraviolet by the uvcs instrument operating aboard soho , has been performed to compute the properties of some important plasma parameters in the middle corona below about 2r. simultaneous observations obtained with the mlso / mk4 white - light coronagraph , providing both the early evolution of the cme expansion in the corona and the pre - shock electron density profile along the cme front , were also used to study this event . by combining the above information with the analysis of the metric type ii radio emission detected by ground - based radio spectrographs
, we finally derive estimates of the values of the local alfvn speed and magnetic field strength in the solar corona . |
when the hiv epidemic peaked in 1993 , aids was the leading cause of death among 25 - 44-year - old men and women and the eighth most common cause of death worldwide ( 1 ) . injection drug use ( idu ) and unprotected sex are considered as major risk factors for the acquisition of retroviral infection ( 2 - 4 ) , and more than half of people living with hiv are women and girls ( 2 , 5 - 7 ) .
because of social , economic , and political differences among regions , the epidemic has not been uniform ( 8 , 9 ) .
the most common route of hiv transmission in high - income countries such as the united states and the united kingdom is homosexuality .
people who engage in high - risk sexual behaviors in sub - saharan africa and idus in eastern europe and southeast asia are the major at - risk groups ( 10 - 14 ) . in 2011 ,
adolescents , women , homosexuals , and serodiscordant couples ( i.e. one partner is hiv positive , and the other is hiv negative ) were reported to be the populations with the highest risk of infection ( 10 ) .
although the aids epidemic is relatively mild in asia , factors such as low condom use , unsafe injections among injection drug users , immigration , lack of education , and misconceptions about hiv / aids could affect the hiv / aids epidemic in all countries that currently have low prevalence ( 15 - 17 ) . according to estimates by the ministry of health and medical education , as of september 21 , 2011
more than 91% of these patients were men , and approximately 4,400 had died ( 18 ) .
needle sharing in idus is the most common route of hiv transmission in men in iran , accounting for approximately 70% of established transmissions .
however , studies show an increased rate of high - risk behaviors for hiv infection , particularly unprotected sex ( 18 - 22 ) . in addition
, it is important to consider the hiv infection prevalence in specific populations in iran , such as patients with tuberculosis ( 23 ) .
rapid changes in the epidemiological characteristics of infectious diseases necessitate health practitioners being aware of the current status of these diseases , especially in cases of pandemic diseases such as aids , whose control requires global efforts ( 24 - 26 ) . in this
regard , research should serve as a tool to improve the effectiveness of healthcare systems .
successful aids prevention and treatment depend on the complete and accurate identification of at - risk populations as well as regular and standardized data collection , analysis , and interpretation ( 1 , 27 ) .
one of the best opportunities to combat aids in iran is the use of the healthcare system .
however , behavioral research in the general population and high - risk groups is not systematic , integrated , or consistent ( 24 , 28 ) .
the present study investigated the epidemiology of hiv / aids in east azerbaijan , iran .
the target population was all patients with hiv / aids in east azerbaijan province , including patients who acquired hiv infection through idu , sexual transmission , mother - to - child transmission ( i.e. vertical transmission ) , and contaminated blood products .
the vice chancellor of research and technology of tabriz university of medical sciences ethical committee approval was obtained for the study ( decision number : 5.4.328).first , necessary supplies including computers , telephone lines , etc .
( table 1 ) were extracted and entered into microsoft excel software for classification and sorting .
next , data were analyzed , and diagrams and tables were created using microsoft excel and spss .
among the 371 patients who were identified by healthcare centers in east azerbaijan from 1987 to 2012 ( figure 1 ) , 91% ( n = 338 ) were men and 9% ( n = 33 cases ) were women .
the prevalence of hiv / aids increased along with the population growth from 1987 to 2012 ( data not shown ) , the current prevalence of hiv / aids in east azerbaijan is 1/10,000 .
the mean ages of the total , male , and female patients were 30.8 12.3 , 31.3 12.1 , and 28 10.3 years , respectively .
approximately 54% ( n = 200 ) of patients were single ( table 1 ) .
more than one - third ( n = 131 ) of patients had died , and 27% ( n = 99 ) were actively undergoing regular consultation ( figure 2 ) .
the most common transmission route was related to idu ( 59% , n = 219 ) ( table 1 ) .
approximately 80% ( n = 208 ) of them had high school or lower ( table 1 ) .
the occupational status of 36% ( n = 102 ) and 17% ( n = 48 ) of patients was recorded as self - employed and unemployed , respectively ( table 1 ) .
the rest of the patients were employed in various jobs ( data not shown ) .
available : patients who actively undergoing regular consultation ; unavailable : patients who havent referred to council centers since they have diagnosed as hiv positive .
the frequencies of coinfection with hepatitis c virus ( hcv ) , hepatitis b virus ( hbv ) , syphilis , and toxoplasmosis with respect to sex are shown in table 2 .
thirty - one of 207 patients ( 15% ) were diagnosed with hbv , and 25 of 371 patients were diagnosed with hiv / hbv / hcv co - infection .
p : positive , n : negative , ud : undetermined . data are presented as no .
the prevalence of hiv infection among injection drug users is reported to be 15.07% ( 18 ) .
the results of the present study show that idu is the most common hiv transmission route in east azerbaijan province , which is similar to national reports on hiv / aids ( 18 ) . in a study conducted in tehran and kermanshah provinces ,
unsafe needle sharing was the reported cause of 83% of hiv infections ( 29 ) .
another study performed in 2007 in imam khomeini hospital in tehran reports idu as the most common hiv transmission route ( 31% ) ( 30 ) .
furthermore , a study conducted by researchers at shiraz university of medical sciences reports 76% of patients had a history of idu .
however , in the present study , idu was not the only route of hiv acquisition ( 31 ) , 15% of patients simultaneously had a history of idu and other high - risk behaviors .
a study conducted in china in 2008 found that the most common hiv transmission route is idu ( > 43% of transmissions ) ( 32 ) .
meanwhile , a study in the us reports the most common hiv transmission route is homosexual contact in men , whereas idu is ranked third ( 1 ) .
thus , it seems that educating prisoners and injection drug users , reducing jail time , focusing on public education , making more efforts to provide methadone for people unable to quit their drug addiction , and doing research counseling methods and behavioral disorders at healthcare centers and prisons may improve the current status of the hiv epidemic ( 24 , 33 - 36 ) .
many studies conducted in iran report the prevalence of hiv / aids is higher in men than in women ( approximately 85 - 95% higher ) ( 29 , 31 , 37 , 38 ) .
however , the 2010 joint united nations programme on hiv / aids global report states that more than 50% of people living with hiv worldwide are women and girls ( 39 ) .
the higher prevalence of hiv infection among men in iran and east azerbaijan could be due to a lack of precise data on the number of sex workers , which may partly explain the low rate of hiv / aids in women .
hbv and hcv coinfection among patients infected with hiv is common ; and severe complications can occur in these patients ( 40 ) .
severe complications can occur in patients with coinfection via the same transmission route of hiv ( 40 - 44 ) .
the present results show that 53% of patients with hiv / aids were coinfected with hcv , hbv , or both . in a study conducted in southwestern iran , hcv or hbv
however , the prevalence of coinfection with hepatitis viruses in patients with hiv / aids varies among studies as well as populations with respect to exposure to different risk factors , geographic region , and ethnicity ( 41 , 46 ) .
one limitation of the present study is that the sample may not be representative of the total population in east azerbaijan province .
furthermore , the paper - based records of some patients were incomplete , preventing us from analyzing some variables ( associated to the clinical features ) ( e.g. some laboratory parameters ) . however , the present study is one of the few studies on this topic that have been conducted in iran ; there are no reports from many other provinces . the findings of this study highlight the information gap in this area and help fill in a small part of it .
it is noteworthy that some countries have had success in controlling and preventing contagious diseases such as hiv through healthcare systems set up for the general population ; thus , the development of effective methods on the basis of evidence and data is clearly possible ( 22 , 24 , 28 , 47 - 50 ) . | background : this study was performed in response to the rapid propagation of hiv / aids across iran and its status in this region .
accordingly , an evidence - based program is required to combat this disease.objectives:the present study estimated the prevalence of hiv / aids in east azerbaijan ( population : 3,724,000).materials and methods : we created a database of all positive cases from 1987 to 2012 .
we also analyzed and described the epidemiological status of hiv / aids during a 25-year period by using spss.results:in east azerbaijan , 371 hiv / aids cases have been reported , i.e. 1 case per 10,000 population .
the vast majority of reported cases ( 91% , n = 338 ) were men , whereas only 9% ( n = 33 ) were women . the mean age of patients was 30.8 12.3 years .
unsafe drug injection ( 59% , n = 219 ) and sexual interaction ( 13% , n = 48 ) were the two major modes of hiv transmission .
in addition , 7% ( n = 25 ) of patients have been diagnosed with hiv , hepatitis b virus , and hepatitis c virus simultaneously .
moreover , 60% ( n = 205 ) of men were infected via drug injection , while 82% ( n = 27 ) of women were infected via unprotected sexual interaction ( p < 0.001).conclusions : the results indicate a rapid increase in the number of hiv / aids cases in east azerbaijan , necessitating immediate attention and strategies to combat the rapid spread of the disease .
development of provincial and national hiv / aids strategies demands more accurate and comprehensive hiv / aids surveillance . |
it is a major cause of disability and higher health care costs in the world .
this clinical condition often results from vertebral injuries , intervertebral disc herniation , intervertebral foramen stenosis , or other disorders affecting the dorsal root ganglion ( drg ) or its near nerve root .
so far , to reveal the underlying mechanism of radicular low back pain , a number of preclinical models have been developed that attempt to mimic the above known causes of low back pain [ 1 , 2 ] . amongst those , chronic compression of the dorsal root ganglion ( ccd ) model in rodents displayed dramatic pain hypersensitivity such as mechanical hypersensitivity ( hyperalgesia and allodynia ) and thermal hyperalgesia that mimic the pain symptom observed in low back pain patients [ 16 ] .
although epidural steroid injection and surgical intervention have been used both clinically and experimentally in many cases , radicular low back pain remains a common chronic pain condition that is sometimes refractory to current treatment modalities [ 7 , 8 ] .
therefore , development of new therapeutics is helpful and in urgent need towards the treatment of radicular low back pain .
much evidence has accumulated that reactive oxygen species ( ros ) play an important role in the development of chronic pain [ 9 , 10 ] .
various ros scavengers and antioxidants provided analgesic effects in animal models of inflammatory and neuropathic pain [ 9 , 1115 ] . in recent years , nitroxide radicals have been extensively studied as a unique and interesting class of antioxidants to protect against ionizing radiation , ischemia / reperfusion injury , neurodegenerative diseases [ 18 , 19 ] , and chronic pain [ 9 , 1114 ] .
some nitroxide radicals , for example , amifostine , are being used in clinical practice . unlike other antioxidants that act in a sacrificial mode , nitroxide radicals act as self - replenishing antioxidants in a catalytic manner .
the tempo and -nitronyl ( nit ) groups are the two major kinds of nitroxide radicals ( figure 1 ) .
tempol ( 4-hydroxy-2,2,6,6-tetramethylpiper - idine - n - oxyl ) , a kind of tempo , is found to alleviate pain in various experimental pain models [ 1215 , 21 ] .
compared with tempol , nit group nitroxide radicals have an extensive distribution of unpaired spin density . as such
, much attention has been levied towards the development of this kind of new nit nitroxyl radicals . however , whether nit group nrs possess antinociceptive action has remained elusive .
in addition to reactive oxygen species , inflammatory processes are thought to play key roles in radicular low back pain ( please see strong et al . for review ) .
attenuation of pain behaviors by nonsteroidal anti - inflammatory drugs ( nsaids ) has been demonstrated in various rodent back pain models [ 2226 ] .
however , long - term use of nsaids causes unwanted gastrointestinal and cardiovascular side effects , which limits its broader applications .
herein , we have for the first time synthesized a novel nit nitroxide radical with salicylic acid framework ( sanr ) exerting the beneficial effects of both the nsaids and the antioxidants ( figure 1 ) .
we demonstrated that both acute and long - term systemic administration of sanr attenuated the mechanical hypersensitivity and thermal hyperalgesia observed in an experimental model of ccd rats .
furthermore , sanr provided more pain relief than either traditional nitroxide compound tempol or nsaids aspirin alone .
evidence has accumulated that abnormal spontaneous discharges of primary afferent sensory neurons may contribute to radicular low back pain [ 3 , 4 ] .
we therefore investigated whether sanr produced analgesia by inhibition of spontaneous discharges of primary afferent sensory neurons in ccd rats .
our results revealed that sanr produced a marked depression of spontaneous discharges of primary afferent a fibers in ccd rats in a dose - dependent manner .
when compared to the same concentration of tempol or aspirin , sanr exhibited a much stronger analgesic effect .
taken together , these findings clearly suggest that synthesis of new nit nitroxyl radical with salicylic acid framework may represent a potential new candidate for the treatment of radicular low back pain .
all other chemical reagents were purchased from the beijing ouhe chemical limited company ( beijing , china ) .
high resolution mass spectroscopy ( hrms ) was carried out on a varian 7.0 t esi - fticr - ms ( varian , usa ) .
elemental analyses were carried out using a perkinelmer analyser model 240c ( perkinelmer , america ) . a solution of 5-formyl-2-hydroxybenzoic acid ( 1.66 g , 10 mmol ) and 2,3-bi ( hydroxyl amino)-2,3-dimethylbutane ( 1.48 g , 10 mmol ) in methanol ( 25 ml ) was heated under reflux for 18 h. after the reaction , the methanol was removed and the residue was suspended in 100 ml ch2cl2 , aqueous naio4 ( 2.14 g , 10 mmol in 50 ml ) was added dropwise over a period of 10 min at 0c , and the mixture was stirred for a further 20 min at 0c .
the organic phase was separated , and the aqueous phase was extracted with ch2cl2 ( 3 20 ml ) .
the crude product was purified by column chromatography on silica gel using absolute ether / ethyl acetate/(1 : 1 ) as eluent , giving a deep blue solid product 1 .
n , 9.55% , found : c , 57.35 ; h , 5.81 ; n , 9.67% .
all experimental protocols were approved by the institutional animal use and protection committee , fourth military medical university .
briefly , under anesthesia ( sodium pentobarbital , 40 mg / kg , i.p . ) , the transverse process and intervertebral foramen at l5 on left side were exposed .
a l - shaped stainless steel rod was inserted into foramen to produce a steady compression on the ganglia .
after the rods were in place , the muscle and skin layers were sutured with administration of about 200 mg antibiotics .
behavioral testing was carried out in habituated mice by an observer blinded to the identity of the groups .
as previously described [ 27 , 28 ] , mechanical sensitivity was tested with manual application of von frey hairs ( north coast ) to the plantar surface of hindpaw .
each filament was applied 10 times and the paw withdrawal response frequency ( the percentage of positive responses to the stimulus ) was recorded .
the force of a particular filament required to elicit 50% frequency of paw withdrawal was expressed as the mechanical threshold .
thermal sensitivity was tested by application of infrared heat to the plantar surface of hindpaw and the response latency was measured from an automated device readout ( iitc life science ) .
the latency for the rat to fall off the rod and the speed of rod at this time were recorded and a mean latency and speed for the 4 trials were calculated .
the acceleration was from 2 to 60 r.p.m . over a 180 s period .
activities of single unit drg a - fibers were recorded 38 days after the ccd surgery . under sodium pentobarbital anesthesia ( 40 mg / kg , i.p . )
, laminectomy was performed at the l1-l2 and l4-l5 levels , and two small pools were formed at the exposure regions , separately . in the l4-l5 pool the stainless steel rod was removed . the spinal nerve was transected 710 mm distal to the drg so that the discharge activities of the dorsal root fibers would originate primarily from the drg region and not from peripheral sensory terminals . during recording , l4-l5 pool ( drug pool )
was filled with warm acsf ( 3537c ) containing ( in mm ) : nacl 150 , kcl 5 , cacl2 2 , mgcl2 1 , d - glucose 10 , and hepes 10 , with the ph adjusted to 7.4 , and l1-l2 pool ( recording pool ) was filled with warm paraffin oil ( 3537c ) . under a microscope , a microfilament ( 1530 m in diameter ) presumably including up to a few nerve fibers was teased from the dorsal root .
the proximal end was placed on a fine platinum electrode ( 29 m in diameter ) for electrophysiological recording of drg single fiber activities .
the firing patterns of a single fiber were displayed on a memory oscilloscope ( vc-11 , japan ) and recorded via an a / d board to a computer hard drive and stored for offline analysis .
unit activities with identical wave forms were selected as single fiber activities as previously described [ 3 , 29 ] .
aspirin was dissolved in dimethyl sulfoxide ( dmso ) as a stock solution and kept frozen ; it was diluted in acsf before the experiments .
single unit discharges were recorded in the presence and absence of these three drugs or vehicle for at least another 5 min .
inhibition rate in discharge were calculated as ( maximal discharge rate after drug using baseline rate)/baseline rate 100% .
analysis of variance ( anova ) for random measures was carried out , followed by either a post hoc fisher 's test or dunnett 's test .
the compound sanr was synthesized according to ullman 's procedure as shown in figure 2 . according to ullman 's pioneering work
followed by condensation of 5-formyl-2-hydroxybenzoic acid with 2,3-bis(hydroxyl amino)-2,3-dimethyl butane in methanol solution at room temperature , stable white solids 1,3-dihydroxyimidazolidine were rapidly obtained .
one of the key steps in the synthesis of nit nitroxide radicals is the oxidation of 1,3-dihydroxyimidazolidines .
we chose the aqueous of naio4 as oxidant to oxidize the 1,3-dihydroxyimidazolidine to obtain the final target compound sanr in yield of 21% . following chronic compression of l5 drg ( ccd )
, the rats appeared in good health and did not show any signs of autotomy throughout the study .
sensitivity of ccd rats to mechanical and thermal stimuli was tested at different time points after operation .
compared to sham controls , ccd rats developed bilateral mechanical hypersensitivity ( allodynia and hyperalgesia ) , which was manifested as a significant decrease in response threshold to von frey hairs application to the bilateral hindpaws ( figures 3(a ) and 3(b ) , n = 10 , p < 0.05 at all time points ) .
this mechanical hypersensitivity appeared on the 1st day after compression , persisting over the entire experimental period . in parallel ,
a dramatic drop in response latency to noxious plantar heat stimuli , reflecting thermal hyperalgesia , was found in bilateral hindpaws of ccd rats ( figures 3(c ) and 3(d ) , n = 10 , p < 0.05 at all time points ) .
therefore , it can be inferred that rats with chronic compression of l5 drgs develop strong mechanical hypersensitivity and thermal hyperalgesia , which is consistent with previous reports in rodents [ 35 ] . to investigate the acute effect of sanr on the pain hypersensitivity observed in ccd rats , we administered sanr via intraperitoneal ( i.p . )
injection once at 3 d after operation when mechanical and thermal hyperalgesia was completely developed . as shown in figure 4(a ) , acute administration of sanr ( 54 , 180 , 540 mol / kg body weight ) dose - dependently elevated the mechanical threshold to von frey hairs in ipsilateral paw as compared to predrug level , reflecting as attenuation of mechanical hypersensitivity ( figure 4(a ) , n = 10 ) . this antinociceptive effect
started from 1 h after drug administration , persisting over the test period , namely , 24 h after sanr delivery .
dose - response curve at 7 h after drug was fitted to a hill equation , which yielded an ic50 of 506.5 14.6 mol / kg for sanr in attenuation of mechanical hypersensitivity ( figure 4(b ) ) .
similarly , ccd - induced ipsilateral thermal hyperalgesia was dramatically reduced by acute sanr in a dose - dependent manner with a similar time course , as measured by a prolongation of response latency to radiant heat stimuli compared to predrug level ( figure 4(c ) , n = 10 ) .
the dose - response curve revealed an ic50 of 119.9 4.1 mol / kg for sanr in inhibition of thermal hyperalgesia ( figure 4(d ) ) .
in contrast , i.p . vehicle did not alter the magnitude of ccd - induced mechanical hypersensitivity and thermal hyperalgesia ( figures 4(a ) and 4(c ) , n = 10 , p > 0.05 ) . in addition , motor coordination was not altered by systemic sanr administration ( 180 mol / kg , i.p . ) , as compared to vehicle group ( figures 4(e ) and 4(f ) , p > 0.05 , n = 6 ) .
we then further compared the analgesic potency of sanr with the same dosing regimen of traditional nitroxide compound tempol and classical nsaids aspirin alone . as shown in figure 5(a ) , sanr at dose of 180 mol / kg significantly inhibited ipsilateral mechanical hypersensitivity .
tempol and aspirin alone at this low concentration produced little effect on the mechanical hypersensitivity .
quantitative analysis at 7 h after drug delivery revealed that the analgesic potency by sanr was significantly higher than tempol and aspirin alone ( figure 5(b ) , p = 0.045 versus tempol , p = 0.006 versus aspirin ) . in parallel , sanr exerted stronger inhibition on ipsilateral thermal hyperalgesia when compared to tempol and aspirin alone ( figures 5(c ) and 5(d ) , n = 10 , p = 0.047 versus tempol , p = 0.025 versus aspirin ) .
in addition to prominent inhibition on ipsilateral pain hypersensitivity , acute sanr produced marked reduction of contralateral spread of mechanical hypersensitivity and thermal hyperalgesia as well ( figures 5(e ) and 5(g ) , n = 10 , p < 0.05 ) . similarly , sanr was found to be more efficacious on depression of both contralateral mechanical ( figure 5(f ) , p = 0.044 versus tempol , p = 0.032 versus aspirin ) and thermal hyperalgesia than tempol and aspirin alone ( figure 5(h ) , p = 0.018 versus tempol , p = 0.046 versus aspirin ) .
we further addressed whether repeated treatment of sanr could reverse the development of ccd - induced pain hypersensitivity . to do this , we administered sanr for 21 d once daily beginning at 3 d after drg compression .
as compared to vehicle group , repeated administration of sanr progressively reversed the development of ipsilateral mechanical hypersensitivity in ccd rats ( figure 6(a ) , n = 10 , p < 0.05 at all time points tested ) . in comparison with tempol and aspirin , sanr produced more pain relief on mechanical hyperalgesia ( figure 6(b ) , n = 10 for each drug ) .
quantitative analysis at 18 d after drug treatment showed that the extent of reversal of mechanical hyperalgesia by sanr was significantly stronger than that of tempol and aspirin ( figure 6(b ) , p = 0.043 versus tempol , p = 0.039 versus aspirin ) .
similarly , progressive and complete reversal of ipsilateral thermal hyperalgesia was seen after treatment with repeated sanr ( figure 6(c ) , n = 10 ) .
furthermore , sanr was much more efficacious than tempol and aspirin alone ( figure 6(d ) , p = 0.029 versus tempol , p = 0.018 versus aspirin ) . figures 6(e ) and 6(g ) show a notable depression of contralateral mechanical hypersensitivity ( figure 6(e ) , n = 10 ) and thermal hyperalgesia by sanr ( figure 6(g ) , n = 10 ) .
although tempol displayed a comparable effect on contralateral mechanical hypersensitivity with sanr when examined at 18 d after drug application ( figure 6(f ) , n = 10 , p = 0.059 ) , its inhibition on thermal hyperalgesia was still weaker than sanr ( figure 6(h ) , n = 10 , p = 0.036 ) .
aspirin was consistently weaker than sanr on attenuation of both contralateral mechanical hypersensitivity ( figure 6(f ) , n = 10 , p = 0.047 ) and thermal hyperalgesia ( figure 6(h ) , n = 10 , p = 0.049 ) .
collectively , these behavioral results uncovered a potential therapeutic value of this newly synthesized compound sanr on the development of hyperalgesia observed in radicular low back pain .
previous studies have shown that abnormal spontaneous discharges of primary afferent sensory neurons may contribute to the development of radicular low back pain [ 3 , 4 ] .
we are therefore interested to know whether sanr - induced analgesia in low back pain was mediated by its suppression on ectopic spontaneous discharges in compressed drg neurons .
single fiber recordings were employed in the dorsal roots of the injured l5 drg derived from 27 ccd rats . upon testing at 35 d after the onset of compression
, we observed that primary afferent a fibers frequently exhibited ectopic spontaneous discharges ( figure 7(a ) , right panel ) .
the conduction velocities were within the range of 2.526 m / s indicating that all the axons were myelinated .
this ectopic spontaneous discharge rarely happens in control rats ( figure 7(a ) , left panel ) .
quantitative analysis showed that the mean frequency of spontaneous firing reached 16.78 2.33 hz ( figure 7(b ) , n = 118 ) .
this spontaneous activity of all the tested units can last for 26 h ( 2.28 1.35 h ) . according to the dynamic features of interspike interval series
, three different firing patterns have been previously reported in the injured lumbar a - fiber drg neurons , which is periodic , nonperiodic ( irregular ) , and bursting activity . in our case , most patterns of spontaneous firing ( 59/118 ) observed fell into regular discharges ( figure 7(c ) , left panel ) .
37/118 were bursting activity ( figure 7(c ) , middle panel ) , and 22 out of 118 fibers displayed irregular firings ( figure 7(c ) , right panel ) . as compared to control
, superfusing sanr at a concentration of 10 mm reversibly inhibited the rate of spontaneous discharges of the primary afferent a fibers from ccd rats ( see figure 7(d ) left and middle panels for original traces and figure 7(d ) right panel for frequency histogram of typical examples ) . when examined in a concentration range of 1100 mm ,
the inhibitory rate of sanr was enhanced in magnitude with increasing concentrations , as seen in figure 7(e ) .
figure 7(f ) illustrates a dose - response curve for the suppressive effect of sanr on spontaneous activity ( figure 7(f ) , n = 7 ) .
the inhibitory rate averaged to be 19.4 5.5% , 31.2 11.6% , 43.7 15.8% , 80.6 5.4% , and 98.4 1.5% for sanr at 1 , 3 , 10 , 30 , and 100 mm , respectively .
analysis of the curve based on the hill plot yielded an ic50 of 18.8 mm for sanr ( figure 7(f ) ) .
these results indicate that sanr - induced analgesia may be at least , partially mediated by its inhibition of ectopic spontaneous activity of injured drg neurons .
we further compared the efficacy of sanr with the same concentration of tempol and aspirin alone on the ectopic spontaneous discharges in the injured drg neurons .
three compounds were tested in the same cell . to exclude the possible influence between each other ,
the drug was applied in a mixed order . as shown in figure 8(a ) , bath - applied aspirin at 10 mm significantly depressed the frequency of ectopic spontaneous discharges compared to control in a reversible manner ( figure 8(a ) , n = 4 , p = 0.014 ) .
a similar effect was obtained upon tempol ( 10 mm ) treatment ( figure 8(a ) , n = 4 , p = 0.019 ) . in striking contrast , administration of sanr ( 10 mm ) produced most potent inhibition of spontaneous activity ( figure 8(a ) , n = 4 , p = 0.013 ) .
quantitative analysis revealed that the averaged inhibition rate by sanr was significantly different from that by tempol and aspirin alone ( figure 8(b ) , n = 4 , p = 0.049 versus tempol , p = 0.047 versus aspirin ) .
oxidative stress and inflammation have long been assumed to be involved in the development and maintenance of chronic pain [ 1 , 9 , 10 ] .
various antioxidants , for example , nitroxides and nsaids , for example , aspirin , have been shown to provide partial relief in some types of chronic pain [ 9 , 1115 , 2225 ] .
however , chronic use of these drugs at high doses often causes severe unwanted side effects , which limits their broader implication . utilizing chemical synthesis ,
the present study for the first time synthesized a new nit nitroxide radical with salicylic acid framework ( sanr ) . in vivo and in vitro results revealed that this new compound produced a remarkable antinociceptive effect in ccd - induced low back pain , which is much more potent than traditional nitroxide compound tempol and classical nsaids aspirin .
these results indicate that nit nitroxide radical with salicylic acid framework might provide a new class of analgesic drug candidates .
a growing body of evidence has accumulated that stable nitroxide radicals have been studied as a unique and interesting class of antioxidants to protect again various oxidant stress including chronic pain [ 1219 , 21 ] .
although tempol has been shown to exert antinociceptive effect in inflammatory and neuropathic pain [ 1215 , 21 ] , whether nit radicals protect against pain has not been fully understood .
compared with other antioxidants , nit group nitroxides have incomparable advantages of scavenging radicals through a rapid catalytic manner .
more importantly , electrochemical properties with more extensive distribution of the unpaired spin density make nit group nitroxides more suitable for structure modification . for example , with the introduction of chirality into nit nitroxides , a series of chiral nit nitroxides were synthesized .
these new compounds have been shown to be able to protect against radiation and memory deficit .
these results inferred that modification of nit nitroxides may represent a direction for the development of new therapeutic drugs against neurological disorders .
however , whether nit nitroxides and its derivatives possess analgesic effect has remained elusive . in this study , we successfully synthesized a new nit nitroxide with salicylic acid framework with both antioxidative and anti - inflammatory action . in support of our assumption , tempo - aspirin and tempo - indomethacin
have been recently synthesized and are found to exert anti - inflammatory and superoxide dismutase scavenging properties in a459 cells .
one of the most striking findings of this study is that sanr eliminated pain hypersensitivity in low back pain produced in an experimental of ccd model .
consistent with previous reports , ccd rats display not only ipsilateral but also contralateral mechanical and thermal hyperalgesia , indicating the involvement of peripheral and central sensitization [ 5 , 35 , 36 ] .
acute treatment with systemic sanr produced dramatic analgesic effect on bilateral mechanical hypersensitivity and thermal hyperalgesia lasting beyond 24 h. following repeated administration of sanr , the development of bilateral pain hypersensitivity was progressively and completely reversed .
this indicates that sanr may play a role in both peripheral and central sensitization induced by chronic compression .
furthermore , we compared the antihyperalgesic potency exerted by sanr with the same concentration of traditional nitroxides tempol and classical nsaids aspirin alone .
it was found that both acute and repeated treatment with sanr exhibited much stronger efficacy than tempol and aspirin in expediting the recovery of low back pain .
this suggests that the addition of nit nitroxides moiety into nsaids salicylic acid framework may provide the additive analgesia via synergistic anti - inflammatory and antioxidative action .
ectopic spontaneous activity of injured drg neurons has been implicated as a key driver of neuropathic pain including radicular low back pain [ 3 , 4 , 37 ] .
previous reports together with our unpublished data showed that chronic neuropathic pain is often associated with spontaneous activity in a - type drg neurons , whereas chronic inflammatory pain mainly induces spontaneous discharges in c - type drg neurons [ 3 , 4 , 37 , 38 ] .
consistent with previous studies , we demonstrated that a - type drg neurons displayed frequent spontaneous firing following chronic drg compression [ 3 , 4 , 30 ] .
another striking finding of the present study is that bath applications of sanr remarkably depressed the spontaneous discharges of a - type drg neurons caused by drg compression in a dose - dependent manner .
although nsaids aspirin and nitroxides tempol have been reported to exert analgesic effect in some types of neuropathic pain [ 9 , 11 , 12 , 2225 ] , the cellular mechanisms underlying this analgesia in ccd - induced low back pain has not been studied in details .
this study showed that tempol and aspirin exhibited significant inhibition on the spontaneous firing in injured a - type drg neurons from ccd rats as well .
however , when evaluating the inhibitory rate of the same concentration of these three compounds , sanr provided the strongest analgesia , which is significant from tempol and aspirin . it can be inferred from the above that sanr - induced analgesia in low back pain in vivo may be mediated , at least in partial by the depression of ectopic spontaneous activity of injured drg neurons .
the primary mechanisms of sanr underlying the behavioral and neuronal antinociceptive actions in vivo and in vitro have remained unclear .
several mechanisms have been implicated in the antinociceptive actions of aspirin , including inhibition of cyclooxygenases [ 39 , 40 ] , nf-b pathway , and acid - sensing ion channels as well as activation of adenosine a2 receptors . under pathological pain states ,
ros has been involved in the activation of trp channels [ 44 , 45 ] , enhancement of nmda receptor phosphorylation , induction of ampa receptor trafficking to the membrane , and activation of map kinases ; delivery of antioxides could reverse the above changes . however , which downstream mechanisms could underlie the analgesic effect of sanr in low back pain needs to be further studied in the future . in conclusion , the present study firstly synthesized a novel compound sanr by introducing functional moiety of nsaids salicylic acid into nit nitroxides and demonstrated the dramatic analgesic effects of this newly synthesized compound in low back pain .
it was further revealed that this analgesia is at least in partial mediated by a reduction of ectopic spontaneous discharges by sanr in injured drg neurons .
salicylic acid framework may represent a novel potential therapeutic candidate for the treatment of chronic pain , including radicular low back pain . | evidence has accumulated that reactive oxygen species and inflammation play crucial roles in the development of chronic pain , including radicular low back pain .
nonsteroid anti - inflammatory drugs ( nsaids ) , for example , salicylic acid , aspirin , provided analgesic effects in various types of pain .
however , long - term use of these drugs causes unwanted side effects , which limits their implication . stable nitronyl ( nit ) nitroxide radicals have been extensively studied as a unique and interesting class of new antioxidants for protection against oxidative damage .
the present study synthesized a novel nit nitroxide radical with salicylic acid framework ( sanr ) to provide synergistic effect of both antioxidation and antiinflammation .
we demonstrated for the first time that both acute and repeated sanr treatment exerted dramatic analgesic effect in radicular low back pain mimicked by chronic compression of dorsal root ganglion in rats .
this analgesic potency was more potent than that produced by classical nsaids aspirin and traditional nitroxide radical tempol alone .
furthermore , sanr - induced behavioral analgesia is found to be mediated , at least in partial , by a reduction of ectopic spontaneous discharges in injured drg neurons .
therefore , the synthesized nit nitroxide radical coupling with salicylic acid framework may represent a novel potential therapeutic candidate for treatment of chronic pain , including radicular low back pain . |
the use of chemotherapeutics to treat malignancies is rooted in the use of mustard gas during the world wars .
sinking of the uss john harvey carrying a cargo of mustard bombs in the port city of bari , italy , in 1943 released the toxic payload and exposed survivors and residents to nitrogen mustard gas . within 34 days , many of those exposed had severe drops in their white blood cell counts.1 hypothesizing that the actions of the mustard gas on white blood cells could potentially be used to treat hematologic diseases , several clinicians studied the effectiveness of intravenously administered nitrogen mustards , tris(-chloroethyl)amine and methyl - bis(-chloroethyl)amine , and published landmark findings in 1946 that nitrogen mustards were effective in treating various forms of leukemia and lymphoma.2 their conclusion heralded in the era of cancer chemotherapeutic discovery :
chemicals discovered to be therapeutically active in neoplastic disease deserve close study [ ] . from this point of view
the heuristic aspects of the actions of the -chloroethylamines here reported may eventually prove of greater importance than the chemical results obtained to date.2 chemicals discovered to be therapeutically active in neoplastic disease deserve close study [ ] .
from this point of view the heuristic aspects of the actions of the -chloroethylamines here reported may eventually prove of greater importance than the chemical results obtained to date.2 cancer is traditionally thought of as unregulated cell replication , where tumor cells acquire mutations allowing them to bypass normal cell cycle checkpoints and apoptosis regulatory pathways .
nitrogen mustards work by alkylating dna , preventing cell division , and causing cell cycle checkpoint - mediated apoptosis .
unfortunately , this mechanism of action is not specific to malignant cells , resulting in chemotherapy - related toxicities to healthy tissue .
the nonspecific effects of most chemotherapeutic agents are the primary limiting factor in the treatment of malignancies .
methods to reduce these toxicities therefore allow for higher dosage regimens of chemotherapeutics and enhance clinical outcomes in many forms of cancer .
the blood - producing hematopoietic system is particularly susceptible to the toxicities of chemotherapeutics given the production demand of approximately 1 trillion blood cells daily.3 the most abundant white blood cells in the blood are neutrophils , which are essential members of the innate immune system and provide protection from a wide variety of bacterial and fungal pathogens . given the abundance of neutrophils and their short half - life in the blood of 68 hours , the production rate from hematopoietic progenitor cells in the bone marrow is 510 to 1010 neutrophils per day.4 the notable amount of cell divisions required to meet this high cellular demand makes the myeloid compartment of the bone marrow hematopoietic system particularly susceptible to chemotherapy .
neutropenia is the most serious hematologic toxicity of chemotherapy and often is the limiting factor in dose escalation or duration of chemotherapeutic administration.5 the national cancer institute s common toxicity criteria rates the severity of neutropenia into five classifications : grade 1 is < 2.0101.510 cells / l ; grade 2 is < 1.5101.010 cells / l ; grade 3 < 1.0100.510 cells / l ; grade 4 is < 0.510 cells / l ; and grade 5 is death.6 neutrophils are the primary cellular mediators of the innate immune system and the first line of defense to numerous pathogens .
neutropenic patients are susceptible to infections from the respiratory tract ( 35%40% of cases ) , bloodstream ( 15%35% ) , urinary tract ( 5%15% ) , skin ( 5%10% ) , gastrointestinal tract ( 5%10% ) , and other sites ( 5%10%).7 many of the symptoms manifested in response to bacterial infection are a consequence of the inflammatory immune response to the pathogen . in neutropenic patients , because they lack neutrophils that are mounting the normal response , they lack many of the normal symptoms of infection and hence it can go unnoticed .
oftentimes , the only clinical sign of infection early on is a fever , which defines a serious clinical outcome of chemotherapy known as febrile neutropenia , having grade 4 neutropenia with a fever greater than 38.3c .
more than 60,000 patients are hospitalized annually as a result of neutropenia , and these hospitalizations are estimated to have resulted in $ 13,400 in medical costs.8 the average costs for febrile neutropenia encounters were reported to be $ 22,086 , with patients who had febrile neutropenia incurring a mean cost difference of $ 1,149 per patient , per month.9 similarly , another study reported that mean hospitalization costs per patient were $ 18,042 for patients with neutropenia , $ 22,839 for patients with neutropenia plus infection or fever , and $ 27,587 for patients with neutropenia plus infection.10 therefore , boosting the bone marrow production of neutrophils is imperative to successful chemotherapy treatment and reduction of medical costs .
the landscape of febrile neutropenia was changed dramatically in the mid-1980s with the molecular cloning of granulocyte colony stimulating factor ( g - csf).1114 initially believed to be a multipotent hematopoietic growth factor as well an inducer of granulocyte differentiation , g - csf was found to stimulate proliferation15,16 and differentiation13 of several types of myeloid progenitor cells alone and in combination with other growth factors,1719 and this stimulation of progenitor cells leads to a significant increase in mature neutrophil output .
the first clinical trials were performed in cancer patients receiving chemotherapy2023 leading to us food and drug administration approval in 1991 of neupogen ( filgrastim ) .
filgrastim is approved to decrease the incidence of neutropenia in patients with nonmyeloid malignancies receiving chemotherapeutics associated with a significant incidence of severe neutropenia with fever ; to reduce incidence and duration of neutropenia in patients with congenital , cyclic , or idiopathic neutropenia ; for reducing the time to neutrophil recovery and the duration of fever , following induction or consolidation chemotherapy treatment of adults with acute myeloid leukemia ( aml ) ; and to reduce the duration of neutropenia and neutropenia - related clinical sequelae , eg , febrile neutropenia , in patients with nonmyeloid malignancies undergoing myeloablative chemotherapy followed by hematopoietic transplantation . while successful , the burden and associated pain of daily injections of filgrastim can result in patient discomfort and/or noncompliance , resulting in missed doses and reduced efficacy .
improved dosage forms that reduce the daily injection requirement can thus lead to enhanced patient care . a longer lasting monomethoxy polyethylene glycol ( peg)conjugated filgrastim ( pegfilgrastim ; neulasta ) having the same mechanism of action24 and clinical effects compared to filgrastim25
was subsequently introduced , but requires only single administration per cycle of chemotherapy rather than multiple daily injections of filgrastim.26 since the introduction of filgrastim and pegfilgrastim to clinical practice , the management of neutropenia has been enhanced , and several biosimilars have recently entered the marketplace and others are in development ( reviewed in hoggatt and pelus27 ) . to date , only a single long - acting biosimilar filgrastim has been introduced . this review will focus on lipegfilgrastim ( lonquex ) approved by the european medicines agency ( ema ) for the treatment of neutropenia .
lipegfilgrastim is a long - acting , site - specific glycol - pegylated r - methu g - csf produced by conjugation of a single 20-kda peg to the natural o - glycosylation site of g - csf ( threonine 134 ) , using a novel glycopegylation technology ( figure 1 ) .
because the recombinant g - csf is produced in escherichia coli , the glycosylation site is empty .
addition of the o - glycan was achieved by enzymatic activity of a truncated n - acetylgalactosaminyltransferase isoform 2 fused with maltose - binding protein at the threonine residue site .
a 20-kda peg - sialic acid derivative was enzymatically transferred to the o - glycan with a sialyltransferase .
in contrast , pegfilgrastim ( neulasta ) is a recombinant methionyl human g - csf with a methoxy - polyethylene glycol propionaldehyde 20-kda peg covalently conjugated to its n - terminus.28,29 the novel pegylation process used in lipegfilgrastim results in different pharmacokinetic and pharmacodynamic profiles than pegfilgrastim , as discussed later . in preclinical studies , lipegfilgrastim and pegfilgrastim binding to the g - csf receptor was evaluated using an nfs-60 cell - based [ i]-g - csf competitive g - csf receptor - binding assay . in the cell - based [ i]-g - csf competitive g - csf receptor - binding assay ,
g - csf receptor binding was equivalent between lipegfilgrastim and pegfilgrastim as indicated by the inhibition of [ i]-g - csf binding ( 0.700.09 nm ic50 versus 0.720.18 nm ic50 [ mean standard deviation]).28
lipegfilgrastim was evaluated in three separate healthy volunteer studies.30,31 in a limited healthy volunteer study , three separate subcutaneous injection sites were evaluated ; upper arm , abdomen , and the thigh .
bioavailability was found to be lower after injection in the thigh compared to the upper arm or abdomen .
however , increases in neutrophil counts were similar across the three injection sites and the recommended administration of lipegfilgrastim is for any of these three sites.30 in additional clinical studies , lipegfilgrastim was administered as a single subcutaneous injection and in some analyses compared to pegfilgrastim . in the dose escalation phase , administration of lipegfilgrastim was not continued beyond 100 g / kg as two subjects experienced ancmax ( maximum absolute neutrophil count ) levels greater than 70 neutrophils / nl , the limit for hyperleukocytosis in healthy subjects.31 the maximal blood concentration was reached after 3036 hours postadministration .
the terminal half - life ranged from approximately 32 to 62 hours , which was 710 hours longer for lipegfilgrastim 100 g / kg compared with pegfilgrastim 100 g / kg .
lipegfilgrastim is mainly internalized and eliminated by neutrophils and can be metabolized via intra- or extracellular degradation by proteolytic enzymes , which becomes saturated at higher doses . in vitro data
suggest there is little to no effects on cyp1a2 , cyp2b6 , cyp2c8 , cyp2c9 , cyp2c19 , and cyp3a4/5 activity , suggesting lipegfilgrastim does not affect human cytochrome p450 metabolism.30
in conventional safety and pharmacology studies in animal models , no special hazard for humans was found . in all healthy volunteer studies , the overall safety and tolerability was good , and no serious adverse events were reported.31 while there have been reports of pegylation - inducing anti - peg antibodies in patients,32,33 no subject tested positive for antibodies against filgrastim , pegfilgrastim , or lipegfilgrastim demonstrating no significant immunogenicity of the compound . in phase iii trials in breast cancer patients ,
the safety of lipegfilgrastim was similar to pegfilgrastim.34,35 the most commonly reported adverse events in both treatment groups were alopecia , nausea , asthenia , bone pain , diarrhea , fatigue , anorexia , vomiting , headache , and myalgia .
the first trial was a randomized , double - blind phase ii study , conducted between june and november of 2008.35 this study included screening of 229 patients and ultimately enrollment of 208 patients at 37 centers in the czech republic , germany , hungary , romania , russia , and ukraine .
a total of 202 patients ( 97.1% ) completed the study , with three patients withdrawing due to adverse events , two withdrew consent , and one was lost to follow - up .
male and female patients ( only three male patients were in the study ) 18 years of age with high - risk stage ii , iii , or iv breast cancer were eligible if they had an eastern cooperative oncology group performance status 2 ; absolute neutrophil count 1.510/l ; platelet count 10010/l ; and adequate cardiac , hepatic , and renal function .
patients received four 3-week chemotherapy cycles and were randomized 1:1:1:1 to receive 3.0- , 4.5- , or 6.0-mg lipegfilgrastim or 6.0-mg pegfilgrastim subcutaneously on chemotherapy day 2 .
chemotherapy consisted of doxorubicin 60 mg / m given as an intravenous bolus injection followed 1 hour later by a 1-hour intravenous infusion of docetaxel 75 mg / m .
no significant difference in the duration of severe neutropenia for the first chemotherapy cycle among the three lipegfilgrastim doses and pegfilgrastim was observed . the percentage of patients who did not experience severe neutropenia was higher in the 6.0-mg lipegfilgrastim dosage group ( 62.0% ) compared with the 3.0- and 4.5-mg dosage groups ( 43.4% and 49.0% , respectively ) .
this percentage of patients was also higher than the 6.0-mg pegfilgrastim dosage group ( 46.3% ) . for chemotherapy cycles 24 ,
the mean duration of severe neutropenia was significantly shorter for the 6.0-mg lipegfilgrastim dosage group versus the pegfilgrastim dosage group .
this study established 6.0-mg lipegfilgrastim as the optimal dose for breast cancer patients receiving chemotherapy and established noninferiority to pegfilgrastim in reducing cycle 1 duration of severe neutropenia .
following this study , a similar experimental approach was taken in a randomized , multicenter phase iii study.34 breast cancer patients receiving chemotherapy were randomized 1:1 to receive either the 6.0-mg lipegfilgrastim dose ( n=101 ) or 6.0-mg pegfilgrastim dose ( n=101 ) .
lipegfilgrastim was found to be noninferior to pegfilgrastim in the duration of severe neutropenia following cycle 1 of chemotherapy , with a 95% confidence interval of 0.498% , 0.062% days ( p=0.1260 ) .
similarly , there was no significant difference in the duration of severe neutropenia between the two groups following cycles 24 .
intriguingly , the absolute neutrophil count nadir after cycles 24 was higher in the lipegfilgrastim group compared to the pegfilgrastim group ( 2.6 vs 2.0 , 2.5 vs 2.0 , and 2.7 vs 2.310/l ; p=0.0189 , p=0.0353 , and p=0.1122 , respectively ) . similarly , the time to neutrophil recovery was ~1.5 days shorter for the lipegfilgrastim group compared to the pegfilgrastim group ( p<0.05 )
. the results of this study further demonstrated noninferiority of 6.0-mg lipegfilgrastim compared to 6.0-mg pegfilgrastim and suggest a possible enhancement over pegfilgrastim in neutrophil recovery following chemotherapy .
recently , the results of a double - blind , randomized , placebo - controlled study in elderly patients with non small - cell lung cancer receiving cisplatin / etoposide chemotherapy were reported at the 12th meeting of the international society of geriatric oncology.36 patients received 6.0-mg lipegfilgrastim or placebo on day 4 of the chemotherapy cycle , and the incidence of febrile neutropenia was assessed .
fewer elderly patients receiving lipegfilgrastim ( 0/53 ; 0.00% ) experienced febrile neutropenia compared to the placebo group ( 4/30 ; 13.33% ) ( p=0.0064 ) .
there was no significant difference in incidence severity or type of adverse events between the treatment and placebo groups .
lipegfilgrastim , in the setting of breast cancer chemotherapy induced neutropenia is at least as equivalent to pegfilgrastim in reducing neutropenic complications .
lipegfilgrastim , lonquex , represents the first long - acting biosimilar filgrastim to reach the market in germany and has received ema approval and will likely be marketed throughout europe .
as the neupogen and neulasta patent lives have ended , and given the large economic market of g - csf therapies in reducing the complications associated with chemotherapy , there has been a large effort in biosimilar development . to date
, several filgrastim biosimilars have received us food and drug administration and/or ema approval and have entered the marketplace ( recently reviewed in hoggatt and pelus27 ) .
while lipegfilgrastim is the first long - acting biosimilar filgrastim to reach the market , additional long - acting biosimilars , some with unique modifications to increase half - life , are currently in late - stage clinical development ( reviewed in hoggatt and pelus27 ) .
to date , no long - acting biosimilar filgrastim has been approved by the us food and drug administration .
additionally , dosage forms that eliminate the need for injection altogether , such as the topical filgrastim product nupen , may enhance patient compliance and resulting care in some clinical applications , although further clinical studies are required .
overall , the use of biosimilar filgrastim and pegfilgrastim products will be dictated by market prices and individual center preferences until further enhancements in efficacy or ease of administration , eg , oral , are identified and developed . in the shorter term , it is expected that biosimilar g - csf agonists may reduce the significant medical costs of neutropenia treatment and facilitate market expansion . | chemotherapy , irradiation , and other agents are widely used to target the process of cell division in neoplastic cells . however , while these therapies are effective against most cancers , the high proliferative rate of the cells of the hematopoietic system that produce billions of blood cells needed daily throughout life is extremely sensitive to these agents , resulting in loss of blood cell populations , which can be life threatening .
neutropenia is the most serious hematologic toxicity of chemotherapy , which can result in patient morbidity and mortality due to opportunistic infection and often is the limiting factor in dose escalation or duration of chemotherapeutic administration .
neutropenic patients often require hospitalization and incur substantial medical costs associated with anti - infective therapy .
treatment of iatrogenic and congenic neutropenia was changed in the early 1990s with the introduction of filgrastim ( neupogen ) and pegfilgrastim ( neulasta ) . with the expiration of patent lives of both of these drugs ,
biosimilars have begun to emerge . in this review , we will summarize the chemical characteristics , pharmacokinetics , safety and efficacy of lipegfilgrastim ( lonquex ) , the first long - acting biosimilar filgrastim to receive regulatory approval and enter the marketplace . |
depressive disorders are characterized by sustained and persistent low mood with loss of interest , easy fatigability , disturbed sleep , decreased concentration and suicidal thoughts .
anxiety disorders have sustained feeling of impending threat with disturbed autonomic system ( ans ) functions . according to national comorbidity survey replication ( ncs - r ) done in the u.s . , the life time prevalence of major depressive disorders is 16.6% and of anxiety disorders is 28.8% , respectively ( 1,2 ) .
panic disorder , post traumatic disorder ( ptsd ) , generalized anxiety disorder ( gad ) , obsessive compulsive disorder ( ocd ) and phobic anxiety disorders are the various subtypes of anxiety disorders ( 1 ) . a variety of genetic , biological , psychological and social factors are postulated to be the causal factors , leading to emergence of anxiety and depressive disorders ( 1 ) .
the human 5- hydroxy tryptamine transporter ( 5 htt ) gene also called slc6a4 has been cloned and mapped on to chromosome 17q11.1-q12 ( 3 ) .
a polymorphism has also been identified in the promoter region of slc6a4 , called 5ht transporter linked promoter region ( 5httlpr ) polymorphism .
5httlpr consists of different lengths of repetitive sequences which contains gc - rich 20 - 23 base pairs long repeat elements .
a deletion / insertion in the promoter region creates a short ( s ) allele and a long ( l ) allele ( 14 and 16 repeat alleles ) , which alters the promoter activity ( 1,4,5 ) .
the 5httlpr polymorphism has been studied in both depressive and anxiety disorders ( 5 ) .
individuals carrying 1 or 2 copies of the s allele were noted to exhibit elevated neuroticism , a personality trait involved in the propensity to depression ( 6 ) .
further , it was reported that s carriers exhibited elevated depressive symptoms , depressive disorders and suicidality after experiencing stressful life events and childhood maltreatment ( 7 ) .
short allele of serotonin transporter gene has been found to be associated with the risk of anxiety disorders in one race ( caucasians ) ; however in another race ( asians ) , long allele was found to be associated with the risk of anxiety disorders ( 8) . killpatrick et al .
( 9 ) found that s - genotype was associated with increased risk of post- traumatic stress disorder , a type of anxiety disorder , but only under high environmental stress .
( 3 ) in a study of 57 subjects having major depression found that l - type homozygotes responded much better to antidepressant treatment .
there are studies showing genetic variation in depressive disorders related to 5htlpr gene responding to antidepressant treatment . in studies conducted on mongoloid races
it has been reported that better response to treatment occurs in those persons with s - allele ( 11 ) .
l - homozygotes are reported to be more in caucasian individuals while s genotypes are more frequent in mongoloid individuals .
the contradictory results of 5httlpr polymorphism studies among different ethnic groups in both depressive and anxiety disorders and their treatment responses appear to be intriguing .
it has been observed that allele frequency in the indian population differed from other populations ( 10 - 12 ) .
this perhaps can be understood from the fact that india has been a melting pot for millennia : caucasoid , mongoloids , australoid races intermingled and intermarried and inhabited the land for eons ( 13 ) . in view of this , a study was conducted to make a modest effort to study 5htt polymorphisms in anxiety and depressive disorders of kashmiri ( indian ) population .
this study was conducted in the post graduate department of psychiatry , government medical college srinagar , india .
thirty unrelated persons suffering from unipolar major depression and anxiety disorders were selected for this study .
diagnoses were confirmed by two experienced psychiatrists independently ; 40 unrelated healthy volunteers ( controls ) belonging to the same state were selected after excluding mental disorders by comprehensive clinical interview by two experienced psychiatrists .
the inclusion criteria included persons suffering from depressive disorders and anxiety disorders , conforming to dsm iv tr criteria , all patients above 18 years of age and patients willing to participate in the study by means of informed consent .
the exclusion criteria included persons below 18 years of age , all those with depressive disorders and anxiety disorders due to general medical conditions and psycho - active substances use and patients not willing to participate .
an observer rating scale such as ham a and ham - d was used to assess the severity of anxiety and depression in these disorders .
for genotyping gene ,
dna was extracted from a portion of whole blood using genei genomic extraction kit supplied by the messers bangalore genei , india .
polymerase chain reaction was carried out with primers 5 ggcgttgccgctctcaatg c-3 ( stpi-5 - 2 ) and 5 gagggactgag ctggacaacccac 3 ( stpi3 - 2 ) .
pcr was performed in 20 ul mixture containing about 50ng genome dna , 10pmol of each primer , tris - hcl , ph 8 , 100um dntp s , 1u of taq polymerase using tech - gene thermal cycle ( uk ) .
the amplification was carried out after an initial incubation at 95 degree celsius for ten minutes , 30 cycles of a denaturation at 94 degree celsius for one minute and an annealing step at 65 degree celsius for one minute followed by an extension step at 72 degree celsius for one minute , and final incubation at 72 degree celsius was done for ten minutes .
after a 1.5 agarose gel electrophoresis , the pcr products were stained with ethidium bromide and then bands were observed under a uv light .
allelic size was determined by the comparison of bands with size standards after electrophoresis in polyacramide gel , using psi x 174 hae iii endonuclease enzyme followed by silver staining , and 3 genotypes i.e. , ll , ls , ss were observed ( fig .
short allele(s ) consisted of 448 base pair and long allele ( l ) of 528 base pair .
electrophoresis of 5 httlppr gene using polyacramide gel and endonuclease enzyme
hardy - weinberg equation was applied to the genotypes of the patients and controls .
all statistical analyses were performed using spss software version 16.0 for windows , and the differences were taken as significant when p - value was less than 0.05 .
this study was conducted in the post graduate department of psychiatry , government medical college srinagar , india .
thirty unrelated persons suffering from unipolar major depression and anxiety disorders were selected for this study .
diagnoses were confirmed by two experienced psychiatrists independently ; 40 unrelated healthy volunteers ( controls ) belonging to the same state were selected after excluding mental disorders by comprehensive clinical interview by two experienced psychiatrists .
the inclusion criteria included persons suffering from depressive disorders and anxiety disorders , conforming to dsm iv tr criteria , all patients above 18 years of age and patients willing to participate in the study by means of informed consent .
the exclusion criteria included persons below 18 years of age , all those with depressive disorders and anxiety disorders due to general medical conditions and psycho - active substances use and patients not willing to participate .
an observer rating scale such as ham a and ham - d was used to assess the severity of anxiety and depression in these disorders .
for genotyping gene ,
dna was extracted from a portion of whole blood using genei genomic extraction kit supplied by the messers bangalore genei , india .
polymerase chain reaction was carried out with primers 5 ggcgttgccgctctcaatg c-3 ( stpi-5 - 2 ) and 5 gagggactgag ctggacaacccac 3 ( stpi3 - 2 ) .
pcr was performed in 20 ul mixture containing about 50ng genome dna , 10pmol of each primer , tris - hcl , ph 8 , 100um dntp s , 1u of taq polymerase using tech - gene thermal cycle ( uk ) .
the amplification was carried out after an initial incubation at 95 degree celsius for ten minutes , 30 cycles of a denaturation at 94 degree celsius for one minute and an annealing step at 65 degree celsius for one minute followed by an extension step at 72 degree celsius for one minute , and final incubation at 72 degree celsius was done for ten minutes .
after a 1.5 agarose gel electrophoresis , the pcr products were stained with ethidium bromide and then bands were observed under a uv light .
allelic size was determined by the comparison of bands with size standards after electrophoresis in polyacramide gel , using psi x 174 hae iii endonuclease enzyme followed by silver staining , and 3 genotypes i.e. , ll , ls , ss were observed ( fig .
short allele(s ) consisted of 448 base pair and long allele ( l ) of 528 base pair .
hardy - weinberg equation was applied to the genotypes of the patients and controls .
all statistical analyses were performed using spss software version 16.0 for windows , and the differences were taken as significant when p - value was less than 0.05 .
the mean ( sd ) age of the control , anxiety and depressive groups were found to be 29.75 10.94 , 32.7 10.99 and 32.5 9.93 , respectively .
the differences were not statistically significant ( p > 0.05 ) ( table 1 ) .
males constituted 65% ( n= 26 ) of the controls while they formed 57% ( n= 17 ) and 50% ( n= 15 ) of the depressive and anxiety groups of patients .
gender differences were not statistically significant ( p>0.05 ) ( table 2 ) . in the depressed group , 53% ( n = 16 ) had severe depression .
panic disorder patients constituted the majority in the anxiety group 56.67% ( n= 17 ) .
patients with generalized anxiety disorder formed 27% of the group ( table 3 ) .
the mean ( sd ) ham - a scores for the anxiety and depressive group were 28.2 5.14 and 16.7 5.61 , respectively .
difference in the mean scores was found to be statistically significant ( p < 0.001 ) .
however , 12 out of the 30 ( 40% ) depressed patients had ham - a scores of mild to moderate anxiety ( 18 to 30 ) ( table 4 ) .
mean ( sd ) depression scores for the depressed and anxiety group of patients were 25.15 5.58 and 15 6.13 , respectively .
however , 16 out of the 30 patients with anxiety ( 53% ) had moderate to severe depression .
an additional 12 patients ( 40% ) had depression in the mild range of 8 to 17 , showing considerable overlap of symptoms between the two groups ( table 4 ) .
5httlpr genotype and allele frequencies were not distributed uniformly between the control and patient groups ( p<0.001 ) ; and this may be due to the significant differences in the genotype frequencies between the anxiety group and controls ( p < 0.001 ) .
the frequency of s allele was significantly high ( 83.3% vs. 60% ) in the group with anxiety when compared to controls ( p < 0.05 ) .
no significant differences were observed in the genotype or allele frequencies between controls and depressed group ( p > 0.05 ) ( table 5 ) .
superscript a denotes comparison of control vs. depression , superscript b denotes comparison of control vs. anxiety
significance at p < 0.05
e ham - a scores in persons carrying ss and ls / ll genotypes of 5httlpr were compared within the anxiety group , but no significant differences were found in ham - a scores between ss and ls / ll genotypes ( p > 0.05 ) .
ham - d scores of ss genotype persons were not significantly different from that of ls / ll genotypes ( table 6 ) .
ss : mean 28.6 , ls / ll : mean 27.62 , ss : mean 28.4 , ls / ll : mean 24.5
* t : 0.48 , df : 28 , p > 0.05 , * * t : 1.62 , df : 28 , p > 0.05
* ham d scores on the depressed group , * * ham a scores on the anxiety group
scores of 30 and above on ham - a is considered as severe anxiety .
there was more number of severe anxiety cases in ss genotype than in ls / ll genotypes ( 13 0ut of 14 versus 9 out of 16 ) .
5httlpr genotypes were not related to the severity of anxiety ( p= 0.107 ) ( table 7 ) .
@ ham - a score of 30 and above is taken as severe anxiety , 5httlpr fisher s exact test : p= 0.0205 *
5httlpr polymorphism is the most widely studied genetic variant in psychiatry ( 14 ) .
a few studies have been carried out on 5httlpr polymorphisms in india ( 14 ) .
the present study is a modest effort at ascertaining the role of polymorphisms in genes of 5ht transporter ( 5httlpr ) in anxiety and depressive disorders of kashmiri ( indian ) population .
though there was no deliberate effort to match the control and patient groups for age , both groups were found to be matched for age . excluding social anxiety disorder ,
depression is also known to be more common in women ( 1 ) . in the present study , gender distribution in anxiety and depressive groups of patients
panic disorder and severe depression appeared to be disproportionally represented in the sample , but perhaps could be understood from the fact that this was a hospital based study .
the mean anxiety score in the anxiety and depression groups was 28.2 and 19.47 , respectively .
a score of 18 and above was considered as mild anxiety ( ham - a ) . though the difference was highly significant
, the fact that a substantial number ( 40% ) of depressive patients have at least mild levels of anxiety lends credibility to the contention that depression and anxiety are two facets of the same disorder ( 15,16 ) .
this argument can be further advanced by the fact that 53% of the anxiety patients in this study had moderate to severe levels of depression and an additional 40% had mild levels of depression .
it has been observed that ethnicity plays an important role in gene related studies and has vital clinical applications .
the risk for anxiety and depressive disorders is linked with short allele of serotonin - transporter gene in the caucasian population ; however , the association is still unclear in the asian population . in a study done by long h et al . in 2013
it was found that in the chinese han population , in contrast to the caucasian population , the l - allele has susceptibility to anxiety or depression disorders ( 8) .
most of the studies reported that polymorphisms in the population of controls and patients were in hardy- weinberg equilibrium ( 14,17 ) . in the present study , controls and patients were in hardy weinberg equilibrium . widely varying 5httlpr genotype frequencies
have been reported by various workers in their control populations ( 18 - 20 ) .
mukherjee et al . commented on the different genotype frequencies in the psychiatric patients of our country ( 12 ) .
the ll , ls and ss genotypes in the controls of the present study were 12.5%,55% and 32.5% while billiviers et al . ( 18 ) reported 32.6% , 52.7% and14.7% ; lee et al .
genotype frequencies of 33.3% , 43.3% and 23.3% in the mood disorder patients of the present study corresponds to those of margoob et al . ( 14 ) .
the genotype frequencies of ll 6.6% , ls 20% and ss 73.3 % in the anxiety group of the present study correspond to those of lee et al .
the ss genotype was significantly high in the anxiety group , and the frequency of s allele was also high in the anxiety group .
the finding of this study is similar to that of other studies ( 7,19,21,22 ) .
there are many studies showing an association of the ss genotype and s allele with depression ( 19,20,22 ) .
no association of the genotypes or allele frequencies with depression was found in the present study .
risch et al . also reported no association of 5httlpr with depression ( 23 ) ; margoob et al . in their review pointed to several studies which reported no association of depression with 5httlpr polymorphism ( 14 ) .
few researchers believe that this disparity in the results is due to the fact that ethnic variation may play a part in it ( 24,25 ) .
the findings of this study which showed 5httlpr association with anxiety and not with depression is intriguing .
as it has been observed , 93% of the patients in the anxiety group had a mild to moderate depression on the one hand and 40% of the depressive patients had significant anxiety on the other hand .
the finding of this study seems to suggest that the biological underpinnings of the two conditions are different .
some studies have suggested that female gender plays a role in the effect of polymorphisms over the emergence or exacerbation of phenotype anxiety and depression ( 17 , 26 - 29 ) . in the present study
, there were no statistically significant differences in genotype frequencies , ham - d scores and ham - a scores between the male and female population .
in the preliminary analysis , ss genotype was found to be significantly high in patients with anxiety .
further comparison of anxiety scores between the 5httlpr genotypes revealed no statistically significant differences in the anxiety scores between ss genotype and ll / ls genotypes .
this finding is rather unexplainable , but one outlier score 13 in ss genotype patients ( s.no .
10 of table 6 ) and outlier 34 in ll / ls genotypes may have something to do with it .
the intriguing finding of this study was that ss genotype of 5httlpr was associated with anxiety disorders and not with their severity ( table 6 ) .
further analysis was made considering the ham - a cutoff score of 30 and above as indicative of severe anxiety . in this study , 13 out of 14 ( 92.8% ) of the severely anxious patients carried the ss genotype whereas only 56% of the patients with mild / moderate anxiety carried ss genotype .
the sample size of this study was too small for an association study and therefore the findings can only be generalized with caution . | background : 5httlpr polymorphism ( 5- hydroxy tryptamine transporter linked promoter region polymorphism )
is the most widely studied genetic variant in psychiatry .
the present study is a modest effort at ascertaining
the role of 5ht transporter linked promoter region polymorphism ( 5httlpr ) in anxiety and depressive
disorders in kashmir ( india).the aim of this study was to examine 5-hydroxy tryptamine transporter ( 5htt )
gene promoter region polymorphism in anxiety and depressive disorders .
methods : thirty patients with unipolar depressive disorders , 30 patients with anxiety disorders and 40 healthy
volunteers ( controls ) were studied on a case control design , using polymerase chain reaction ( pcr ) and agarose
gel electrophoresis after digestion with endonuclease enzyme .
genotypes and allele frequencies were compared
using chi square tests , and p value of < 0.05 was considered as statistically significant .
results : the mean ( sd ) ham - a ( hamilton rating scale for anxiety ) scores for anxiety and depressive groups
were 28.25.14 and 175.61 , respectively ( p < 0.001 ) .
the mean ( sd ) ham - d ( hamilton rating scale for depression )
scores for depressive and anxiety groups were 255.58 and 156.13 , respectively .
( p < 0.001 ) .
the
frequency of s allele was significantly high ( 83.3% vs 60% ) in the group with anxiety ( p < 0.05 ) compared to
the control group ( p > 0.05 ) .
conclusion : the genetic studies are still evolving as pathogenesis of anxiety and depressive disorders and involve
the interaction of environmental factors with various genes .
genetic variation in different populations and
hence different environments is important for elucidation of the mechanisms of these disorders .
however , the
study concludes that the locus under study is not shared between the two disorders . |
hypoxic - ischemic encephalopathy ( hie ) is a serious brain injury due to birth asphyxia [ 1 , 2 ] .
hie has a variety of perinatal causes of interrupted uterine and fetal blood flow and/or hypoxia , such as uterine rupture , placental abruption , and cord prolapse .
however , most often , the exact timing and underlying cause remain unknown [ 35 ] .
its severity is also variable . despite major advances in monitoring technology and knowledge of fetal and neonatal pathophysiology ,
birth asphyxia remains a serious condition that causes significant mortality and long - term morbidity .
causing 23% of all neonatal deaths worldwide [ 6 , 7 ] , it is one of the top 20 leading causes of burden of disease in all age groups ( in terms of disability life adjusted years ) by the world health organization .
most deaths occur in the first week of life due to multiple organ failure or withdrawal of care due to the prospect of severe neurological impairments .
moreover , children who survive birth asphyxia develop problems such as cerebral palsy , mental retardation , learning difficulties , and other disabilities .
in the past several years , studies have been carried out to address the safety and efficacy of cooling in hie [ 1018 ] .
unfortunately , this neuroprotective strategy seems efficient in preventing brain injury in some asphyxiated newborns , but not in all of them .
thus , there is an increasing interest to rapidly understand the degree of brain injury and to possibly refine individual neuroprotective strategies or add neurorestorative strategies [ 1923 ] . in this paper ,
the physiopathology behind hie , the current available treatment , the potential alternatives , and the next steps before implementation of these other treatments are reviewed .
neonatal brain hypoxic - ischemic injury is an evolving process initiated by the hypoxic - ischemic insult leading to decreased blood flow to the brain ( primary lesions ) , followed by the restoration of blood flow in an injured brain and the initiation of a cascade of pathways ( secondary lesions , reperfusion injury ) .
this cascade includes accumulation of extracellular glutamate with excessive activation of glutamate receptors , calcium influx , and generation of reactive oxygen and nitrogen species , leading to cell death and definitive brain injuries .
this cascade is a primary target for neuroprotective interventions , but such interventions are currently limited by insufficient knowledge of the timing and duration of the so - called therapeutic window in newborns [ 19 , 21 , 22 ] .
as the only neuroprotective treatment to have been clinically tested in large randomized controlled trials to minimize brain injury in asphyxiated term newborns [ 10 , 11 , 13 , 14 , 17 , 18 ] , cooling is thought to be protective in newborns ( table 1 ) mainly by reducing brain perfusion and metabolism , as well as mitigating reperfusion injury , reducing excessive free radical production , depressing the immune response and various potentially harmful proinflammatory reactions , and suppress epileptic activity .
some results also suggest that moderate hypothermia can prevent or delay apoptosis and might be able to extend the therapeutic window for other interventions .
results of these clinical trials in newborns have shown that moderate hypothermia after birth asphyxia reduces the risk of death and disability at 18 months , with a number needed to treat of nine asphyxiated newborns to prevent brain injury in one of them [ 12 , 15 , 16 ] . there was no real difference in any of the outcomes between whole - body cooling versus selective head cooling [ 12 , 15 , 16 ] .
only little adverse affects have been observed with this therapy ; the main ones were arrhythmia and thrombocytopenia [ 12 , 15 , 16 ] . however , it remains unclear why induced hypothermia seems effective in preventing brain injury and improving neurological outcome in some asphyxiated newborns ( mainly those with moderate hie ) , but not in others ( mainly those with severe hie ) [ 11 , 14 ] .
a better understanding of why brain injury continues to develop despite hypothermia treatment in some asphyxiated newborns is required in order to create and test some more specifically adjunctive therapies .
furthermore , early identification of patients who will develop further brain injury despite this treatment would be of utmost importance in order to determine those who would benefit most from adjustments in their hypothermia therapy or from adjunctive neuroprotective or neurorestorative therapies [ 20 , 23 ] . according to the trials described above , mild hypothermia to an esophageal temperature of 33.5c
must be started in an asphyxiated newborn within 6 hours of life and continued for 72 hours .
this temperature and this timing are based on animal data ; for example the 6-hour limit for cooling initiation comes from data suggesting that the effectiveness of cooling diminishes as time increases from the hypoxic - ischemic event to initiation of cooling , with the closing of the window at a time between 5.5 and 8 hours after the event .
some authors are thus starting to suggest that there might be a benefit from later initiation of cooling and increased depth or duration of cooling , as well as prolonged rewarming strategies .
additional randomized controlled trials are required to test if adjustments of current guidelines of moderate hypothermia for asphyxiated newborns might be useful to prevent even more efficiently brain injury in these patients .
such trials are currently ongoing ( e.g. , late hypothermia for hypoxic - ischemic encephalopathy ,
one of the main challenges of the current guidelines for cooling newborns with hie is to initiate the hypothermia within the first 6 hours of birth . in many places around the world
, newborns may be born at great distances from neonatal intensive care units offering cooling , preventing them from arriving there within the good timing .
specific protocols have to be developed in collaboration with the cooling centers of reference in order to start cooling safely and reliably prior to the transport at the birthing center and/or during transport , so that these patients can also benefit from this treatment [ 16 , 19 , 28 ] .
in particular , continuous measurement of central temperature ( esophageal or rectal ) should be carefully monitored , in order to target the recommended temperature [ 16 , 19 , 28 ] .
studies of alternative treatments for neonatal hie are becoming available , with the goal of further increasing the proven benefits of hypothermia , that is , to decrease eventual neonatal brain injury associated with neonatal asphyxia and thus improve future neurodevelopmental outcome .
these alternatives include specific brain - oriented therapies , which are targeted to the critical events contributing to tissue damage in neonatal hie ( table 1 ) .
they usually involve molecules that cross the blood - brain barrier and target mechanisms of injury , which are not ( or are not completely ) inhibited by hypothermia .
these include anticonvulsant or antiexcitatory drugs ( e.g. , phenobarbital , topiramate , bumetanide , magnesium sulphate , and xenon ) , antiapoptotic drugs ( e.g. , erythropoietin ) , anticalcium drugs ( e.g. , xenon ) , or anti - inflammatory or antioxidative drugs ( e.g. , melatonin and n - acetylcysteine ) [ 3033 ] .
the goal of these experimental therapies is to produce a synergistic neuroprotective effect with hypothermia [ 3033 ] .
most research efforts in neonatal neurology are currently targeted to neuroprotective strategies , but another concept is slowly emerging from the adult stroke literature . in adults , neurorestorative therapies ( table 1 )
are currently being developed to treat ischemic brain injury , including cerebral vasculature - based therapy combined with neuroprotection , as the more conventional strategies ( antiapoptosis , anticalcium , anti - inflammation , and antioxidative injury ) have been disappointingly ineffective in adult clinical trials .
these neurorestorative therapies target the neurovascular unit , composed of functionally integrated cellular ( including brain endothelial cells , astrocytes , pericytes and smooth muscle cells , neural stem cells , oligodendrocytes , and neurons ) and acellular elements that form the basement membrane .
thus , they lead to enhancement of restorative events , such as endogenous neurogenesis , angiogenesis , axonal sprouting , and synaptogenesis in the ischemic brain , explaining the potential for stimulation of brain plasticity and improvement in functional recovery [ 36 , 37 ] . in adult models of stroke , these therapies have been shown to activate these processes and successfully reduce the size of the infarcted areas [ 38 , 39 ] and are now being actively tested in human patients . the immature brain of a newborn is not simply a small adult brain , and the mechanisms underlying neonatal hie is certainly very unique compared to the ones underlying adult stroke .
however , evidence is growing in the literature that such a systemic approach might also be useful to better treat neonatal brain injury .
one such potential neurorestorative drug in newborns is erythropoietin , which has a recognized role in promoting neural regeneration and neurovascular remodeling .
a randomized controlled clinical trial is currently ongoing in human newborns to assess its efficiency ( i.e. , neonatal erythropoietin in asphyxiated term newborns ,
another promising alternative treatment of neonatal hie utilizes cord blood and mesenchymal stem cells to replace neurons lost due to brain injury , as well as activate endogenous stem cells , and release growth factors , thus minimizing brain damage and promoting restoration [ 42 , 43 ] .
these adjunctive therapies have until now mostly been tested only in animal models of hie or in different populations of newborns ( e.g. , erythropoietin to reduce the need for blood transfusions in very low birthweight infants ) . only a few randomized - controlled studies using these treatments are currently available in human newborns with hie , and they usually include only small numbers of patients . before implementation
, these alternative therapies will need to prove their efficiency and safety in randomized - controlled clinical trials with appropriate sample size consideration and outcome variables .
determining the best timing and the best dosage range for these alternative treatments is also crucial to obtain their maximum efficiency .
it has been hypothesized that hypothermia may buy additional time for neuroprotective drugs to act within an expanded therapeutic window .
however , for most of these alternative treatments , it remains to be determined when they will have the maximum synergistic effect with hypothermia treatment .
for example , in the first randomized trial of erythropoietin in term newborns with hie , the medication was given first within the first 24 hours of birth and thereafter every other day for 2 weeks ; the results showed a significantly improved primary outcome ( death or disability rate ) at 18 months , suggesting that this medication may provide therapeutic effect over a longer period than the first 6 hours of life .
in addition , appropriate selection of the newborns most likely to respond to these adjunctive treatments is of utmost importance to demonstrate more quickly their efficacy . planning for such trials should include early identification of newborns at risk of developing brain hi injury ( whether or not hypothermia is administered ) . to this end ,
early biomarkers of future brain hi injury in newborns are required to adjust existing cooling guidelines or to add other therapeutic interventions .
ideally , regular evolution of such biomarkers should be known , so that the impact of these newer treatments may be assessed as soon as possible .
current biomarkers included clinical signs , neurological exam outcomes , and assessment of brain activity by amplitude - integrated electroencephalogram [ 14 , 46 , 47 ] .
recent studies suggest some other potential biomarkers , such as brain magnetic resonance imaging , near - infrared spectroscopy , or biochemical markers , which would help guide the adjustment of therapies .
adjustment of the hypothermia treatment or addition of new treatments should be performed according to the specific clinical and imaging findings of these patients .
how to treat newborns with hie following birth asphyxia has become one of today 's hottest topics in neonatal medicine . over the past decade
however , much remains to be done to improve these early positive results and reduce further the eventual neonatal brain injury and improve future neurodevelopmental outcome of newborns with a hypoxic - ischemic event at birth .
several promising new treatments for the prevention of the devastating neurological consequences of birth asphyxia are currently being tested , but further detailed animal and clinical studies are required before their safe application in human newborns with hie .
define early biomarkers of brain injury to target the newborns with hie most vulnerable to brain injury despite hypothermia treatment .
determine if adjustments to current guidelines for mild hypothermia ( i.e. , cool later , lower , or longer ) might be useful to further prevent brain injury .
determine the best timing and the best dosage range of administration of adjunctive treatments , in order to get their maximum efficiency .
study in more detail the synergistic effects of these various treatments to better target the different and specific needs of each asphyxiated newborn infant and decrease the eventual neonatal brain injury and thus improve future neurodevelopmental outcome . perform safety studies in human newborns with hie for all these potential alternative treatments . | despite major advances in monitoring technology and knowledge of fetal and neonatal pathophysiology , neonatal hypoxic - ischemic encephalopathy ( hie ) remains one of the main causes of severe adverse neurological outcome in children . until recently
, there were no therapies other than supportive measures . over the past several years
, mild hypothermia has been proven to be safe to treat hie .
unfortunately , this neuroprotective strategy seems efficient in preventing brain injury in some asphyxiated newborns , but not in all of them .
thus , there is increasing interest to rapidly understand how to refine hypothermia therapy and add neuroprotective or neurorestorative strategies . several promising newer treatments to treat birth asphyxia and prevent its devastating neurological consequences are currently being tested . in this paper , the physiopathology behind hie , the currently available treatment , the potential alternatives , and the next steps before implementation of these other treatments are reviewed . |
its prevalence ranges from 1% to 3% in the western world.1,2 genetic and enviromental factors are relevant in the pathogenesis of psoriasis.3 in psoriasis , the inflammatory cytokine network is deregulated , leading to the excessive release of proinflammatory mediators from immune cells and increase in the proliferation of keratinocytes.4 in particular , th1 and th17 cell populations produce different cytokines ( interleukin [ il]-6 , il-17 , and il-22 , interferon- , and tumor necrosis factor [ tnf]- ) , causing a change in differentiation and hyperproliferation of keratinocytes , dilatation of blood vessels , and infiltration of leukocytes into the dermis and epidermis.5 the hallmarks of psoriasis are raised and clearly delimited erythematous lesions covered by silver scales ( figure 1a ) .
psoriatic lesions are commonly localized on the elbows , knees , trunk , sacrum , and scalp ; the involvement of the face , genitals , nails , palmoplantar regions is associated with higher impact on quality of life ( figure 1b and c).6 in most patients , the lesions cover < 10% of the body surface area ( bsa ) , but very rarely , psoriasis could involve the whole bsa , leading to erythroderma .
psoriatic lesions are frequently symptomatic with pruritus , followed by scaling and flaking.7 psoriasis may affect many facets of life , including emotional , social , work , and leisure .
approximately one - third of patients present signs / symptoms of concomitant psoriatic arhtritis ( psa),8 besides , several metabolic diseases , such as obesity , diabetes , fatty liver disease , metabolic syndrome , and cardiovascular diseases ( psoriasis itself could be an independent cardiovascular risk factor ) , are frequently associated with psoriasis.912 the concept of severity relates to many different aspects of psoriasis , including the extent of disease , location of lesions , degree of inflammation , responsiveness to treatment , and impact on patient quality of life .
generally , several assessment tools are used to assess the severity of chronic plaque psoriasis , including the psoriasis area and severity index ( pasi ) score that is based on the intensity of redness , thickness , and scaling of the representative lesions , and it ranges from 0 to 72 .
the bsa estimates the percentage of body surface affected by psoriasis ranging from 0% to 100% .
the physician global assessment ( pga ) grades disease severity in six categories including clear , almost clear , mild , moderate , severe , and very severe.13 the multifaceted nature of psoriasis burden drives the need for a specific focus on health - related quality of life and patient - reported outcome measures.14 patient s quality of life is commonly assessed by questionnaires , including dermatology life quality index ( dlqi ) and short form-36 health survey .
dlqi is a dermatology - specific tool aimed at assessing itch , pain , embarrassment , and interference of skin disease in the patient s daily activities , relationships , and sexual activity .
short form-36 is a nondermatological questionnaire that investigates the physical functioning , bodily pain , general health perceptions , vitality , social functioning , and mental health .
although none of the mentioned severity scores meets all the validation criteria required for an ideal score , pasi is the most commonly used , being the gold standard tool in clinical trials as well as in daily practice .
according to the european s3 guidelines on the systemic treatment of psoriasis vulgaris , moderate - to - severe disease is defined as a pasi score > 10.15 pasi 75 and pasi 90 responses are dynamic parameters that indicate the percentage of patients who have achieved at least a 75% or 90% improvement , respectively , in their baseline pasi score during treatment .
the scottish intercollegiate guidelines for the diagnosis and management of psoriasis in adults differentiate between mild and severe psoriasis for the purposes of referral and selection of treatments .
mild psoriasis is defined as dlqi 5 , while severe psoriasis , for which systemic or biological therapy may be appropriate , is defined as pasi and dlqi scores 10.16 the guidelines on clinical investigation of medicinal products indicated for the treatment of psoriasis , edited by the european medicines agency , proposes the following operational definition of psoriasis severity , which can be used to describe patient population in clinical trials.17 mild - to - moderate psoriasis : good control of lesions with topical therapy alone .
bsa involvement < 10% or pasi < 10 . category mild - to - moderate on pga .
. moderate - to - severe psoriasis : topical therapies fail to control the disease .
very thick lesions located in difficult to treat regions ( eg , palmoplantar ) with bsa involvement < 10% may also be considered .
very important local signs with very thick lesions with bsa involvement > 10% may also be considered .
finally , a european consensus was achieved to define goals for treatment of plaque psoriasis with systemic therapy and improve patient care.18 severity of plaque psoriasis was graded into mild and moderate - to - severe disease .
mild disease was defined as bsa 10% , and pasi and dlqi scores 10 ; and moderate - to - severe psoriasis as bsa > 10% , and pasi and dlqi scores
, it is recommended to treat mild psoriasis with topical agents . in refractory cases ,
the addition of phototherapy should be considered . however , psoriasis could be graded as moderate - to - severe also in cases of bsa 10% and/or pasi 10 but dlqi > 10 because of a significant impact on the quality of life and systemic therapy could be indicated .
however , patients with mild psoriasis , as indicated by the bsa and pasi scores , may present with disease manifestations not adequately controlled by topical therapy alone , which may lead to a significantly impaired quality of life .
these manifestations can include the involvement of visible areas ( ie , face , scalps , and hands ) , genitals , palms and/or soles , nails , intense pruritus , and presence of single recalcitrant plaques .
the consensus group recognized that the presence of disease manifestations listed earlier may alter the classification of mild disease ( pasi 10 , bsa 10 , dlqi 10 ) to moderate - to - severe disease that warrants phototherapy , systemic treatment , and/or combination therapy.18 the main outcome of psoriasis therapy is to safely achieve remission of psoriasis , that is , complete or almost complete clearance of skin lesions with no impairment of the disease on the quality of life.19 treatments of psoriasis are numerous and they can be topical , systemic , or phototherapy ( table 1 ) .
the topical therapies include keratolytics , corticosteroids , vitamin d analogs , retinoids , dithranol , and topical calcineurin inhibitors .
first - line treatments are vitamin d derivatives and corticosteroids , usually given in combination schedules . for topical treatments ,
the selection of the most appropriate vehicle is of major importance , thus improving adherence to the treatment , which is frequently impaired by the complexities of topical therapeutic choices .
evidence for efficacy and safety of topical treatments is readily available for vitamin d and short - term treatments with corticosteroids .
data on long - term use of topical therapies are scarce . new small molecules , including janus kinase - signal transducer and activator of transcription and phosphodiesterase type 4 ( pde4 ) inhibitors , are under clinical development.20 a major issue with topical therapy is adherence , which may reduce dramatically in the long term , rendering topical treatments poorly accepted and ineffective .
phototherapy , which includes either narrow - band ultraviolet b light or photochemotherapy ( ie , psoralen plus ultraviolet a light ) , and systemic agents , such as cyclosporine , methotrexate , fumaric acid esters , and acitretin , are indicated for moderate - to - severe patients . according to the european guidelines , in case of intolerance , inefficacy , or contraindication to phototherapy or conventional systemic treatments , the patients are eligible for biological agents , which include tnf- antagonists ( adalimumab , etanercept , and infliximab ) , anti - il12/23 monoclonal antibody ustekinumab , and anti-17 monoclonal antibodies secukinumab and ixekizumab.15 the efficacy of biologic drugs for the treatment of skin signs of psoriasis and psa is well known .
the retention rate of biological agents is higher than conventional drugs because they are better tolerated in the long term.21 treatment decisions are based on clinical status , patient treatment history , disease - related psychosocial burden , comorbidities , safety considerations , and patient s preferences . in particular
, disease severity is the leading factor for the choice of a systemic therapy . according to the rule of tens proposed by finlay,22 a systemic treatment
is indicated when the pasi score and/or bsa involvement and/or dlqi > 10 . indeed , a systemic therapy could also be appropriate in the case of involvement of high impact areas ( ie , scalp , genitals , palms and/or soles and nails ) , high intensity of symptoms such as pruritus , or presence of lesions not responsive to topical therapy .
treatment choice is also influenced by the concomitance of comorbidities , because methotrexate , cyclosporine , and acitretin may be harmful in some cases .
for example , cyclosporine could cause arterial hypertension , alter glucose tolerance , and/or favor dyslipidemia.23 consequently , cyclosporine is contraindicated in patients with metabolic syndrome and reduced kidney function .
similarly , acitretin could induce or worsen hyperc - holesterolemia and hypertriglyceridemia.23 methotrexate and acitretin have a documented teratogenic effect and can not be administered to females with child - bearing potential or those who wish to become pregnant .
methotrexate is also contraindicated in patients with a history of alcohol abuse or significant liver impairment .
females who have undergone acitretin treatment must avoid getting pregnant for at least 3 years following its withdrawal.15 the major safety concern of biologics emerging from registries or long - term studies is an increased risk of infections.24 in a large study based on cumulative incidence , the rate of serious infections was 1.45 per 100 patient - years ( n = 323 ) across treatment cohorts ; 0.83 , 1.47 , 1.97 , and 2.49 per 100 patient - years in the ustekinumab , etanercept , adalimumab , and infliximab cohorts , respectively ; and 1.05 and 1.28 per 100 patient - years in the nonmethotrexate / nonbiologics and methotrexate / nonbiologics cohorts , respectively . the most commonly reported types of serious infections across the registry were pneumonia and cellulitis . increasing age , diabetes mellitus , smoking , significant infection history , infliximab and adalimumab exposure were each associated with an increased risk of serious infection.25 more importantly , a significant proportion of patients ( 10%20% every year ) develop treatment resistance , which may impair the long - term effectiveness of biologic agents . in treatment decision , it is also very important to take into account several patient - related factors , including the age and sex , likelihood of adherence , expectation of remission , and fear of side effects .
recent large surveys reported that many patients with psoriasis discontinue their treatment because of lack / loss of the therapeutic efficacy , intolerance , and perceived safety concerns.7,26 undertreatment of moderate - to - severe psoriasis is quite common .
a recent population - based , multinational survey of 3,426 patients from 139,948 screened households in north america and europe found that only 11% of patients with bsa > 10% were receiving a systemic agent for psoriasis , with 52% receiving a topical treatment alone , and 37% being untreated.7 indeed , despite all the medications available , new drugs for psoriasis and psa are urgently needed to guarantee better disease control
. in particular , oral drugs would be very much appreciated by some patients because they overcome the injection - related issues that may be observed with biologic therapies .
the concept of severity relates to many different aspects of psoriasis , including the extent of disease , location of lesions , degree of inflammation , responsiveness to treatment , and impact on patient quality of life .
generally , several assessment tools are used to assess the severity of chronic plaque psoriasis , including the psoriasis area and severity index ( pasi ) score that is based on the intensity of redness , thickness , and scaling of the representative lesions , and it ranges from 0 to 72 .
the bsa estimates the percentage of body surface affected by psoriasis ranging from 0% to 100% . the physician global assessment ( pga ) grades disease severity in six categories including clear , almost clear , mild , moderate , severe , and very severe.13 the multifaceted nature of psoriasis burden drives the need for a specific focus on health - related quality of life and patient - reported outcome measures.14 patient s quality of life is commonly assessed by questionnaires , including dermatology life quality index ( dlqi ) and short form-36 health survey .
dlqi is a dermatology - specific tool aimed at assessing itch , pain , embarrassment , and interference of skin disease in the patient s daily activities , relationships , and sexual activity .
short form-36 is a nondermatological questionnaire that investigates the physical functioning , bodily pain , general health perceptions , vitality , social functioning , and mental health .
although none of the mentioned severity scores meets all the validation criteria required for an ideal score , pasi is the most commonly used , being the gold standard tool in clinical trials as well as in daily practice . according to the european s3 guidelines on the systemic treatment of psoriasis vulgaris
, moderate - to - severe disease is defined as a pasi score > 10.15 pasi 75 and pasi 90 responses are dynamic parameters that indicate the percentage of patients who have achieved at least a 75% or 90% improvement , respectively , in their baseline pasi score during treatment .
the scottish intercollegiate guidelines for the diagnosis and management of psoriasis in adults differentiate between mild and severe psoriasis for the purposes of referral and selection of treatments .
mild psoriasis is defined as dlqi 5 , while severe psoriasis , for which systemic or biological therapy may be appropriate , is defined as pasi and dlqi scores 10.16 the guidelines on clinical investigation of medicinal products indicated for the treatment of psoriasis , edited by the european medicines agency , proposes the following operational definition of psoriasis severity , which can be used to describe patient population in clinical trials.17 mild - to - moderate psoriasis : good control of lesions with topical therapy alone .
bsa involvement < 10% or pasi < 10 . category mild - to - moderate on pga .
. moderate - to - severe psoriasis : topical therapies fail to control the disease .
very thick lesions located in difficult to treat regions ( eg , palmoplantar ) with bsa involvement < 10% may also be considered .
very important local signs with very thick lesions with bsa involvement > 10% may also be considered .
finally , a european consensus was achieved to define goals for treatment of plaque psoriasis with systemic therapy and improve patient care.18 severity of plaque psoriasis was graded into mild and moderate - to - severe disease .
mild disease was defined as bsa 10% , and pasi and dlqi scores 10 ; and moderate - to - severe psoriasis as bsa > 10% , and pasi and dlqi scores > 10 . in accordance with existing guidelines
, it is recommended to treat mild psoriasis with topical agents . in refractory cases ,
the addition of phototherapy should be considered . however , psoriasis could be graded as moderate - to - severe also in cases of bsa 10% and/or pasi 10 but dlqi > 10 because of a significant impact on the quality of life and systemic therapy could be indicated .
however , patients with mild psoriasis , as indicated by the bsa and pasi scores , may present with disease manifestations not adequately controlled by topical therapy alone , which may lead to a significantly impaired quality of life .
these manifestations can include the involvement of visible areas ( ie , face , scalps , and hands ) , genitals , palms and/or soles , nails , intense pruritus , and presence of single recalcitrant plaques .
the consensus group recognized that the presence of disease manifestations listed earlier may alter the classification of mild disease ( pasi 10 , bsa 10 , dlqi 10 ) to moderate - to - severe disease that warrants phototherapy , systemic treatment , and/or combination therapy.18
the main outcome of psoriasis therapy is to safely achieve remission of psoriasis , that is , complete or almost complete clearance of skin lesions with no impairment of the disease on the quality of life.19 treatments of psoriasis are numerous and they can be topical , systemic , or phototherapy ( table 1 ) .
the topical therapies include keratolytics , corticosteroids , vitamin d analogs , retinoids , dithranol , and topical calcineurin inhibitors .
first - line treatments are vitamin d derivatives and corticosteroids , usually given in combination schedules . for topical treatments ,
the selection of the most appropriate vehicle is of major importance , thus improving adherence to the treatment , which is frequently impaired by the complexities of topical therapeutic choices .
evidence for efficacy and safety of topical treatments is readily available for vitamin d and short - term treatments with corticosteroids .
data on long - term use of topical therapies are scarce . new small molecules , including janus kinase - signal transducer and activator of transcription and phosphodiesterase type 4 ( pde4 ) inhibitors , are under clinical development.20 a major issue with topical therapy is adherence , which may reduce dramatically in the long term , rendering topical treatments poorly accepted and ineffective .
phototherapy , which includes either narrow - band ultraviolet b light or photochemotherapy ( ie , psoralen plus ultraviolet a light ) , and systemic agents , such as cyclosporine , methotrexate , fumaric acid esters , and acitretin , are indicated for moderate - to - severe patients . according to the european guidelines , in case of intolerance , inefficacy , or contraindication to phototherapy or conventional systemic treatments , the patients are eligible for biological agents , which include tnf- antagonists ( adalimumab , etanercept , and infliximab ) , anti - il12/23 monoclonal antibody ustekinumab , and anti-17 monoclonal antibodies secukinumab and ixekizumab.15 the efficacy of biologic drugs for the treatment of skin signs of psoriasis and psa is well known .
the retention rate of biological agents is higher than conventional drugs because they are better tolerated in the long term.21 treatment decisions are based on clinical status , patient treatment history , disease - related psychosocial burden , comorbidities , safety considerations , and patient s preferences .
in particular , disease severity is the leading factor for the choice of a systemic therapy . according to the rule of tens proposed by finlay,22 a systemic treatment
is indicated when the pasi score and/or bsa involvement and/or dlqi > 10 . indeed , a systemic therapy could also be appropriate in the case of involvement of high impact areas ( ie , scalp , genitals , palms and/or soles and nails ) , high intensity of symptoms such as pruritus , or presence of lesions not responsive to topical therapy .
treatment choice is also influenced by the concomitance of comorbidities , because methotrexate , cyclosporine , and acitretin may be harmful in some cases .
for example , cyclosporine could cause arterial hypertension , alter glucose tolerance , and/or favor dyslipidemia.23 consequently , cyclosporine is contraindicated in patients with metabolic syndrome and reduced kidney function .
similarly , acitretin could induce or worsen hyperc - holesterolemia and hypertriglyceridemia.23 methotrexate and acitretin have a documented teratogenic effect and can not be administered to females with child - bearing potential or those who wish to become pregnant .
methotrexate is also contraindicated in patients with a history of alcohol abuse or significant liver impairment .
females who have undergone acitretin treatment must avoid getting pregnant for at least 3 years following its withdrawal.15 the major safety concern of biologics emerging from registries or long - term studies is an increased risk of infections.24 in a large study based on cumulative incidence , the rate of serious infections was 1.45 per 100 patient - years ( n = 323 ) across treatment cohorts ; 0.83 , 1.47 , 1.97 , and 2.49 per 100 patient - years in the ustekinumab , etanercept , adalimumab , and infliximab cohorts , respectively ; and 1.05 and 1.28 per 100 patient - years in the nonmethotrexate / nonbiologics and methotrexate / nonbiologics cohorts , respectively . the most commonly reported types of serious infections across the registry were pneumonia and cellulitis . increasing age , diabetes mellitus , smoking , significant infection history , infliximab and adalimumab exposure were each associated with an increased risk of serious infection.25 more importantly , a significant proportion of patients ( 10%20% every year ) develop treatment resistance , which may impair the long - term effectiveness of biologic agents . in treatment decision , it is also very important to take into account several patient - related factors , including the age and sex , likelihood of adherence , expectation of remission , and fear of side effects .
recent large surveys reported that many patients with psoriasis discontinue their treatment because of lack / loss of the therapeutic efficacy , intolerance , and perceived safety concerns.7,26 undertreatment of moderate - to - severe psoriasis is quite common .
a recent population - based , multinational survey of 3,426 patients from 139,948 screened households in north america and europe found that only 11% of patients with bsa > 10% were receiving a systemic agent for psoriasis , with 52% receiving a topical treatment alone , and 37% being untreated.7 indeed , despite all the medications available , new drugs for psoriasis and psa are urgently needed to guarantee better disease control .
in particular , oral drugs would be very much appreciated by some patients because they overcome the injection - related issues that may be observed with biologic therapies .
apremilast , an oral , small molecule pde4 inhibitor , was approved for the management of psoriasis and psa in 2014 by the us food and drug administration and in 2015 by the european commission . in eu ,
apremilast is indicated for adult patients with moderate - to - severe plaque psoriasis who have failed to respond or have a contraindication to , or are intolerant to other systemic therapy . in active psa ,
apremilast is administered alone or in combination with conventional drugs to adult patients who have had an inadequate response or have been intolerant to a prior conventional therapy .
apremilast is the first small - molecule inhibitor of pde4 , an enzyme involved in the chronic inflammatory pathways , including those associated with psoriasis.27 pde4 is the main camp - specific pde in inflammatory cells , including macrophages , monocytes , mast cells , dendritic cells , eosinophils , and t cells .
the main mechanism of action of apremilast is the inhibition of pde , which consequently increases the intracellular levels of camp and modulates the signaling pathways , by the activation of protein kinase a and phosphorylation of camp - response element binding protein , that inhibit the secretion of inflammatory cytokines ( eg , tnf- , interferon- , il-2 , il-12 , and il-23 ) and stimulate the production of anti - inflammatory cytokines , such as il-6 and il-10 ( figure 2).28 preliminary studies on patients with severe psoriasis showed that apremilast is able to effectively modulate the inflammatory outcomes in psoriatic lesions.29 a phase ii study on 30 patients with recalcitrant psoriasis confirmed this activity of apremilast , as it showed a significant reduction in the number of immune cells ( myeloid dendritic cells , t - cells , nk - cells ) infiltrating the psoriatic lesions of the dermis and epidermis.7 after 4 and 12 weeks , other inflammatory mediators ( il-8 , defb4 , mx-1 , k16 , inducible nitric oxide synthase , il-12/23 p40 , il-17a ) also showed reduced levels .
furthermore , a relationship between the median change from the baseline pasi scores at week 12 and decrease in inducible nitric oxide synthase , il-17a , defensin beta 4 and keratin 16 levels was observed .
these results highlight that the wider modulation of the inflammatory response by apremilast versus other drugs targeting a single proinflammatory mediator is potentially responsible for its biologic effects.29 apremilast in the treatment of chronic plaque psoriasis has been investigated in the efficacy and safety trial evaluating the effects of apremilast in psoriasis ( esteem ) phase iii clinical trial program .
esteem 1 and 2 are two multi - center , randomized , double - blind , placebo - controlled studies aimed at analyzing the efficacy and safety of apremilast in the management of patients with moderate - to - severe plaque psoriasis ( n=844 in esteem 1 and n=411 in esteem 2).30,31 both trials had similar design .
first , the patients were randomized in two groups : apremilast 30 mg twice daily or placebo for 16 weeks ; in the second phase of the study , all the patients received apremilast for another 16 weeks ( until week 32 ) .
then , patients initially randomized to apremilast with a 75% ( esteem 1 ) or 50% ( esteem 2 ) reduction in pasi score ( pasi 75 or 50 response ) were rerandomized to continuation of apremilast or placebo ( in this group , apremilast was restarted in case of loss of response ) . in both the esteem trials ,
the primary end point was the proportion of patients who achieved pasi 75 at week 16 . in esteem 1 , significantly more patients taking apremilast than placebo achieved pasi 75 at week 16 , that is , 33.1% versus 5.3% , p<0.0001 .
pga score of 0 or 1 at week 16 , the major secondary end point of the study , was achieved by significantly more patients receiving apremilast than placebo , that is , 21.7% versus 3.9% , p<0.01 .
after 16 weeks , the mean change in pasi score from baseline was 52.1% versus 16.7% in apremilast and placebo groups , respectively ( p<0.0001 ) .
the improvement in pasi was retained over 52 weeks in those responders.30 apremilast reduced the severity of pruritus .
indeed , the severity of pruritus was reduced in patients receiving apremilast compared with placebo ( 33.8 mm vs 7.7 mm in esteem 1 ; 36.4 mm vs 12.9 mm in esteem 2 ; p<0.0001 for both studies ) at week 16.32 improvement in the severity of pruritus with apremilast was correlated with improvement in the quality of life as measured by dlqi change from baseline.30 in the esteem 1 study , a reduction in itching > 20% ( ie , minimal clinically important difference ) was more frequently observed in subjects treated with apremilast than with placebo ( 70.6% vs 33.7% , p<0.0001).33 apremilast was effective in treating patients with psoriasis located in nail , scalp , and palmoplantar areas .
in particular , at week 16 , a napsi 50 response ( eg , 50% improvement from baseline in target nail napsi score ) was observed in 33% and 45% of patients with nail psoriasis in esteem 1 and 2 , respectively.34 a greater proportion of patients treated with apremilast than placebo achieved a scalp pga score of 01 ( clear / almost clear ) at week 16 ( 46.5% vs 17.5% and 40.9% vs 17.2% ) in esteem 1 and 2 , respectively.34 among patients with moderate - to - severe palmoplantar psoriasis at baseline , palmoplantar pga score of 0 or 1 achievement at week 16 was significantly greater in patients who received apremilast versus placebo ( 65.4% vs 31.3% , p=0.03 ) and it was maintained at week 32 in 53.8% of patients.34 finally , apremilast was able to significantly improve patient - reported outcomes versus placebo , as assessed by the dlqi , sf-36 item health survey version 2 , and the work limitations questionnaire-25 index.35 a pooled analysis of eet eem 1 and 2 trials showed that apremilast significantly improved work limitations , as well as increased work productivity compared with placebo.35 esteem 1 and 2 trials confirmed the well - known and manageable safety and tolerability profile of apremilast . in these studies ,
adverse reactions were mostly mild - to - moderate in severity in all the study periods ( short- and long - term ) .
the most frequent adverse events were diarrhea ( 17.8% ) , nausea ( 16.6% ) , and upper respiratory tract infections ( 8.4% ) .
the incidence of diarrhea and nausea was higher during the first 2 weeks of treatment compared with other study periods , and commonly resolved within 4 weeks.36 long - term safety data ( 52 weeks of exposure ) did not show a higher incidence of adverse events ( including serious adverse events ) based on exposure - adjusted incidence rates per 100 patient - years . during the esteem program , only three deaths were observed : one in the placebo group for suicide and two in the apremilast group , both for cardiovascular accidents .
discontinuations due to adverse events occurred in 6.1% and 4.1% of patients receiving apremilast and placebo , respectively , during weeks 016 ( mainly due to diarrhea 0.9% and nausea 1.4% ) and did not increase with longer apremilast exposure .
incidence of major adverse cardiac events , serious infections , opportunistic infections , or malignancies in esteem 1 and 2 ( pooled analysis ) trials were comparable to placebo.36 no reactivations of tuberculosis were noted , even if eight patients ( n=5 in esteem 1 and n=3 in esteem 2 ) treated with apremilast had a positive anamnesis for tuberculosis .
abnormal laboratory test results in apremilast - treated patients were rare , transient , and not clinically significant .
furthermore , patients treated with apremilast in esteem studies showed a weight loss . during the period from week 0 to 16 , weight loss of > 5%
was experienced by 13.7% of patients receiving apremilast and 5.5% of patients receiving placebo ; and from week 0 to 52 by 19.2% of patients receiving apre - milast . at week 52 , the mean ( median ) change from baseline weight was 1.99 ( 1.40 ) kg in patients taking apremilast . weight loss was not associated with any overt medical consequences , including diarrhea or nausea / vomiting.37 according to the analysis of clinical trials and published literature , there is no evidence of an increase in the risk of psychiatric events , including suicidality , with the use of apremilast.38 finally , apremilast showed an acceptable safety profile and was well tolerated for up to 104 weeks of exposure.39 the clinical presentation of psoriasis could widely vary among patients because of different ages of onset , types of symptoms , areas of involvement , comorbidities , and disease severity .
the choice of the treatment of psoriasis should be personalized according to the specific needs of the patients in order to optimize the outcome .
apremilast is a well - tolerated and effective pde4 inhibitor , indicated in the treatment of moderate - to - severe plaque psoriasis and psa .
the clinical development program of apremilast includes other potential indications , including ankylosing spondylitis , behet s disease , ulcerative colitis , and atopic dermatitis .
apremilast will have an important place in the treatment of chronic plaque psoriasis because of oral administration , very favorable safety profile , lack of label - required screening or ongoing laboratory monitoring , efficacy in difficult - to - treat areas including nails , scalp , and palmoplantar regions , and rapid improvement in pruritus .
safety is a major unmet need in the therapy of psoriasis.40 in the long term , conventional systemic treatments could cause adverse effects , including renal and liver toxicity , myelosuppression , impairment of metabolic comorbidities , and necessitate periodic laboratory monitoring .
the major concerns of biologics are the onset of infections and malignancies , and immunogenicity , which can cause a progressive loss or reduction of clinical efficacy .
apremilast does not formally require any screening to assess latent tuberculosis and chronic viral infections , or laboratory monitoring , and thus is perceived as extremely safe .
to what extent the transient gastrointestinal side effects ( nausea and vomiting ) will limit its use will be addressed only with its use in clinical practice .
pasi 75 response observed with apremilast is lower compared with biological treatments , including the novel il-17 inhibitors.41 however , it should be considered that patients enrolled in esteem studies had a high degree of severity of psoriasis according to the baseline pasi score of 19.4 7.4 ( mean standard deviation ) , and a significant proportion of them had been pretreated with phototherapy ( 30% ) and standard systemic and/or biologic therapy ( 54% ) .
apremilast could also be adopted as intermittent treatment , as most patients who lost pasi 75 response after discontinuing it because of rerandomization to placebo regained it after reinitiation of apremilast . as patients with psoriasis
are frequently overweight or obese , it is of interest that > 20% of patients treated with apremilast showed a decrease in weight > 5% .
the relevance of this effect of apremilast on metabolic parameters associated with obesity needs to be investigated in further clinical studies . in conclusion
, psoriasis has a complex and substantial impact on a patient s health - related quality of life across physical , psychological , social , and economic domains .
many of the currently available systemic treatments for psoriasis have limitations with respect to efficacy and/or safety .
new biologic drugs ( including il-17 or -23 inhibitors and new small molecules ) are currently under investigation in phase iii studies .
apremilast is a novel and valuable option for its safety profile and for refractory psoriatic lesions in difficult - to - treat regions such as scalp , nails , and palmoplan - tar areas .
moreover , apremilast could be indicated for those patients with a pasi score < 10 but with a significant impact on quality of life ( ie , dlqi > 10 ) as well as in those with concomitant psa . given the favorable safety profile and the low risk of drug interactions
the long - term safety and efficacy of apremilast must be further investigated , and more data are needed to guide clinicians in the use of this drug in clinical practice .
apremilast in the treatment of chronic plaque psoriasis has been investigated in the efficacy and safety trial evaluating the effects of apremilast in psoriasis ( esteem ) phase iii clinical trial program .
esteem 1 and 2 are two multi - center , randomized , double - blind , placebo - controlled studies aimed at analyzing the efficacy and safety of apremilast in the management of patients with moderate - to - severe plaque psoriasis ( n=844 in esteem 1 and n=411 in esteem 2).30,31 both trials had similar design .
first , the patients were randomized in two groups : apremilast 30 mg twice daily or placebo for 16 weeks ; in the second phase of the study , all the patients received apremilast for another 16 weeks ( until week 32 ) .
then , patients initially randomized to apremilast with a 75% ( esteem 1 ) or 50% ( esteem 2 ) reduction in pasi score ( pasi 75 or 50 response ) were rerandomized to continuation of apremilast or placebo ( in this group , apremilast was restarted in case of loss of response ) . in both the esteem trials ,
the primary end point was the proportion of patients who achieved pasi 75 at week 16 .
in esteem 1 , significantly more patients taking apremilast than placebo achieved pasi 75 at week 16 , that is , 33.1% versus 5.3% , p<0.0001 .
pga score of 0 or 1 at week 16 , the major secondary end point of the study , was achieved by significantly more patients receiving apremilast than placebo , that is , 21.7% versus 3.9% , p<0.01 .
after 16 weeks , the mean change in pasi score from baseline was 52.1% versus 16.7% in apremilast and placebo groups , respectively ( p<0.0001 ) .
the improvement in pasi was retained over 52 weeks in those responders.30 apremilast reduced the severity of pruritus .
indeed , the severity of pruritus was reduced in patients receiving apremilast compared with placebo ( 33.8 mm vs 7.7 mm in esteem 1 ; 36.4 mm vs 12.9 mm in esteem 2 ; p<0.0001 for both studies ) at week 16.32 improvement in the severity of pruritus with apremilast was correlated with improvement in the quality of life as measured by dlqi change from baseline.30 in the esteem 1 study , a reduction in itching > 20% ( ie , minimal clinically important difference ) was more frequently observed in subjects treated with apremilast than with placebo ( 70.6% vs 33.7% , p<0.0001).33 apremilast was effective in treating patients with psoriasis located in nail , scalp , and palmoplantar areas . in particular , at week 16 , a napsi 50 response ( eg , 50% improvement from baseline in target nail napsi score ) was observed in 33% and 45% of patients with nail psoriasis in esteem 1 and 2 , respectively.34 a greater proportion of patients treated with apremilast than placebo achieved a scalp pga score of 01 ( clear / almost clear ) at week 16 ( 46.5% vs 17.5% and 40.9% vs 17.2% ) in esteem 1 and 2 , respectively.34 among patients with moderate - to - severe palmoplantar psoriasis at baseline , palmoplantar pga score of 0 or 1 achievement at week 16 was significantly greater in patients who received apremilast versus placebo ( 65.4% vs 31.3% , p=0.03 ) and it was maintained at week 32 in 53.8% of patients.34 finally , apremilast was able to significantly improve patient - reported outcomes versus placebo , as assessed by the dlqi , sf-36 item health survey version 2 , and the work limitations questionnaire-25 index.35 a pooled analysis of eet eem 1 and 2 trials showed that apremilast significantly improved work limitations , as well as increased work productivity compared with placebo.35 esteem 1 and 2 trials confirmed the well - known and manageable safety and tolerability profile of apremilast . in these studies ,
adverse reactions were mostly mild - to - moderate in severity in all the study periods ( short- and long - term ) .
the most frequent adverse events were diarrhea ( 17.8% ) , nausea ( 16.6% ) , and upper respiratory tract infections ( 8.4% ) .
the incidence of diarrhea and nausea was higher during the first 2 weeks of treatment compared with other study periods , and commonly resolved within 4 weeks.36 long - term safety data ( 52 weeks of exposure ) did not show a higher incidence of adverse events ( including serious adverse events ) based on exposure - adjusted incidence rates per 100 patient - years . during the esteem program ,
only three deaths were observed : one in the placebo group for suicide and two in the apremilast group , both for cardiovascular accidents .
discontinuations due to adverse events occurred in 6.1% and 4.1% of patients receiving apremilast and placebo , respectively , during weeks 016 ( mainly due to diarrhea 0.9% and nausea 1.4% ) and did not increase with longer apremilast exposure .
incidence of major adverse cardiac events , serious infections , opportunistic infections , or malignancies in esteem 1 and 2 ( pooled analysis ) trials were comparable to placebo.36 no reactivations of tuberculosis were noted , even if eight patients ( n=5 in esteem 1 and n=3 in esteem 2 ) treated with apremilast had a positive anamnesis for tuberculosis .
abnormal laboratory test results in apremilast - treated patients were rare , transient , and not clinically significant . furthermore , patients treated with apremilast in esteem studies showed a weight loss . during the period from week 0 to 16 , weight loss of > 5%
was experienced by 13.7% of patients receiving apremilast and 5.5% of patients receiving placebo ; and from week 0 to 52 by 19.2% of patients receiving apre - milast . at week 52 , the mean ( median ) change from baseline weight was 1.99 ( 1.40 ) kg in patients taking apremilast . weight loss was not associated with any overt medical consequences , including diarrhea or nausea / vomiting.37 according to the analysis of clinical trials and published literature , there is no evidence of an increase in the risk of psychiatric events , including suicidality , with the use of apremilast.38 finally , apremilast showed an acceptable safety profile and was well tolerated for up to 104 weeks of exposure.39
apremilast in the treatment of chronic plaque psoriasis has been investigated in the efficacy and safety trial evaluating the effects of apremilast in psoriasis ( esteem ) phase iii clinical trial program .
esteem 1 and 2 are two multi - center , randomized , double - blind , placebo - controlled studies aimed at analyzing the efficacy and safety of apremilast in the management of patients with moderate - to - severe plaque psoriasis ( n=844 in esteem 1 and n=411 in esteem 2).30,31 both trials had similar design .
first , the patients were randomized in two groups : apremilast 30 mg twice daily or placebo for 16 weeks ; in the second phase of the study , all the patients received apremilast for another 16 weeks ( until week 32 ) .
then , patients initially randomized to apremilast with a 75% ( esteem 1 ) or 50% ( esteem 2 ) reduction in pasi score ( pasi 75 or 50 response ) were rerandomized to continuation of apremilast or placebo ( in this group , apremilast was restarted in case of loss of response ) . in both the esteem trials ,
the primary end point was the proportion of patients who achieved pasi 75 at week 16 .
in esteem 1 , significantly more patients taking apremilast than placebo achieved pasi 75 at week 16 , that is , 33.1% versus 5.3% , p<0.0001 .
pga score of 0 or 1 at week 16 , the major secondary end point of the study , was achieved by significantly more patients receiving apremilast than placebo , that is , 21.7% versus 3.9% , p<0.01 .
after 16 weeks , the mean change in pasi score from baseline was 52.1% versus 16.7% in apremilast and placebo groups , respectively ( p<0.0001 ) .
the improvement in pasi was retained over 52 weeks in those responders.30 apremilast reduced the severity of pruritus .
indeed , the severity of pruritus was reduced in patients receiving apremilast compared with placebo ( 33.8 mm vs 7.7 mm in esteem 1 ; 36.4 mm vs 12.9 mm in esteem 2 ; p<0.0001 for both studies ) at week 16.32 improvement in the severity of pruritus with apremilast was correlated with improvement in the quality of life as measured by dlqi change from baseline.30 in the esteem 1 study , a reduction in itching > 20% ( ie , minimal clinically important difference ) was more frequently observed in subjects treated with apremilast than with placebo ( 70.6% vs 33.7% , p<0.0001).33 apremilast was effective in treating patients with psoriasis located in nail , scalp , and palmoplantar areas . in particular , at week 16 , a napsi 50 response ( eg , 50% improvement from baseline in target nail napsi score ) was observed in 33% and 45% of patients with nail psoriasis in esteem 1 and 2 , respectively.34 a greater proportion of patients treated with apremilast than placebo achieved a scalp pga score of 01 ( clear / almost clear ) at week 16 ( 46.5% vs 17.5% and 40.9% vs 17.2% ) in esteem 1 and 2 , respectively.34 among patients with moderate - to - severe palmoplantar psoriasis at baseline , palmoplantar pga score of 0 or 1 achievement at week 16 was significantly greater in patients who received apremilast versus placebo ( 65.4% vs 31.3% , p=0.03 ) and it was maintained at week 32 in 53.8% of patients.34 finally , apremilast was able to significantly improve patient - reported outcomes versus placebo , as assessed by the dlqi , sf-36 item health survey version 2 , and the work limitations questionnaire-25 index.35 a pooled analysis of eet eem 1 and 2 trials showed that apremilast significantly improved work limitations , as well as increased work productivity compared with placebo.35
esteem 1 and 2 trials confirmed the well - known and manageable safety and tolerability profile of apremilast . in these studies , adverse reactions were mostly mild - to - moderate in severity in all the study periods ( short- and long - term ) .
the most frequent adverse events were diarrhea ( 17.8% ) , nausea ( 16.6% ) , and upper respiratory tract infections ( 8.4% ) .
the incidence of diarrhea and nausea was higher during the first 2 weeks of treatment compared with other study periods , and commonly resolved within 4 weeks.36 long - term safety data ( 52 weeks of exposure ) did not show a higher incidence of adverse events ( including serious adverse events ) based on exposure - adjusted incidence rates per 100 patient - years . during the esteem program ,
only three deaths were observed : one in the placebo group for suicide and two in the apremilast group , both for cardiovascular accidents .
discontinuations due to adverse events occurred in 6.1% and 4.1% of patients receiving apremilast and placebo , respectively , during weeks 016 ( mainly due to diarrhea 0.9% and nausea 1.4% ) and did not increase with longer apremilast exposure .
incidence of major adverse cardiac events , serious infections , opportunistic infections , or malignancies in esteem 1 and 2 ( pooled analysis ) trials were comparable to placebo.36 no reactivations of tuberculosis were noted , even if eight patients ( n=5 in esteem 1 and n=3 in esteem 2 ) treated with apremilast had a positive anamnesis for tuberculosis .
abnormal laboratory test results in apremilast - treated patients were rare , transient , and not clinically significant .
furthermore , patients treated with apremilast in esteem studies showed a weight loss . during the period from week 0 to 16 , weight loss of > 5%
was experienced by 13.7% of patients receiving apremilast and 5.5% of patients receiving placebo ; and from week 0 to 52 by 19.2% of patients receiving apre - milast . at week 52 , the mean ( median ) change from baseline weight was 1.99 ( 1.40 ) kg in patients taking apremilast . weight loss was not associated with any overt medical consequences , including diarrhea or nausea / vomiting.37 according to the analysis of clinical trials and published literature , there is no evidence of an increase in the risk of psychiatric events , including suicidality , with the use of apremilast.38 finally , apremilast showed an acceptable safety profile and was well tolerated for up to 104 weeks of exposure.39
the clinical presentation of psoriasis could widely vary among patients because of different ages of onset , types of symptoms , areas of involvement , comorbidities , and disease severity .
the choice of the treatment of psoriasis should be personalized according to the specific needs of the patients in order to optimize the outcome .
apremilast is a well - tolerated and effective pde4 inhibitor , indicated in the treatment of moderate - to - severe plaque psoriasis and psa .
the clinical development program of apremilast includes other potential indications , including ankylosing spondylitis , behet s disease , ulcerative colitis , and atopic dermatitis .
apremilast will have an important place in the treatment of chronic plaque psoriasis because of oral administration , very favorable safety profile , lack of label - required screening or ongoing laboratory monitoring , efficacy in difficult - to - treat areas including nails , scalp , and palmoplantar regions , and rapid improvement in pruritus .
safety is a major unmet need in the therapy of psoriasis.40 in the long term , conventional systemic treatments could cause adverse effects , including renal and liver toxicity , myelosuppression , impairment of metabolic comorbidities , and necessitate periodic laboratory monitoring .
the major concerns of biologics are the onset of infections and malignancies , and immunogenicity , which can cause a progressive loss or reduction of clinical efficacy .
apremilast does not formally require any screening to assess latent tuberculosis and chronic viral infections , or laboratory monitoring , and thus is perceived as extremely safe .
to what extent the transient gastrointestinal side effects ( nausea and vomiting ) will limit its use will be addressed only with its use in clinical practice .
pasi 75 response observed with apremilast is lower compared with biological treatments , including the novel il-17 inhibitors.41 however , it should be considered that patients enrolled in esteem studies had a high degree of severity of psoriasis according to the baseline pasi score of 19.4 7.4 ( mean standard deviation ) , and a significant proportion of them had been pretreated with phototherapy ( 30% ) and standard systemic and/or biologic therapy ( 54% ) . moreover ,
apremilast could also be adopted as intermittent treatment , as most patients who lost pasi 75 response after discontinuing it because of rerandomization to placebo regained it after reinitiation of apremilast . as patients with psoriasis
are frequently overweight or obese , it is of interest that > 20% of patients treated with apremilast showed a decrease in weight > 5% .
the relevance of this effect of apremilast on metabolic parameters associated with obesity needs to be investigated in further clinical studies . in conclusion
, psoriasis has a complex and substantial impact on a patient s health - related quality of life across physical , psychological , social , and economic domains .
many of the currently available systemic treatments for psoriasis have limitations with respect to efficacy and/or safety .
new biologic drugs ( including il-17 or -23 inhibitors and new small molecules ) are currently under investigation in phase iii studies .
apremilast is a novel and valuable option for its safety profile and for refractory psoriatic lesions in difficult - to - treat regions such as scalp , nails , and palmoplan - tar areas .
moreover , apremilast could be indicated for those patients with a pasi score < 10 but with a significant impact on quality of life ( ie , dlqi > 10 ) as well as in those with concomitant psa
. given the favorable safety profile and the low risk of drug interactions , apremilast seems an appropriate treatment also for elderly patients .
the long - term safety and efficacy of apremilast must be further investigated , and more data are needed to guide clinicians in the use of this drug in clinical practice . | chronic plaque psoriasis presents clinically as an inflammatory disease of the skin , which is often associated with comorbidities and responsible for a poor quality of life .
it can widely vary among patients because of different age of onset , type of symptoms , areas of involvement , and disease severity .
the choice of the treatment of psoriasis should be personalized according to the specific needs of the patients .
apremilast is a well - tolerated and effective phosphodiesterase type 4 inhibitor that is indicated for the treatment of moderate - to - severe plaque psoriasis and psoriatic arthritis . in this article ,
the pharmacological , clinical , and safety aspects of apremilast are reviewed .
based on these data , apremilast could be indicated for patients with a psoriasis area and severity index score < 10 but with a significant impact on quality of life and seems to be an appropriate treatment for elderly patients also . |
takayasu 's arteritis ( ta ) and crohn 's disease ( cd ) are chronic inflammatory granulomatous autoimmune disorders that lead to characteristic manifestations .
cd predominantly affects the gastrointestinal tract , and various hypotheses concerning the underlying pathology have been proposed , ranging from environmental , genetic , infections , autoimmune processes to a defect in autophagy .
cd appears to be more prevalent in caucasian populations , but recent reports have shown a rising incidence in asian populations .
the chronic inflammation of unknown etiology affecting the arteries may lead to stenosis , occlusions , dilatations and aneurysms of affected vessels .
cell - mediated autoimmunity leading to granulomatous inflammation is believed to represent the most probable pathologic cause of the disease .
a 16-year - old male with a past medical history of thalassemia trait and recent diagnosis of hypertension presented with complaints of abdominal pain , nausea , vomiting , constipation and weight loss .
the work - up for abdominal pain prompted ct angiogram imaging that revealed marked thickening of the terminal ileum and decreased uptake and atrophy of the left kidney , significant wall thickening and narrowing of the left common carotid , right common and internal carotid and subclavian and axillary arteries , and diffuse thickening of aortic arch and descending thoracic and upper abdominal aorta , with left renal artery occlusion ( fig .
an mr enterography of the abdomen confirmed the mucosal thickening of the terminal ileum without bowel wall edema or additional sites of stenosis , hyperenhancement or fistulas .
a colonoscopy was performed which revealed stenotic ileocecal valve with friable and inflamed terminal ileal mucosa and an ulceration ( fig .
biopsy from the terminal ileum confirmed the diagnosis of cd showing moderate active chronic ileitis .
his laboratory studies were significant for elevated sedimentation rate of 23 and crp of 27 , positive antiflagellin antibody ( cbir1 ) , but negative antisaccharomyces antibody ( asca ) , outer membrane porin to escherichia coli antibody ( anti ompc ) , lupus anticoagulant and anticardiolipin antibodies .
meanwhile , he developed retinal and cerebral ischemic episodes , leading to transient visual loss and syncope , for which he underwent ascending aorta to left common carotid artery bypass grafting .
he did not have any significant relapse or symptoms of active cd other than intermittent abdominal pain .
however , he suffered another episode of syncope with dehydration leading to thrombotic occlusion of his aortocarotid bypass graft requiring thrombectomy and revision of the graft .
he remained asymptomatic as both his cd and ta remained under good control for twelve months .
a 38-year - old male with a past medical history of hypertension and mild renal insufficiency presented with abdominal pain , diarrhea and chest pain .
cd had been diagnosed 15 years prior to presenting to our institution by means of colonoscopy with biopsy .
review of reports from prior examinations revealed that cd was initially active in the colon , but later involved the small intestine as well .
there was no previous history of strictures or fistulas or such findings during further evaluation in our institution . during the past 15 years
he had been on infliximab for 1 year when he developed chest , upper back and abdominal pain and noted to have ectasia of the descending aorta .
infliximab was discontinued 6 months later due to fever , bacteremia , and acute renal failure .
his abdominal imaging revealed thickening and paraaortic stranding of the abdominal aorta and wall thickening of the sigmoid colon , but also bilateral renal artery stenosis , which prompted further imaging of the chest and neck .
ta was diagnosed when the ct angiogram showed wall thickening of the aortic arch , abdominal aorta , left subclavian and common carotid arteries , with sigmoid colon wall thickening ( fig .
his laboratory studies were remarkable for leukocytosis , elevated crp and normal sedimentation rate , but negative antinuclear antibody ( ana ) , p - anca ( antinuclear cytoplasmic antibody ) , antiflagellin antibody ( cbir1 ) , antisaccharomyces antibody ( asca ) , outer membrane porin to escherichia coli antibody ( antiompc ) , hepatitis a , b and c serologies .
when follow - up imaging showed severe bilateral renal artery stenoses and renal atrophy , an aorto - birenal bypass grafting was performed .
adalimumab was started 6 months later due to persistent inflammation and elevated esr and crp despite prednisone and azathioprine therapy .
three weeks later he sustained sepsis , thrombosis of the graft , and renal failure requiring dialysis .
cd , being an inflammatory bowel disease , is associated with extraintestinal manifestations and other autoimmune disorders with a higher prevalence than ulcerative colitis .
vasculitis , predominantly of the mesenteric vessels , has been suggested as a possible etiologic cause for intestinal manifestations , leading to ischemic damage and multifocal infarctions of the bowel wall , although granulomatous inflammation of the bowel wall generally does not correspond to the site of vasculitis .
increased thrombophilia associated with inflammatory bowel disease can lead to regional or systemic complications of infarctions , thrombosis and embolism .
histopathological diagnosis of ta is difficult due to the involvement of major blood vessels . hence imaging modalities like angiography , ct and
pathology of a resected artery was helpful in one of our patients , while the imaging studies were the main modes of diagnosis in both patients .
simultaneous occurrence of these two diseases in the same individual is rare , but is being increasingly reported in part owing to heightened awareness and better access to superior diagnostic imaging .
similarities in the presentation of abdominal symptoms , especially abdominal pain and gastrointestinal bleeding in both these diseases might delay or confound the diagnosis of one or the other .
granulomatous vasculitis due to cell - mediated injury is the common pathophysiological mechanism seen in both conditions , suggesting that simultaneous occurrence is more than just a coincidence .
the underlying inflammatory process in both diseases appears to be influenced by cytokines such as tumor necrosis factor a ( tnf- ) and interleukins including il-6 , il-8 , il-12 and il-18 .
hla genotype links and even infectious agents such as mycobacterium tuberculosis have been implicated as common etiologies , but to date no definite etiological associations have been identified .
review of published reports of coexisting cd and ta points to cd preceding ta in 88% of cases in the literature , with the age at diagnosis of cd ranging from 10 to 31 years and ta from 14 to 33 years . while age was similar in both of our patients , cd preceded ta in one patient , but was diagnosed simultaneously in the other .
corticosteroids , immunosuppressive agents and tnf- inhibition have been effective in the management of both cd and ta
. however , the current literature does not provide enough information about the management outcomes of this group of patients with coexisting diseases . in our patients , prednisone
stenotic and thrombotic complications appear to develop not infrequently in this group of patients as evidenced by graft thrombosis and occlusion in both of our patients .
while our first case had an excellent response to biologic therapy and remained asymptomatic , the second case responded poorly to tnf- therapies and was associated with recurrent infectious complications . in summary , in these two patients with both cd and ta the diagnoses were confirmed by imaging and pathologic findings .
cytokine- and interleukin - mediated granulomatous inflammation appears to be the likely common pathologic pathway in these two diseases .
tnf- inhibition targeting this common pathway was effective in one patient but discontinued in the other due to infection .
further research into the association of cd and ta may provide clues to their etiologies and guide effective interventions . | crohn 's disease ( cd ) and takayasu 's arteritis ( ta ) are inflammatory granulomatous autoimmune disorders . simultaneous occurrence of cd and ta in the same individual is rare .
we report two cases treated with biologic agents .
case 1 : a 16-year - old male presented with abdominal pain , nausea , vomiting .
ct angiogram showed thickening of the terminal ileum , wall thickening and narrowing of multiple large and medium arteries including aorta and left common carotid .
colonoscopy with biopsy of the stenotic ileocecal valve confirmed cd .
resected carotid artery pathology was consistent with ta .
treatment was initially begun with prednisone , then methotrexate was started followed by infliximab .
due to side effects , methotrexate was switched to azathioprine .
he remained asymptomatic .
case 2 : a 38-year - old male with well - characterized crohn 's ileocolitis for 15 years , who had been treated with prednisone , mesalamine , sulfasalazine , and azathioprine presented with chest , upper back and abdominal pain .
ct angiogram showed vasculitis of large and medium arteries , with stenosis of the right renal artery , and wall thickening of the sigmoid colon .
he was diagnosed with ta .
he underwent treatment with infliximab and adalumimab on different occasions , which were later discontinued due to fever , bacteremia and complications from sepsis .
he remained on prednisone and azathioprine .
in these two patients with both cd and ta the diagnoses were confirmed by imaging and pathologic findings .
both patients developed vascular complications .
tumor necrosis factor inhibitor therapy was effective in one patient but discontinued in the other due to infection .
further research into the association of cd and ta may provide clues to their etiologies and guide effective interventions . |
ingestion of caustic agents leads to injuries in
the esophagus , pharynx , larynx , and mouth , which often lead to death or other adverse
effects on the gastrointestinal and respiratory tracts .
ingestion of caustic agents are frequently reported in children , especially when children
start to walk or crawl .
therefore these accidents were considered as one of the main causes
of death in children less than 5 years with the peak incidence at 2 years of age .
however , these incidents also were observed in lower than one years old
children .
annually , more than 40,000 cases of caustic ingestion in children are reported in england
and wales.in galsya , with a population of
half a million children under the age of 14 years , 4.8% of the annual medical service
admissions were associated with caustic ingestion in children .
it is still common in children of the united states , despite the
declining of caustic ingestion in ( 5,000 to 15,000 reductions per year ) , with incidence of
15.8 cases in every 100,000 persons.from 2005 to 2006 , 10% of the 51 children
admitted to the department of pediatrics , children s hospital of tabriz , iran , had stenosis
and gastric outlet obstruction following ingestion of caustic agents.however , prevalence of these accidents in our
area is unknown . in a retrospective study conducted between 2002 and 2004 , 74.7% of the 72
children admitted at the department of pediatrics , loghman hakim hospital of iran ,
there are more than 500 toxic substances at home that most of them were kept at the
kitchen without considering appropriate storing points.additionally , most of ingestions are due to consumption of household
chemicals , such as detergents and bleaches . on the other hand , the
swallowing of these agents could led harmful health consequences and impose an economic
burden for measurement , treatment , follow up and caring.esophageal stricture is
considered a short - term effect , but esophageal perforation , esophageal obstruction and
cancer could be some of the long - term effects of ingestion of caustic agents . although the ingestion of caustic materials is a simple preventable problem , but it has
continued in most developing countries .
families , manufacturing , government and health
staff have a major role in the control and management of these accidents .
persist and ongoing communication among these teams could reduce the rate of accidents or
modify the course of the disease .
the aim of study was to evaluate age
distribution , sex ratio type of caustic agents , signs and symptoms , complications , and
management of caustic ingestion in children by a systematic review and meta - analysis
research method .
also , we are going to determine the maximum events according to age and sex
and the adverse agents and toxic effect that utilizing appropriate preventive strategies
would be effective against accidental caustic ingestion .
this study was performed to review the studies on caustic ingestion in children to
determine the prevalence of age , sex ratio , and complications in children .
we conducted a
systematic review of papers published since 1980 , in four phases .
these involved planning
the search strategy and performing the search , collection of articles and systematic review ,
evaluation of inclusion and exclusion criteria , and data analysis .
these phases are
described in the following sections . in the first step , springer , science direct , proquest , google scholar , and pubmed search
randomly following the prisma guidelines
studies published in english or persian from 1980 to july 14 , 2013 were reviewed .
the keywords used were caustic , corrosive , ingestion , and children , and by medical subject
headings ( mesh ) for medical science databases with the cooperation of a gastroenterology
pediatric subspecialist , physiologist , phd by research , and clinical librarian .
the
following search strategy was employed for pubmed : caustics " [ pharmacological action ] or " caustics " [ mesh terms ] or " caustics " [ all fields ]
or " caustic " [ all fields ] or " caustics " [ pharmacological action ] or " caustics " [ mesh terms ]
or " caustics " [ all fields ] or " corrosive " [ all fields ] and " eating " [ mesh terms ] or " eating "
[ all fields ] or " ingestion " [ all fields ] and " child " [ mesh terms ] or " child " [ all fields ] or
" children " [ all fields ] .
the focus question was established according to the pico ( population , intervention ,
comparison , and outcome ) format : in patients with caustic ingestion in children p ( population ) : patients with caustic ingestion in children .
i ( intervention ) : - c ( comparison ) : age , sex , and complications in children o ( outcome ) : patients who referred to hospital - inclusion criteria : in the second phase , all eligible studies were separately surveyed by two expert
appraisers to evaluate article equality , minimize bias , and to excluded irrelevant studies
( e.g. , related to adults , swallowing batteries , and drug abuse , which were excluded due to
low quality ) based on agreement between the appraisers . related studies were included , and
then debatable articles were referred to the third appraiser .
finally , kappa agreement was considered as inclusion criterion for the next
phase . - exclusion criteria : in the third phase , irrelevant studies , systematic articles , case control articles , cohort
articles , clinical trial articles , animal studies , kappa agreement rate less than 84% ,
duplicated articles , and letters were excluded from the sample . in addition , the articles
which obtained manually from the references of the previously selected articles were added
to the set .
summary of studies contributing to the systematic review in the fourth phase , all papers were divided in two categories ( quantitative and
qualitative).the summery of the four phases are shown in table 1 .
the data was summarized in an extraction table using the microsoft office excel software ,
and were then analyzed using comprehensive meta - analysis ( cma ) and pasw statistics 18 and a
meta - analysis was performed to obtain the measure of an overall effect ( odds ratio , or ) . a
forest plot was used to report the results .
a forest plot is made up of squares in which
each square represents the sample size and the lines beside each square represent the 95%
confidence interval for each study .
the methods of handling data and statistical methods as fixed or random effects models
were used for combining results of studies .
i2 values of 25% , 50% , and 75% correspond to cut - of points for low ,
moderate , and high degrees of heterogeneity .
if condition was homogeneity , fixed model was
used and a heterogeneity condition was used random effects models .
a total of 187 studies were identified based on the exclusion and inclusion criteria . out
of them , 76 papers were excluded in phase 2 , and 56 articles in phase 3 .
further , nine
manually selected articles were added in phase 3 , leading to the final sample of 64 articles
from which data were extracted ( table 2 ) . from 64 ( 11,345 participants ) articles
that were included in our study , 52 articles were
cross - sectional studies ( 81.5% ) , eight articles were case reports ( 12.5% ) , and four articles
were case series ( 6% ) .
most of the studies were conducted in turkey with 14 articles ( 21.8
% ) .
the sample of children comprised 4,632 boy ( 55.33% ) and 3,756 girl ( 44.77% ) . with
reference to distribution of gender , based on the results of different studies and the
random effect model , we found that the frequency of caustic agent ingestion was more common
in boys ( 42% , 95% ci : 0.290.57 ) ; however , this difference was not statistically
significant . the mean age of the children with reported cases of ingestion was 2.78 ( 2.02 )
years ( age range : 12 days10 years ) .
the mean time of endoscopy after ingestion was 30 hours ( range : 1108 hours ) .
the most common caustic substance was alkaline , reported in 41.5% patients , followed by
acid ( 30.7% ) , while most of the cases were exposed to household cleaning agents ,
particularly bleaches and cleaners .
with reference to industrial agents , the most abused
agents were industrial detergents and soda .
various factors were found to have an impact on the severity of the injuries presented .
most common were those related to the physical characteristics of agents , such as
concentration , amount of ingested agent , type of agent , and interval between swallowing and
referral to emergency department .
a majority of the patients were symptomatic at admission , and the most common symptoms
included gastric , respiratory , and ent - related symptoms .
vomiting , abdominal pain ,
dysphagia , respiratory distress , drooling , and burns of the oral cavity were the most
frequently observed symptoms ( table 2 ) .
the majority of the lesions observed in digestive , respiratory , and ent tracts .
the
injuries of digestive included esophageal damage , nutritional problems , gastro - esophageal
reflux , and gastric ulceration , respectively .
skin sore , mouth sore , and sore throat were
reported as lesions of ent . esophageal cancer and even death
the summery of articles based on injuries and complications are shown in table 3 .
our review revealed that physical examination and invasive techniques were carried out
for detection and evaluation of digestive tract lesions .
endoscopy was frequently performed
in addition to other diagnostic methods , such as esophagoscopy and barium swallows .
serum therapy , antibiotics , systemic steroids , anti - acid , h2
blockers , protein pump inhibitors , and topical injection of mitomycin and steroid were used .
implantation of a stent and catheter in the esophagus were also mentioned in some of the
articles . on selecting for the meta - analysis the 64 articles that showed the caustic
ingestion in children , the forest plot stated or=0.53 ( figure
2 ) with a 95% confidence interval ( 95% ci ) of 0.49 - 0.57 ( p=0.08 ) for boys and
or=0.38 ( figure .
2 ) with a 95% confidence interval ( 95%
ci ) of 0.35 - 0.42 ( p<0.001 ) for girls . therefore ,
the studies were non - homogeneous ( i2=83.68% and p<0.001 for boys
& i2=82.44% and p<0.001 for girls ) , so for estimate risk was used from random results .
meta - analysis for boys and girls the extracted data are shown in the funnel plot to evaluate the publication bias for
sample size and effect size ( figure 3 ) . regarding the
funnel plot view , it may be noted that the publication bias in the process of search and
selection in the present study was at an acceptable level ( table 1 ) .
the goal of this systematic review was to evaluate previous literature and examine the age
distribution , sex ratio , and the types of ingested agents , symptoms and signs , management ,
and the impact of chemical factors on the complications caused by the ingestion of caustic
agents in children because of highlighting on primary preventive programs .
the widespread
use , easy availability , low costs of cleaners and detergents especially in asiaas well as other products have led to an
increase in the occurrence of swallowing of agents among children . in term of demographic characteristics , we found that the group at the highest risk for
accidental caustic ingestion comprised children of preschool ages ( mean age = 1.74 ( 3.38 )
years ) , and mostly boys were involved in such incidents . in a study by seluck otcu in
turkey , 58% of the children who ingested caustic substances were boys , and 42% were girls ,
with a mean age of 3.6 ( 2.8 ) years.other
related studies had similar results .
children , especially boys , have well - developed skills and are curious to examine substances
and drink them .
this
implies that boys in preschool stage ( 34 years ) need more attention and caring by parents
to avoid such ingestion incidents .
moreover , parents behaviors are affected further by
culture.it is recommended to consider these points by families : - the large amounts of detergent must not be kept at the home , especially at kitchen . - chemical substances should be placed on the locked cabinet and the upper shelves and were
immediately put in its place after utilization . - chemical materials should be kept in the labeled containers with tight door .
- when a drug is given as a medicine , it not be mentioned as candy .
- the phone number of emergency department and poisoning control center should be recorded
in notebook at home . in this present review
, we found that most accidents were due to alkaline agents , such as
bleach products , oven cleaners , and soda .
these products are often used to clean the house ; however , they can cause various types of
poisoning . in a study conducted in
australia ,
76% of the accidents were reported to have occurred at home , where 74% were
caused by alkaline , 6% by acidic agents , and 20% were caused by contamination with other
agents .
parents should avoid storing such substances in everyday , unmarked drinking bottles ,
especially in containers that are known to hold edible substances .
it was reported in iran
and turkey that in most cases such caustic agents had been stored in accessible places in
the house and in containers without labels.most of the studies emphasized the impact of educating parents by
multimedia for raising their awareness and self efficacy in their care of
children .
the present study revealed that the most common signs and symptoms of caustic ingestion
were related to digestive , respiratory , and ent tracts . in a study conducted in turkey
in addition ,
respiratory symptoms were observed in 13% of the cases . in order to detect lesions and evaluate the severity of the damage , physical examination ,
non - invasive techniques , and invasive technique especially endoscopy was used for nearly all
patients .
ferguson et al . , believed that the first step in the treatment of these patients
is physical examination.in this present
review , the mean of initiating time of the endoscopic examination was 30 hours . in different
studies ,
managing and following of
patients result in the early diagnosis of complications and the application of continues
therapeutic recommendations and health care.the increasing of parent awareness toward preliminary canings after agent
swallowing and the handling of health and caring centers on proper admission , management and
care could be helpful about this episode .
esophageal damage , gastro - esophageal reflux , and gastric ulceration , respectively were
common injuries of corrosive substance ingestion in children .
malignant transformation was reported
during long - term follow - ups . in the study conducted in turkey ,
esophageal stricture observed
in 24.2% ( 16 patients ) by endoscopy . in
another study in south korea ,
thus , it is clear that although ingestion of
caustic substances is relatively rare , its effects are debilitating . in the present study
, we found those medical therapies such as oral antibiotics , systemic
steroids , and h2 blockers ; surgical therapy , such as esophageal dilatation ; and conservative
treatments were used .
assessment of involvement of other organs and recovery through massage could help to
prevent secondary problems . in our study ,
medical therapies such as oral antibiotics ,
systemic steroids and h2 blockers , surgical therapies such as esophageal
dilatation and conservative treatments were utilized to limit the severity of inflammation
and infection .
although the benefit of each treatment have not been determined by
investigators , therapeutic methods should be able to develop as the child growth .
the major advantage of the present study was the use of an appropriate number of authentic
studies .
this advantage , along with the large sample size across all the selected studies ,
enabled us to use the funnel plot to assess publication bias .
however , a limitation in this
study was the poor accessibility of european and american papers because of problem
sanctions on iran .
finally , we recommend that more case control or clinical trial studies
should be performed to provide guidelines and protocols .
this study implies the role of families , manufacturers , media , health care systems , and
government in these accidents .
thus , the primary prevention and reduction of undesirable health effects of accidental
ingestion of caustic substances is practical and simple . also , we recommend that continues management and caring health programs should be applied
because of the effects range from an asymptomatic state to intractable complications . with
reference to treatment strategies ,
thus , comprehensive and continues treatment should be
carried out in all cases of caustic ingestion ; consist of medical therapies such as oral
antibiotics , systemic steroids and h2 blockers , surgical therapies such as esophageal
dilatation and conservative treatments were utilized to limit the severity of inflammation
and infection .
| introduction : caustic ingestion that occurs accidently is one
of the most common problems in children .
methods : this systematic review has been performed by
searching the databases including science direct , proquest , google scholar , and pubmed .
a
strategic search was performed with keywords including caustic , corrosive , ingestion , and
children , and was limited to articles in english and persian .
data were analyzed using
comprehensive meta - analysis2 and pasw statistics 18 .
results : we selected 64 articles regarding caustic
ingestion with a total sample of 11,345 cases .
the data analysis indicated a higher
consumption in young boys ( age range 2.78 ( 2.02 ) years ( or=0.53 with a 95% confidence
interval of 0.49 - 0.57 ( p=0.08 ) ) .
the most common caustic substances were household
cleaning agents , particularly bleaches and cleaners .
esophageal cancer and death were
reported as well as digestive and respiratory complications .
invasive and expensive
techniques are frequently used for diagnosis , treatment and follow up .
conclusion : the results demonstrated that although caustic
ingestion is a serious problem among children , it is a preventable and manageable issue .
therefore , appropriate efforts by families , government , factories , health team and media
should be made to handle adequately this matter . |
cerebrospinal fluid ( csf ) leaks complicate operations of the cranial base , particularly those involving the anterior skull base ( asb ) .
kassam et al reported on their experience with almost 800 asb procedures and reported a postoperative csf leak , the most common complication they encountered , in 15.9% of patients.1 all cases of postoperative csf leak resolved with placement of a lumbar drain ( ld ) supplemented at times by an endoscopic repair and , in one instance , with open cranial surgery .
other authors have reported a similar salutary effect of ld placement , and it has become a widely accepted strategy in managing postoperative csf leaks.2345678910111213141516 lumbar drains are at times inserted prior to surgery . for open cranial asb procedures , the presumed benefit of an ld is to decrease brain swelling and improve access to the tumor or floor of the anterior cranial fossa .
in addition , it allows controlled drainage of csf postoperatively , which is purported to reduce intracranial pressure and thereby facilitate adequate healing of the dural repair . for endoscopic asb procedures ,
the ld allows delivery of positive pressure via injection of preservative - free saline to facilitate delivery of tumor from the suprasellar compartment , allow injection of fluorescein to detect occult csf leaks , and facilitate healing of dural defect repair techniques .
lee et al reported the effectiveness of ld placement along with endoscopic repair as an effective strategy for csf rhinorrhea related to defects of the frontal and sphenoid sinuses that are associated with meningocele or encephalocele.17 on the other hand , casiano and jassir questioned the utility of routine ld placement for the management of csf rhinorrhea.18 in addition , other authors caution about the risk of neurological injury with the use of ld in asb procedures.19 this paper presents our institution 's experience with preoperative ld placement prior to open and endoscopic asb surgery and evaluates the efficacy and safety of this strategy in preventing postoperative csf leak .
we performed a retrospective chart review of all patients in the past 5 years who underwent ld placement at our institution to identify patients without evidence of a preoperative csf leak who had a ld placed prior to elective asb surgery . since 2006 , 93 patients had lds placed for a variety of indications : to facilitate anterior cranial fossa ( acf ) tumor resection by open ( n = 20 ) or endoscopic ( n = 23 ) approaches ; prior to encephalocele repair for injection of fluorescein ( n = 21 ) , for traumatic csf leaks ( n = 13 ) ; before craniotomy for aneurysm clipping ( n = 9 ) ; or as part of an ld trial related to normal pressure hydrocephalus ( n = 7 ) ( table 1 ) . table 2 delineates the incidence of asb tumors by pathology
. we also analyzed whether there was any correlation between smoking , hypertension , or diabetes and the incidence of postoperative csf leak .
of the 93 patients , 43 underwent elective ld placement prior to open ( n = 20 ) or endoscopic ( n = 23 ) asb surgery . of these 43 patients , 2 had evidence of csf rhinorrhea prior to elective tumor resection ; in both cases the ld and subsequent surgical repair was effective in eliminating a postoperative csf leak .
abbreviations : acf , anterior cranial fossa ; csf , cerebrospinal fluid ; ld , lumbar drain ; nph , normal pressure hydrocephalus .
the technique of ld placement is well described.13 briefly , following intubation , with the patient in the lateral decubitus position , a catheter is inserted into the lumbar subarachnoid cistern at the l4 - 5 interlaminar space using a 14-gauge tuohy needle .
cefazolin ( ancef ; glaxosmithkline plc , london , united kingdom ) is administered prior to insertion and continued until the drain is removed .
the ld is connected to a closed system drainage bag , and csf is drained in 10-cc aliquots during open cranial procedures to facilitate brain relaxation ( up to a maximum of 30 to 50 cc ) . for endoscopic procedures , a 10-cc aliquot of preservative - free saline is injected to increase pressure in the subarachnoid space and facilitate delivery of tumor from the suprasellar compartment . at times ,
, fort worth , texas , usa ) diluted in 9.9 cc of preservative - free saline is injected through the ld to identify areas of dural dehiscence and csf leaks . following surgery ,
the ld is used to drain csf at 10 to 15 cc / hr to facilitate healing of the dural closure by reducing hydrodynamic stress . for open , asb craniotomy , watertight dural closure is achieved either by primary repair or with the use of a pedicled pericranial graft augmented with autologous fat and/or a synthetic polymer glue ( duraseal , covidian , mansfield , massachusetts , usa ) . for endoscopic procedures , autologous fat buttressed by gelfoam ( baxter international , inc . , deerfield , illinois , usa ) and duraseal is used .
significant defects in the floor of the acf or high - flow csf fistulae that may result from entry into the floor of the third ventricle are repaired with a vascularized nasoseptal flap augmented by the above mentioned closure materials .
postoperatively , the ld is positioned at the level of the patient 's shoulder and permitted to drain 10 to 15 cc / hour in the neurosurgical intensive care unit under close monitoring .
patients remain flat while the drain is in place but are permitted to move around when the drain is clamped .
generally , drainage is performed for the first 3 days following surgery , after which the ld is clamped for 24 hours and , if there was no evidence of a csf leak , the ld is removed .
postoperative csf leaks are defined as any extraventricular extravasation of clear fluid such as rhinorrhea or a subgaleal collection .
the cumulative percentage of patients who experienced a postoperative csf leak or other postoperative complication was calculated .
statistical analysis related to patient comorbidities was performed using fischer 's exact test ( p < 0.05 ) .
approval was obtained from the loyola university medical center institutional review board prior to initiating this study .
surgical routes to the asb included transnasal , transsphenoidal endoscopic approaches ( n = 23 ) , bifrontal craniotomy ( n = 11 ) , pterional craniotomy ( n = 6 ) and cranio - orbito - zygomatic ( n = 3 ) approaches . two patients with esthesioneuroblastomas had preoperative csf rhinorrhea ; in both instances , preoperative ld placement and closure with a nasoseptal flap augmented by synthetic polymer glue was effective in eliminating a postoperative csf leak .
of the remaining 41 patients without signs or symptoms of a preoperative csf leak who underwent ld placement prior to elective asb tumor surgery , two patients developed postoperative csf leaks ( 2/41 = 4.9% overall postoperative csf leak rate ) that required a return to the operating room for definitive closure ; both cases were in our endoscopic patient population ( n = 21 ; 2/21 = 9.5% endoscopic postoperative csf leak rate ) .
one of the patients had a recurrent leak after the first postoperative repair and is described below in detail ( case report 1 ) .
no postoperative csf leaks in the open asb cohort were noted ( n = 20 ) .
other postoperative complications included headache , nausea , vomiting , deep vein thrombosis , and myocardial infarction ( table 3 ) : the most common complication noted was nausea ( n = 8 ; 8/43 = 18.6% ) .
we encountered no postoperative drain site hematomas , lower extremity weakness or paresthesias , infections , or retained catheter tips on attempted removal .
abbreviations : csf , cerebrospinal fluid ; dvt , deep vein thrombosis ; stemi , st segment elevation myocardial infarction . of the 41 patients without preoperative csf leaks who had a ld placed prior to surgery , there were 20 males and 21 females . although all patients had some degree of intracranial air on postoperative imaging , no instances of tension pneumocephalus were noted .
hypertension ( n = 15 ) , diabetes mellitus ( n = 5 ) , and the use of tobacco products ( generally cigarette smoking ; n = 22 ) were confounding variables in 76% ( n = 31/41 ) of patients ; no correlation was noted between these variables and the occurrence of a postoperative csf leak .
we did , however , encounter two significant postoperative complications in patients who had preoperative ld placement prior to craniotomy for asb tumor resection .
this 73-year - old caucasian woman with a history of diabetes mellitus , hypertension , and significant cardiac disease ( for which she had previously undergone coronary artery bypass surgery ) presented with headaches and blurry vision in her left eye .
magnetic resonance imaging ( mri ) revealed a homogenously enhancing , sellar lesion with suprasellar extension and compression of the optic chiasm ( fig .
1 ) . she had a ld placed prior to undergoing an uncomplicated endoscopic , transnasal , transsphenoidal resection of a benign pituitary adenoma with suprasellar extension and invasion of the floor of the third ventricle . at the conclusion of the case ,
the patient had a sellar floor reconstruction with abdominal fat bolstered by gelfoam covered by duraseal with nasal packing .
postoperatively , the patient remained flat with the ld set at shoulder level set to drain 10 to 15 cc / hr .
postoperative cranial computed tomography ( ct ) revealed minimal intracranial air and no evidence of hemorrhage ( fig .
1 ) . postoperative polyuria and transient hypernatremia ( peak na 147 mmol / l ) required one dose of desmopressin ( ddavp ; sanofi - aventis , bridgewater , new jersey , usa ) .
approximately 300 cc of csf was drained via the ld over 2 days , the nasal packing was removed , and the ld was clamped and then removed on the fourth postoperative day with no evidence of a csf leak .
preoperative t1-weighted , gadolinium - enhanced axial and sagittal images ( top ) demonstrating an isointense , poorly enhancing sellar mass with suprasellar extension causing mass effect on the optic chiasm ( a ) .
postoperative noncontrast computed tomography ( ct ) head with minimal pneumocephalus noted in the resection cavity and adjacent to the bilateral frontal lobes .
she had acute onset of nausea and a bout of emesis , after which brisk csf rhinorrhea was noted .
she was taken to the operating room for an endoscopic repair of the sella floor with abdominal fat graft and replacement of the ld .
following surgery , the ld was again set to drain at 10 to 15 cc / hr . on the evening of postoperative day 2 ,
the patient acutely became hypotensive , requiring fluid resuscitation and intravenous phenylephrine ( neo - synephrine ; hospira , inc .
an echocardiogram revealed mild right heart strain , and a pulmonary embolism protocol ct revealed a linear filling defect in the right lower lobe .
the ld was removed , an inferior vena cava filter placed , and a heparin drip initiated .
she was taken back to surgery and repeat endoscopy permitted visualization of the floor of the third ventricle and csf leakage draining from the suprasellar cistern .
this was repaired endoscopically with the placement of an abdominal fat graft and a nasoseptal vascularized flap .
she returned to the neurological intensive care unit ( icu ) with a third ld that drained between 275 and 350 cc of csf daily until the drain was clamped on postoperative day 4 .
the ld remained clamped through postoperative day 6 , at which time there were no signs or symptoms of a csf leak , and the ld was removed .
she was subsequently discharged home and has remained asymptomatic without evidence of a csf leak .
this 39-year - old otherwise healthy african american woman presented with a generalized tonic - clonic seizure and intermittent blurry vision .
an mri of the brain revealed a 3 4 cm olfactory groove lesion suggestive of a meningioma ( fig .
she was started on levetiracetam ( keppra ; ucb pharmaceuticals , brussels , belgium ) and had no subsequent seizures .
preoperative t2-weighted , noncontrast axial and coronal images revealing a well - circumscribed , homogenously contrast - enhancing , dural - based olfactory groove lesion measuring 3 4 cm with evidence of peritumoral vasogenic edema .
she underwent a bifrontal craniotomy , cranialization of the frontal sinus , and a simpson grade 3 resection of the tumor .
reconstruction of the dura of the floor of the asb and sequestration of the frontal sinus from the intracranial space was performed using a pedicled , pericranial graft augmented by duraseal .
prior to surgery , an ld was inserted but kept clamped , and 10 mg of dexamethasone ( decadron ; merck and co. , inc . , whitehouse station , new jersey , usa ) , 1 g of keppra , and 25 g of mannitol were administered intravenously ( iv ) .
following dural opening , an additional 25 g of mannitol was administered iv and 10 cc of csf was siphoned from the ld . during the subfrontal / interhemispheric approach ,
an additional 25 g of iv mannitol was given and another 10 cc of csf drained via the ld .
near - total surgical resection was achieved and a small portion of the tumor encasing the left anterior cerebral artery was left intentionally . following surgery , the patient was extubated without incident and had a normal neurological examination .
a postoperative cranial ct obtained within 6 hours of surgery showed an appropriate resection cavity without evidence of hemorrhage or ventricular dilatation .
, the ld was leveled at the shoulder and set to drain 10cc / hr .
tumor pathology was consistent with a world health organization ( who ) ii atypical meningioma with a ki-67 proliferation index of 4% that was progesterone receptor positive and featured prominent nucleoli and several foci of necrosis .
immediate postoperative , noncontrasted computed tomography ( ct ) head with scant pneumocephalus and trace extra - axial hemorrhage ( a ) . left greater than right frontal hypodensity , representing persistent edema , continued mass effect , and reduced subfalcine herniation .
postoperative day 1 , noncontrasted ct head with persistent bifrontal edema , worsened mass effect on the left lateral ventricle ( b ) .
evidence of extensive cerebral edema , effacement of the sulcal - gyral pattern with loss of gray - white differentiation . slit - like ventricles .
mmol / l ) required close fluid and electrolyte monitoring and one dose of iv ddavp .
she was also reported to be intermittently confused oriented only to name and place but the csf drainage through the ld was continued as per the original protocol , draining 10 cc / hr . over the next 10 to 12 hours , she was reported to be awake and alert , with a nonfocal , normal neurological examination . on the afternoon of postoperative day 1 , she had a brief episode of unresponsiveness and was noted to have sluggish , 6 mm dilated pupils bilaterally .
the ld was immediately clamped and an emergent cranial ct scan revealed significant bifrontal cerebral edema with crowding of the basal cisterns ( fig .
she was administered 100 g of iv mannitol , 20 mg of iv decadron , and 1 g of iv keppra .
electroencephalography ( eeg ) did not reveal any epileptiform discharges or subclinical seizures . within an hour ,
the patient regained her normal neurological examination , an examination that she maintained until the evening of postoperative day 3only requiring intermittent iv ddavp to control elevated urine output . on the evening of postoperative day 3 ,
the patient became acutely unresponsive . on examination , she was noted to have 6 mm , nonreactive pupils with a dysconjugate gaze and flexor posturing . she was given 50 g of iv mannitol .
she then proceeded to have a generalized , tonic - clonic ( gtc ) seizure which required administration of 4 mg of iv lorazepam ( ativan , baxter international , inc . ,
deerfield , illinois , usa ) 1 g of iv keppra , and 1 g of iv fosphenytoin ( cerebyx ; pfizer inc .
, new york , new york , usa ) before her tonic - clonic activity ceased .
subsequent cranial ct scan revealed persistent bifrontal cerebral edema with crowding of the basal cisterns .
she gradually regained consciousness , had intermittent eye opening with spontaneous movement of all four extremities , and spontaneous , normal respirations , but she did not follow commands . on the morning of postoperative day 4 , despite adequate antiepileptic medications , she had another gtc seizure , this time requiring intubation .
the patient 's cranial ct scan showed no change from the previous afternoon . a right frontal external ventricular drain ( evd )
was placed with an opening pressure of 25 cm of h2o with an appropriate waveform .
after 20 cc of csf drained briskly , the catheter stopped draining , and the monitor showed a poor waveform .
a left - sided intracranial pressure monitor was then placed and confirmed elevated pressures of between 21 and 24 mm hg .
the patient showed no signs of neurological improvement and was found to have fixed and dilated pupils , absent brain stem reflexes , and no movement of the extremities .
she was taken to the operating room emergently for removal of her cranial bone flap and resection of swollen and infarcted brain tissue .
a cranial ct scan showed progression of the already severe bifrontal edema with evidence of bilateral anterior cerebral artery infarctions ( fig .
3 ) . the next morning , eeg and cerebral blood flow studies confirmed brain death , and the family withdrew care .
this 68-year - old caucasian woman who had previously undergone radical hysterectomy and bilateral salpingo - oophorectomy for stage ii - a cervical carcinoma presented with a seizure prompting an mri that demonstrated a 3 3 2 cm contrast - enhancing planum sphenoidale lesion suggestive of a meningioma ( fig .
prior to her scheduled admission for surgery , she developed pulmonary complaints and was diagnosed with a pulmonary embolus requiring a 6-month course of warfarin ( coumadin ; bristol - myers squibb company , new york , new york , usa ) .
preoperative axial t2-weighted , noncontrasted enhanced ( a ) and coronal t1 gadolinium - enhanced ( b ) images showing a 3 3 cm , dural - based planum sphenoidale mass with local mass effect .
, she underwent an uncomplicated bifrontal craniotomy and gross total resection of the tumor that was found to be a who grade i meningioma without atypical features . at surgery , the frontal sinus was cranialized and then sequestered from the intracranial compartment by a pedicled pericranial graft that was also used to reconstruct the dural defect augmented by abdominal fat and duraseal . prior to surgery
, she received a loading dose ( 15 mg / kg ) of iv phenytoin ( dilantin , pfizer inc .
, new york , new york , usa ) , in addition to her maintenance dose of keppra and 10 mg of iv decadron .
following surgery , she was extubated uneventfully and had a normal neurological examination ; postoperative ct scan revealed a complete tumor resection with minimal , nontension pneumocephalus ( fig .
immediate postoperative , noncontrasted computed tomography ( ct ) head with bifrontal hypodensity representing persistent cerebral edema ( a ) . local mass effect .
postoperative day 1 , noncontrasted ct head with persistent bifrontal edema with continued mass effect on the lateral ventricles ( b ) . improved pneumocephalus , extra - axial hemorrhage .
postoperative day 6 , noncontrasted ct head with resolving bifrontal cerebral edema , decreased mass effect , and more appropriately demarcated sulcal - gyral pattern ( c ) . on the morning of postoperative day 1 ,
the patient was neurologically intact , and her ld was opened to drain 10 to 15cc / hr . through the afternoon and evening , she reported intermittent nausea and had several episodes of emesis .
on postoperative day 2 , the patient became acutely somnolent with a labored breathing pattern requiring emergent intubation .
the ld was clamped , and a cranial ct revealed bilateral frontal edema ( fig .
her neurological examination steadily improved , returning to normal and permitting successful extubation 12 hours later .
she remained on a low dose of mannitol for 2 days and her decadron was tapered over 1 week .
she was discharged 6 days after surgery with a normal neurological examination and continues to do well at follow up more than 12 months after surgery .
this 73-year - old caucasian woman with a history of diabetes mellitus , hypertension , and significant cardiac disease ( for which she had previously undergone coronary artery bypass surgery ) presented with headaches and blurry vision in her left eye .
magnetic resonance imaging ( mri ) revealed a homogenously enhancing , sellar lesion with suprasellar extension and compression of the optic chiasm ( fig .
1 ) . she had a ld placed prior to undergoing an uncomplicated endoscopic , transnasal , transsphenoidal resection of a benign pituitary adenoma with suprasellar extension and invasion of the floor of the third ventricle . at the conclusion of the case ,
the patient had a sellar floor reconstruction with abdominal fat bolstered by gelfoam covered by duraseal with nasal packing .
postoperatively , the patient remained flat with the ld set at shoulder level set to drain 10 to 15 cc / hr .
postoperative cranial computed tomography ( ct ) revealed minimal intracranial air and no evidence of hemorrhage ( fig .
1 ) . postoperative polyuria and transient hypernatremia ( peak na 147 mmol / l ) required one dose of desmopressin ( ddavp ; sanofi - aventis , bridgewater , new jersey , usa ) .
approximately 300 cc of csf was drained via the ld over 2 days , the nasal packing was removed , and the ld was clamped and then removed on the fourth postoperative day with no evidence of a csf leak .
preoperative t1-weighted , gadolinium - enhanced axial and sagittal images ( top ) demonstrating an isointense , poorly enhancing sellar mass with suprasellar extension causing mass effect on the optic chiasm ( a ) .
postoperative noncontrast computed tomography ( ct ) head with minimal pneumocephalus noted in the resection cavity and adjacent to the bilateral frontal lobes .
on postoperative day 5 she had acute onset of nausea and a bout of emesis , after which brisk csf rhinorrhea was noted .
she was taken to the operating room for an endoscopic repair of the sella floor with abdominal fat graft and replacement of the ld . following surgery ,
the ld was again set to drain at 10 to 15 cc / hr . on the evening of postoperative day 2 ,
the patient acutely became hypotensive , requiring fluid resuscitation and intravenous phenylephrine ( neo - synephrine ; hospira , inc .
an echocardiogram revealed mild right heart strain , and a pulmonary embolism protocol ct revealed a linear filling defect in the right lower lobe .
the ld was removed , an inferior vena cava filter placed , and a heparin drip initiated .
she was taken back to surgery and repeat endoscopy permitted visualization of the floor of the third ventricle and csf leakage draining from the suprasellar cistern .
this was repaired endoscopically with the placement of an abdominal fat graft and a nasoseptal vascularized flap .
she returned to the neurological intensive care unit ( icu ) with a third ld that drained between 275 and 350 cc of csf daily until the drain was clamped on postoperative day 4 .
the ld remained clamped through postoperative day 6 , at which time there were no signs or symptoms of a csf leak , and the ld was removed .
she was subsequently discharged home and has remained asymptomatic without evidence of a csf leak .
this 39-year - old otherwise healthy african american woman presented with a generalized tonic - clonic seizure and intermittent blurry vision .
an mri of the brain revealed a 3 4 cm olfactory groove lesion suggestive of a meningioma ( fig .
she was started on levetiracetam ( keppra ; ucb pharmaceuticals , brussels , belgium ) and had no subsequent seizures .
preoperative t2-weighted , noncontrast axial and coronal images revealing a well - circumscribed , homogenously contrast - enhancing , dural - based olfactory groove lesion measuring 3 4 cm with evidence of peritumoral vasogenic edema .
she underwent a bifrontal craniotomy , cranialization of the frontal sinus , and a simpson grade 3 resection of the tumor .
reconstruction of the dura of the floor of the asb and sequestration of the frontal sinus from the intracranial space was performed using a pedicled , pericranial graft augmented by duraseal . prior to surgery ,
an ld was inserted but kept clamped , and 10 mg of dexamethasone ( decadron ; merck and co. , inc .
, whitehouse station , new jersey , usa ) , 1 g of keppra , and 25 g of mannitol were administered intravenously ( iv ) .
following dural opening , an additional 25 g of mannitol was administered iv and 10 cc of csf was siphoned from the ld . during the subfrontal / interhemispheric approach ,
an additional 25 g of iv mannitol was given and another 10 cc of csf drained via the ld .
near - total surgical resection was achieved and a small portion of the tumor encasing the left anterior cerebral artery was left intentionally . following surgery ,
a postoperative cranial ct obtained within 6 hours of surgery showed an appropriate resection cavity without evidence of hemorrhage or ventricular dilatation .
, the ld was leveled at the shoulder and set to drain 10cc / hr .
tumor pathology was consistent with a world health organization ( who ) ii atypical meningioma with a ki-67 proliferation index of 4% that was progesterone receptor positive and featured prominent nucleoli and several foci of necrosis .
immediate postoperative , noncontrasted computed tomography ( ct ) head with scant pneumocephalus and trace extra - axial hemorrhage ( a ) . left greater than right frontal hypodensity , representing persistent edema , continued mass effect , and reduced subfalcine herniation .
postoperative day 1 , noncontrasted ct head with persistent bifrontal edema , worsened mass effect on the left lateral ventricle ( b ) .
evidence of extensive cerebral edema , effacement of the sulcal - gyral pattern with loss of gray - white differentiation . slit - like ventricles .
immediate postoperative diabetes insipidus and transient hypernatremia ( peak na 153 mmol / l ) required close fluid and electrolyte monitoring and one dose of iv ddavp .
she was also reported to be intermittently confused oriented only to name and place but the csf drainage through the ld was continued as per the original protocol , draining 10 cc / hr . over the next 10 to 12 hours , she was reported to be awake and alert , with a nonfocal , normal neurological examination . on the afternoon of postoperative day 1 , she had a brief episode of unresponsiveness and was noted to have sluggish , 6 mm dilated pupils bilaterally .
the ld was immediately clamped and an emergent cranial ct scan revealed significant bifrontal cerebral edema with crowding of the basal cisterns ( fig .
she was administered 100 g of iv mannitol , 20 mg of iv decadron , and 1 g of iv keppra .
electroencephalography ( eeg ) did not reveal any epileptiform discharges or subclinical seizures . within an hour
, the patient regained her normal neurological examination , an examination that she maintained until the evening of postoperative day 3only requiring intermittent iv ddavp to control elevated urine output . on the evening of postoperative day 3 ,
on examination , she was noted to have 6 mm , nonreactive pupils with a dysconjugate gaze and flexor posturing . she was given 50 g of iv mannitol .
she then proceeded to have a generalized , tonic - clonic ( gtc ) seizure which required administration of 4 mg of iv lorazepam ( ativan , baxter international , inc .
, deerfield , illinois , usa ) 1 g of iv keppra , and 1 g of iv fosphenytoin ( cerebyx ; pfizer inc . , new york , new york , usa )
subsequent cranial ct scan revealed persistent bifrontal cerebral edema with crowding of the basal cisterns .
she gradually regained consciousness , had intermittent eye opening with spontaneous movement of all four extremities , and spontaneous , normal respirations , but she did not follow commands . on the morning of postoperative day 4 , despite adequate antiepileptic medications , she had another gtc seizure , this time requiring intubation .
the patient 's cranial ct scan showed no change from the previous afternoon . a right frontal external ventricular drain ( evd )
was placed with an opening pressure of 25 cm of h2o with an appropriate waveform .
after 20 cc of csf drained briskly , the catheter stopped draining , and the monitor showed a poor waveform .
a left - sided intracranial pressure monitor was then placed and confirmed elevated pressures of between 21 and 24 mm hg .
the patient showed no signs of neurological improvement and was found to have fixed and dilated pupils , absent brain stem reflexes , and no movement of the extremities .
she was taken to the operating room emergently for removal of her cranial bone flap and resection of swollen and infarcted brain tissue .
a cranial ct scan showed progression of the already severe bifrontal edema with evidence of bilateral anterior cerebral artery infarctions ( fig .
eeg and cerebral blood flow studies confirmed brain death , and the family withdrew care .
this 68-year - old caucasian woman who had previously undergone radical hysterectomy and bilateral salpingo - oophorectomy for stage ii - a cervical carcinoma presented with a seizure prompting an mri that demonstrated a 3 3 2 cm contrast - enhancing planum sphenoidale lesion suggestive of a meningioma ( fig .
, she developed pulmonary complaints and was diagnosed with a pulmonary embolus requiring a 6-month course of warfarin ( coumadin ; bristol - myers squibb company , new york , new york , usa ) .
preoperative axial t2-weighted , noncontrasted enhanced ( a ) and coronal t1 gadolinium - enhanced ( b ) images showing a 3 3 cm , dural - based planum sphenoidale mass with local mass effect .
, she underwent an uncomplicated bifrontal craniotomy and gross total resection of the tumor that was found to be a who grade i meningioma without atypical features . at surgery , the frontal sinus was cranialized and then sequestered from the intracranial compartment by a pedicled pericranial graft that was also used to reconstruct the dural defect augmented by abdominal fat and duraseal . prior to surgery
, she received a loading dose ( 15 mg / kg ) of iv phenytoin ( dilantin , pfizer inc .
, new york , new york , usa ) , in addition to her maintenance dose of keppra and 10 mg of iv decadron .
following surgery , she was extubated uneventfully and had a normal neurological examination ; postoperative ct scan revealed a complete tumor resection with minimal , nontension pneumocephalus ( fig .
immediate postoperative , noncontrasted computed tomography ( ct ) head with bifrontal hypodensity representing persistent cerebral edema ( a ) . local mass effect .
postoperative day 1 , noncontrasted ct head with persistent bifrontal edema with continued mass effect on the lateral ventricles ( b ) .
postoperative day 6 , noncontrasted ct head with resolving bifrontal cerebral edema , decreased mass effect , and more appropriately demarcated sulcal - gyral pattern ( c ) .
on the morning of postoperative day 1 , the patient was neurologically intact , and her ld was opened to drain 10 to 15cc / hr . through the afternoon and evening , she reported intermittent nausea and had several episodes of emesis .
on postoperative day 2 , the patient became acutely somnolent with a labored breathing pattern requiring emergent intubation .
the ld was clamped , and a cranial ct revealed bilateral frontal edema ( fig . 5 ) .
her neurological examination steadily improved , returning to normal and permitting successful extubation 12 hours later .
she remained on a low dose of mannitol for 2 days and her decadron was tapered over 1 week .
she was discharged 6 days after surgery with a normal neurological examination and continues to do well at follow up more than 12 months after surgery .
a variety of surgical approaches to address pathology of the asb are described and are effective.20212223242526 however , despite advances in technology and refinement of technique , postoperative csf leaks are still observed in 5 to 20% of patients undergoing cranial base procedures.1427282930 inserting a drain into the lumbar subarachnoid cistern has become an acceptable strategy for mitigating postoperative csf leak following cranial base procedures.2345678910111213141516 in 2007 , bien et al performed a retrospective analysis of 150 posterior fossa craniotomy surgeries and noted that preoperative ld placement decreased the incidence of postoperative csf leak by 23%.13 similar practice is extrapolated to and has been found effective with asb procedures.1 however , some authors question the utility of an ld altogether and others have challenged the prudence of routine , prophylactic ld placement prior to asb surgery , describing an increased incidence of postoperative intracranial complication , in particular tension pneumocephalus.1215 some authors even present data suggesting that particular types of csf leak , csf rhinorrhea for example , are comparatively less likely to respond to ld placement.31 weaver et al reported on 62 patients who underwent preoperative ld placement prior to thoracoabdominal aortic aneurysm repair.32 the ld was inserted at the l2 - 3 level in 21% , l3 - 4 level in 53% , and l4 - 5 level in 25% of patients .
two patients developed intradural hematomas at the insertion site ; one of them had lower extremity neurological deficits as a result .
yates et al did a retrospective review of 22 craniofacial procedures in 18 patients over a 54-month period and noted the development of pneumocephalus in 7 patients and tension pneumocephalus in 2 of these 7 patients.33 both instances of tension pneumocephalus occurred in patients undergoing ld placement and was further complicated by the development of transient diabetes insipidus .
ransom et al retrospectively evaluated 65 patients undergoing ld placement prior to endoscopic asb surgery.19 the ld remained in place an average of 2 to 3 days .
nine ld complications occurred in eight patients . a persistent lumbar csf leak in five patients required epidural blood patch placement .
significant overdrainage , inadvertent disconnection , inappropriate opening , and a retained intradural catheter tip were noted each in one patient .
roland et al retrospectively reviewed the charts of 32 patients undergoing ld placement for csf fistula over a 3-year period.10 minor complaints included headache , nausea , and vomiting and were noted in 59% of patients .
major complications were noted in 12.5% of patients and included unilateral occlusion of the posterior cerebral artery and unilateral vocal cord palsy .
all complications , major and minor , were relieved by cessation of lumbar csf drainage .
pepper et al reported on data gathered from their population of 161 patients who underwent transglabelar , subcranial approaches to the asb over a 14-year period.15 of these , 41 patients had an ld placed prior to surgery .
they noted that routine placement of a ld was associated with an increased risk of tension pneumocephalus and intracranial complications and did not significantly reduce the incidence of csf leak as a result of the operations .
grady et al retrospectively reviewed the charts of 530 patients undergoing ld placement prior to transsphenoidal surgery.12 no new neurological deficits attributable to insertion of the ld were noted .
thirteen patients developed a spinal headache ; seven of these required an epidural blood patch to relieve their symptoms .
their conclusion was that insertion of an ld in an anesthetized patient prior to surgery was a safe procedure .
this study revealed that ld insertion prior to surgery is an effective way to mitigate the risk of postoperative csf leaks following open and endoscopic asb procedures .
the two cases of postoperative csf leaks despite ld placement occurred in the endoscopic population and , in one instance , was easily remedied by a return to the operating room and careful reconstruction of the floor of the anterior cranial fossa .
* however , the patient with a recurrent csf leak following initial repair merits careful analysis . as noted in this patient , in cases where the floor of the third ventricle is violated , a high - flow csf fistula results that may not be eliminated by autologous fat and synthetic polymer glue packing even when augmented by a ld .
such instances and recurrent csf rhinorrhea cases require a vascularized nasoseptal flap closure of the asb augmented by autologous fat , gelfoam , and synthetic glue .
as kassam et al noted , and as done in this case , the use of a vascularized nasoseptal flap is a significant factor in achieving a successful closure of the csf fistula.1 the second and third case vignettes highlight the problem of cerebral edema that accompanies some tumors such as meningiomas , particularly atypical or anaplastic meningiomas , and the risk of lumbar csf drainage in these patients following open asb procedures .
one patient had a who grade ii atypical meningioma , whereas the other patient had a who grade i meningioma but with significant vasogenic edema , as is sometimes noted with the secretory variant of the tumor
. similar problems of cerebral edema may be observed in kadish stage c esthesioneuroblastomas that invade the dural and pial boundaries .
cerebral edema may exist prior to surgery in these patients and may be exacerbated by surgery , particularly if venous structures are sacrificed to get access to the tumor .
lumbar csf drainage in these cases can result in significant neurological compromise due to downward herniation syndromes either through the tentorial incisura or the foramen magnum . in patient # 2 ,
the ct obtained after neurological decline did indeed demonstrate crowding of the cerebellar tonsils into the foramen magnum that was not previously noted .
patient # 2 also developed problems with seizures , which are frequently seen with atypical or anaplastic meningiomas .
although this decline could be attributed to seizures and cerebral edema , it is also possible that the ld played a role .
bloch and regli described a case of a patient who developed brainstem and cerebellar dysfunction 3 days after removal of a ld that was placed following a pterional craniotomy for clipping of an aneurysm.34 an mri scan demonstrated both tentorial and foramen magnum herniation .
the patient was successfully treated with ventricular drainage , an epidural blood patch , and being placed in a trendelenburg position . presumably , a persistent csf fistula existed even after the ld was removed , allowing csf egress through the drain site , and some element of postoperative cerebral edema or swelling existed that led to the herniation syndrome described .
as with any nonrandomized , retrospective review without a control arm , there are limitations to this study .
in addition , the three cases described may represent anecdotal evidence at best . however , the lessons are germane to the practice of all practicing skull base surgeons and consistent with the experience of other surgeons .
it has also caused us to alter our practice , and this has resulted in excellent outcomes with no further ld - induced complications . at present , we insert a ld only in endoscopic cases with suprasellar or limited intracranial tumor extension without mass effect .
we do not insert a ld for large , intracranial asb lesions with significant mass effect or vasogenic edema .
drainage of csf is only commenced after a 12-hour observation period in the neurosurgical icu with a ct scan that demonstrates no evidence of cerebral edema , tension pneumocephalus , or hydrocephalus and with open basal cisterns .
drainage is performed at 10 to 15 cc / hour during daytime hours from 8:00 am to 5:00 pm with close neurological monitoring for 3 days .
the drain is clamped on the fourth day and then , after 24 hours , removed in the absence of a csf leak .
our experience demonstrates that ld placement is generally well - tolerated by patients we had no postoperative complications directly attributable to drain placement and is an effective means by which to minimize postoperative csf leak after asb craniotomy
. recurrent or high - flow csf leaks require a vascularized pedicled nasoseptal flap reconstruction .
the risk of brain herniation syndromes with ld placement is present in patients with large intracranial tumors , especially those prone to generating significant vasogenic edema , and ld placement is avoided in these patients .
neurological decline in a patient with an ld in place necessitates an immediate iv bolus of mannitol and a cranial ct to rule out tension pneumocephalus or intracranial hypertension . | this study assesses the efficacy of preoperative lumbar drain ( ld ) placement prior to elective open cranial and endoscopic anterior skull base ( asb ) surgery in reducing postoperative cerebrospinal fluid ( csf ) leak .
a retrospective review of 93 patients who underwent ld placement at our institution between 2006 and 2011 was performed . of these patients ,
43 underwent elective ld placement prior to asb surgery ; 2 patients had evidence of csf rhinorrhea prior to surgery , and 41 had no evidence of a preoperative csf leak .
of those 41 patients , 2 developed csf rhinorrhea ( 2/41= 4.9% ) as a result of surgery all in our endoscopic patient population ( n = 21 ; 2/21= 9.5% ) .
no postoperative csf leaks were noted in our open asb surgery cohort ( n = 20 ) .
other complications were rare , but we encountered two instances of delayed malignant cerebral edema in the open asb cohort that are discussed in detail .
overall , preoperative ld placement was found to be an effective means of preventing postoperative csf leaks after asb approaches , but potential and significant intracranial complications may occur in select patients that merit careful consideration prior to ld placement . |
neuroleptic malignant syndrome ( nms ) is a rare but the most severe side effect associated mostly with the use of antipsychotics.1 the most frequent signs and symptoms of nms include fever , muscle rigidity , autonomic dysfunction ( eg , tachycardia , labile blood pressure , tachypnea ) , confused mental state , and changes in laboratory findings , and untreated nms can lead to a life - threatening coma.2 clozapine - withdrawal effects are characterized by psychotic and somatic symptoms .
delusions , hallucinations , altered consciousness , insomnia , motor restlessness , and autonomic dysfunction have been reported.3,4 rapid withdrawal effects of clozapine present usually in the first 2 weeks .
we report a case of a patient who developed nms following the use of a combination of clozapine and haloperidol .
recognition of sudden clozapine - withdrawal effects is important during prolonged clozapine therapy in the psychiatric clinic .
ethical written informed consent was obtained from the patient in accordance with protocols approved by the clinical research ethics committee of the third affiliated hospital of sun yat - sen university .
the patient , who met the diagnostic and statistical manual of mental disorders , fourth edition ( dsm)-iv criteria for schizophrenia , was a 37-year - old female .
treatment with antipsychotics was initiated at 18 years of age , and the patient had been treated in our department since that time .
the patient had been treated with clozapine ( 350 mg / day ) for more than 5 years . with the exception of slight hypotension
seven days before admission to hospital , she was reported to present with anxiety and insomnia , because her only daughter would leave her to go elsewhere .
she presented exacerbation of the psychotic symptomatology with delusions and hallucinations , which required her to be readmitted to our department .
she had no family history of psychiatric illness , and no alcohol or substance abuse was reported .
physical and neurological examinations were normal ( pulse rate 90 per minute , body temperature 36.4c , and blood pressure 110/70 mmhg ) .
initial laboratory tests , which included erythrocyte - sedimentation rate , glucose levels , cholesterol levels , thyroid - function tests , rheumatoid factor , complete blood count , kidney and liver assessments , and urinalysis , were within normal ranges .
clozapine was subsequently increased to 425 mg / day upon her admission and increased to 475 mg / day after 5 days of hospitalization . at the time , she had suicidal ideas and impulsive behavior .
after 6 days of hospitalization , the patient presented with agitation , diaphoresis , finger tremor , and mild muscle rigidity .
these symptoms were thought to be due to extrapyramidal symptoms . at that time , trihexyphenidyl ( 4 mg / day ) was added to control her extrapyramidal symptoms . after 7 days of hospitalization
her vital signs at the time included a heart rate of 170 bpm , blood pressure of 160/70 mmhg , respiratory rate of 38 , and temperature of 39.5c .
laboratory investigations revealed a serum creatine kinase ( ck ) level of 1,166 u / l ( normal range : 24184
u / l ) with a normal myocardial component ( ck - mb ) and a white blood - cell count of 12.4110 cells / mm .
apart from treatment with the previous drugs , there were no other risk factors for conscious disturbance identified in the patient .
subsequently , her medications , including clozapine , haloperidol , and trihexyphenidyl , were stopped .
she received supportive therapy and additional treatments , including a urapidil hydrochloride injection to control pulse rate and blood pressure , an ice blanket to reduce her temperature , intravenous administration of diazepam ( 50 mg / day ) for muscular rigidity and agitation , and mannitol to reduce brain edema .
after 14 days of hospitalization , a marked improvement in nms symptoms was noted , and her vital signs included normal heart rate , blood pressure , respiratory rate , and temperature . however , the patient presented in a confused state with insomnia and auditory hallucinations .
this condition remained unimproved over the course of 2 months , despite her being treated with olanzapine ( daily dose of 20 mg ) and risperidone ( daily dose of 6 mg ) for 4 weeks .
afterward , she was given only a rechallenge with clozapine at a starting dosage of 25 mg / day .
at the beginning , the patient s condition was recognized as nms caused by the combination of clozapine and haloperidol .
the following nms symptoms were observed in the case : impaired consciousness , rigidity , autonomic dysfunction ( eg , tachycardia , labile blood pressure , tachypnea , diaphoresis ) , tremor , and delirium .
the patient described fully met the criteria for nms as proposed in the dsm - iv .
although the precise pathophysiological mechanism underlying nms remains unknown , it is believed to be related to striatal dopaminergic hypofunction caused by a blockade of striatal dopamine receptors .
the antidopaminergic properties of antipsychotics could cause nms , which is supported by the disorder s response to dopamine agonists , such as bromocriptine.5 a genetic association study has sought to identify polymorphisms influencing susceptibility to nms , especially with respect to the dopamine d2 receptor , serotonin receptor , and cytochrome p450 2d6.6 it has been suggested that serotonergic and noradrenergic receptors might have a role in the pathophysiology of nms.7 sympathoadrenal hyperactivity has been reported as one of the types of pathophysiology of nms.8 nms symptoms are remitted only by drug discontinuation and supportive care.9 diazepam is useful for treating muscular rigidity and agitation.10 after an episode of nms , the patient presented with altered mental status , and two principal hypotheses were considered : 1 ) the condition was due to reexacerbation of chronic schizophrenia and 2 ) the condition was due to clozapine withdrawal .
there was , especially after an episode of nms , a therapeutic dilemma about the treatment of this case .
at least 2 weeks should elapse before a rechallenge with antipsychotic medications , and the second medication should be different from the medication implicated in the initial nms episode.11 nms is the one extreme of neuroleptic - related toxicity.12 it was questionable whether it was proper to give a patient who presented with symptoms of nms a medication that could cause such a condition or even worsen it , especially because of its unclear clinical picture and lack of drug concentrations in the serum .
we propose that abrupt clozapine withdrawal induced mental status changes after nms - symptom resolution in the patient .
the fact that it persisted despite her receiving other medications , and the rapid improvement observed after clozapine readministration , further supports this contention .
clinicians need to be aware of the withdrawal effects of neuroleptics when they are suddenly discontinued .
cholinergic overdrive -aminobutyric acid supersensitivity may contribute to many of the symptoms of acute clozapine withdrawal.3 clinically , whenever possible , clozapine should be slowly tapered .
if side effects necessitate rapid withdrawal , the use of anticholinergics would help to improve at least some of the symptoms.13
in conclusion , psychiatrists not aware of possible clozapine - withdrawal effects may misdiagnose as a part of the primary mental illness or initial symptoms worsening if unrecognized . | here , we report a female patient developing neuroleptic malignant syndrome following the use of a combination of clozapine and haloperidol .
subsequently , the patient presented withdrawal effects after an abrupt discontinuation of clozapine .
psychiatrists not aware of possible clozapine - withdrawal effects may misdiagnose as a part of the primary mental illness or as the initial symptoms worsening , if unrecognized . |
high caffeine containing energy drinks are widely consumed by adolescents . due to the risk - taking behavior of adolescents and their lack of education on the negative effects of caffeine , cases of toxicity due to the consumption of energy drinks are likely to continue to increase [ 13 ] .
in particular , there is growing concern about a decrease in male reproductive health , because a number of laboratory animal studies and human cases have pointed to toxic effects of caffeine on the male reproductive system [ 37 ] .
for instance , caffeine induced testicular atrophy in adult animals , resulting in decreased spermatogenesis . in addition , gestational and lactational exposure of rats to caffeine had adverse effects on the size and structure of the testis , serum testosterone levels , and the fertility of male offspring . since the adolescent gonad is not fully mature either anatomically or histochemically , it would not be surprising if chronic exposure to caffeine during this vulnerable period were to interfere with normal gonad maturation . though detrimental effects of caffeine on the development of the reproductive system during prenatal period have been widely demonstrated ,
data on the effect on the reproductive system during the prepubertal period are relatively sparse and conflicting .
the purpose of this study was to investigate the effects of caffeine on the parameters of growth and maturation of the testis in prepubertal male rats throughout most of their rapid growth period .
we assessed changes in the gross and microscopic structure of the testis along with changes in body composition caused by caffeine exposure .
in addition , effects of caffeine on serum testosterone , known to be important for pubertal sexual maturation , were evaluated , and ex vivo testosterone production was measured in leydig cells retrieved from testes after caffeine exposure to assess their secretory activity .
sprague - dawley ( sd ) male rats ( n = 45 ) aged two weeks were obtained from samtako biokorea ( kyunggi , south korea ) .
the rats were allowed to acclimate until 21 days of age before being used in experiments .
each animal was housed in a separate plastic cage under controlled conditions ( 2224c , humidity 4050% , and 12-h light - dark cycle ) with free access to food and water .
animal care was consistent with institutional guidelines , and the hanyang university acuc committee approved all procedures involving animals ( hy - iacuc-2013 - 0110a ) .
the experiment was started when the rats were 22 days of age , because postnatal days 2225 are considered the beginning of puberty in the rat .
rats were assigned to groups by the stratified randomization method based upon body weight on the day before the start of treatment , in order to eliminate variation in mean body weight between the groups .
rats weighed approximately 50 2 g on the first day of the experiment .
louis , mo , usa ) was dissolved in distilled water ( 10 ml / kg ) at concentrations calculated to deliver 120- and 180-mg / kg body weight / day ( designated as cf1 and cf2 , n = 15/group ) .
animals were fed caffeine dissolved in water by gavage to ensure complete consumption of the established dose , once daily in the morning ( 9 to 11 am ) .
fifteen animals in the control group ( ctrl ) received the same volume of distilled water used as vehicle .
a treatment duration of 4 weeks was selected to cover the time of rapid growth of the reproductive system of rats ( from 22 days to 55 days ) .
animals were examined for clinical signs and weighed on a daily basis , and food intake was also monitored .
body weight was measured to the nearest 0.1 g with an electronic scale ( dretec corp . ,
seoul , korea ) and recorded on the first day before feeding caffeine ( initial ) until the day they were killed ( final ) .
all the animals were killed 24 h after the last treatment following protocols and ethical procedures .
blood samples were collected at 4 weeks by heart puncture , and serum samples were stored at 70c until further analysis .
the body composition of all the animals was evaluated at the end of the experiment by dual - energy x - ray absorptiometry ( discovery w qdr series , hologic inc . ,
application of this software had been validated in very young animals as an excellent noninvasive technique for measuring fat and lean tissue in the rat .
total body mass ( tbm ) , lean body mass ( lbm ) , and total body fat were measured .
animals were anesthetized by isoflurane inhalation during examination . to elucidate the effects of caffeine on gonad growth ,
both testes were dissected from each rat and cleaned of fat and attached tissues at necropsy .
they were then weighed to the nearest 0.001 g with an electronic scale ( adventure electronic balances , ar1530 , ohaus corp . ,
usa ) and their morphology was grossly evaluated . to measure testosterone ( t4 ) production ex vivo , one testis from each male rat
was sliced into four pieces weighing approximately 50~100 mg each . isolation and purification of leydig cells followed a similar protocol to that previously described .
briefly , the testis was minced under sterile conditions and the tissue was then dissociated in dissociation buffer containing collagenase d ( roche applied science , indianapolis , in ) , dnase i ( roche ) , and dispase ii ( sigma - aldrich , st .
the tubules were then rinsed in dmem / f12 and allowed to settle and the supernatant was filtered through 100-m nylon mesh and centrifuged in percoll gradient buffer .
the resulting pellet , containing leydig cells , was resuspended in dmem / f12 containing 2% bsa , placed in 24-well plates ( 3 10 cells / well ) and cultured with or without luteinizing hormone ( 200 ng / ml ) for 24 h at 37c , in a 5% co2 incubator . after 24 h of incubation , the medium was collected for t4 assay . immediately after removal ,
serial sections of 5 m thickness were taken from the midportion of each testis and stained with hematoxylin and eosin .
all histomorphometric evaluations were performed by the same trained , calibrated , and blinded examiner using an image analysis system ( leica las software ) coupled to a light microscope ( dm4000b , leica , heidelberg , germany ) with final magnifications of 100x or 200x .
four serial sections were traced for each testis per animal , and eight measurements per section were made of the number of seminiferous tubules within two defined regions ( 1.23 mm ) at 100-fold magnification and these were combined to obtain a mean value per animal .
eight measurements per section were made for analyzing the diameter of seminiferous tubules or area of intertubular space within the same defined regions ( 0.307277 mm ) at 200-fold magnification , and mean value was calculated .
the thickness of the seminiferous epithelium was obtained by subtracting the luminal diameter from the tubule diameter in the same seminiferous tubule and combining the measurements to obtain a mean value per animal .
t4 levels were analyzed in serum samples and in the conditioned media collected from the cultured leydig cells using a commercially available enzyme - linked immunosorbent assay ( elisa ) kit ( cusabio biotech co. , ltd . , china ) .
intra- and interassay coefficients of variance were less than 15% , and the limit of detection was 0.06 ng / ml under our conditions .
the absorbance was read at 450 nm within 15 min , against a blanking well in elisa reader ( biorad , hercules , ca ) .
all samples were run in duplicate in the same assay and each elisa was repeated twice .
statistical significance was determined by kruskal - wallis one - way analysis of variance for multiple group comparisons .
as shown in figure 1(a ) , all groups gained weight throughout the experimental period .
although there were no differences in starting body weights between the groups ( ctrl , 53.1 1.5 g ; cf1 , 52.6 1.6 g ; cf2 , 52.6 2.1 g ) , the caffeine - fed groups weighed significantly less than the controls .
the difference was significant from the first week of the experiment and it persisted throughout the experiment .
the final body weights of the treatment groups were significantly less than those of the controls ( ctrl , 242.2 12.2 g ; cf1 , 190.5 14.8 g ; cf2 , 183.8 11.7 g , p < 0.001 versus ctrl ) .
likewise , food consumption was decreased in the caffeine - fed groups from the 1st week of caffeine exposure , although a significant reduction was only attained in the cf2 ( p < 0.05 versus ctrl ) .
the significant reduction persisted until the end of experiment ( p < 0.001 versus ctrl ) ( figure 1(b ) ) .
parallel with tbm , body fat and lbm also declined in the caffeine - fed groups compared to the control ( p < 0.05 ) .
however , the percentage of body fat was not significantly different ( ctrl versus cf1 versus cf2 ; 17.9 2.2% , 16.2 2.6% , and 16.1 2.5% ) , although some decrease was observed in the caffeine - fed groups .
likewise , the percentage of lbm did not differ between the control and caffeine - fed groups , pointing to a proportional reduction in body fat and lbm in the caffeine - fed groups . to investigate the effect of caffeine on the reproductive organ in vivo ,
the testes were weighed ( figure 3 ) . as shown in figure 3(a ) , absolute testis weight was significantly reduced in the caffeine - fed groups compared to the control ( ctrl , 1.56 0.09 g ; cf1 , 1.27 0.23 g ; cf2 , 1.27 0.09 g ) ( p < 0.001 versus ctrl ) . on the contrary ,
testis weights relative to body weight were significantly increased in caffeine - fed groups ( ctrl , 6.35 0.34 ; cf1 , 6.78 0.57 ; cf2 , 7.01 0.25 mg / g body weight ) ( p < 0.001 versus ctrl ) ( figure 3(b ) ) .
as clearly shown in the representative picture ( figure 3(c ) ) , caffeine feeding caused a significant reduction in testes size .
the histomorphometric parameters analyzed on histological sections of the testes are summarized in table 1 .
caffeine consumption caused a dose - dependent reduction in the mean diameter of the seminiferous tubules ( ctrl , 295.60 14.03 ; cf1 , 280.12 9.05 ; cf2 , 271.82 11.58 ) ( m ) ( p < 0.01 versus ctrl ) .
although there was no statistically significant difference between the caffeine - fed groups , mean diameters in cf2 were smaller than in cf1 .
also , the height of the germinal epithelium was significantly reduced in cf2 compared to both the control ( p < 0.001 ) and cf1 ( p < 0.01 ) . on the other hand ,
the numbers of seminiferous tubules counted within the same defined areas were similar in the control and caffeine - fed groups .
in contrast , the intertubular area increased dose - dependently in the caffeine - fed groups ( p < 0.01 , ctrl versus cf1 ; p < 0.001 , ctrl versus cf2 ) . the histomorphometric changes noted in the caffeine - fed animals most likely contributed to the reduction in testes size and weight .
there were lower levels of t4 in both caffeine - fed groups , although a significant reduction was attained only at the 180-mg dose ( p < 0.05 versus ctrl ) ( figure 5(a ) ) .
serum levels of t4 could be highly variable depending on releasing pulses from the testes into the blood ; thus we also analyzed ex vivo t4 production from primary leydig cells retrieved from the caffeine - fed animals ( figure 5(b ) ) .
basal - t4 productions were not different between the control and caffeine - fed groups , but stimulated - t4 productions were profoundly decreased in leydig cells retrieved from the caffeine - fed animals compared to those from the control . on the other hand , serum lh levels
were also measured at the end of experiment ( ctrl : 4.64 2.68 , cf1 : 6.33 4.28 , and cf2 : 11.72 14.94 miu / ml ; mean sd ) and the differences were not significant due to variability .
our data demonstrate that chronic high doses of caffeine exposure during the pubertal growth period interfere with testis growth and testosterone secretion , accompanied by histomorphometric changes in the testis . considering that male offspring are more susceptible than females to caffeine treatment during both gestation and lactation [ 3 , 16 ]
, we chose male rats to evaluate whether pubertal caffeine exposure has effects similar to prenatal exposure .
the choice of dose levels was based on the literature coupled with range finding studies to avoid sublethal effects at the highest dose .
the median lethal dose of caffeine administered orally to rats is 192 mg / kg .
the doses ( 120- or 180-mg ) chosen here , which have been reported to interfere with components of the endocrine system such as the pituitary and adrenal glands in the fetuses of treated pregnant rats , were within the range found to have no undue systemic toxicity in our preliminary study . currently , body surface area- ( bsa- ) based dose calculations are the most appropriate method for extrapolating from animals to humans and they are far superior to simple conversions based on body weight . using dose conversion based on the bsa in humans and rats ,
the dosages employed in this study were equivalent to approximately 19.4- and 29.1-mg / kg in humans .
therefore , one or two energy drinks can easily lead to the above doses of caffeine .
a number of human and animal studies have demonstrated that gestational and lactational caffeine exposure causes fetal growth retardation and low birth weight .
likewise , caffeine - fed immature male rats showed a significant reduction in body weight .
consistent with this , we noted a significant reduction in body weight gain in the caffeine - fed animals after only one week ( p < 0.001 versus ctrl ) and these reductions persisted throughout experimental period ( figure 1(a ) ) .
in contrast , another study found no difference between the control and caffeine - fed groups . this discrepancy may result from differences in doses , duration , administration route , and age between the studies . because food intake was persistently decreased after 1 week of caffeine exposure ( figure 1(b ) ) and caffeine is known to increase metabolic rate and fat oxidation
, these effects could contribute to the lower body weight gain in the caffeine - fed animals .
also , since body weight and food intake influence the onset of puberty , reduced body weight resulting from caffeine exposure could contribute to the delayed pubertal growth . increased muscle mass and body fat are two of the major physical changes characterizing normal pubertal development .
previous studies suggest that , more than weight , an appropriate body composition or a certain percentage of fat is needed to start puberty in rats . along with the poor weight gain in the caffeine - fed groups , lbm and
many human studies have pointed to a major influence of body fat on the onset of puberty [ 25 , 26 ] .
although the reductions of muscle and body fat in the caffeine - fed groups were proportional to the tbm , body fat percentage declined more in the caffeine - fed groups than in the control ( ctrl = 17.9 2.2% , cf1 = 16.2 2.6% , and cf2 = 16.1 2.5% ) .
this result suggested that caffeine may inhibit the fat deposition and muscle growth normally occurring during puberty . among the major physical alterations of normal pubertal development ,
although appropriate weight and adiposity might be necessary for puberty to occur in girls , previous reports showed that a critical body weight does not seem to be essential in males [ 28 , 29 ] .
in addition , rapid testicular growth is stimulated by adequate bioavailability of gonadotropin and testosterone [ 3032 ] .
thus , a negative influence of caffeine on testis growth during the pubertal period may be related to disrupted hormonal milieu .
consistent with our result , maternal caffeine exposure causes a significant dose - dependent reduction in the testis weight of offspring .
in contrast , no effect of caffeine exposure on testis weight was noted in adult rats . considering that puberty is a critical period of sexual maturation , caffeine exposure may cause more adverse effects in this period than in adulthood when
the testis is largely composed of tightly coiled seminiferous tubules , which contain differentiated germ cells [ 34 , 35 ] .
the size or number of seminiferous tubules could be a main factor to determine the weight of the testis . as expected from the reduction in testis size , the diameter of seminiferous tubules decreased in the caffeine - fed groups ( table 1 ) .
similarly , prenatal caffeine exposure significantly reduced seminiferous tubule diameter and inhibited differentiation of the seminiferous cords . on the other hand
, we did not find any difference in the number of seminiferous tubules between groups and this could be explained by the fact that the pubertal period is a period of organic maturation rather than morphogenetic reorganization .
several animal studies have also demonstrated a negative influence of caffeine on the seminiferous tubules , although the dose , duration , administration route , and animal strains were different [ 36 , 37 ] .
as the seminiferous tubule consists of the germinal epithelium including differentiated spermatogenic cells [ 34 , 35 ] , a reduction in seminiferous tubule size points to detrimental changes in the germinal epithelium .
in fact , adult animal studies revealed marked damage to the germinal epithelium in the form of degeneration or disruption .
our results also showed a reduction in germinal epithelium height , which may reflect a decreased number of germ cells or differentiated spermatogenic cells .
it is known that optimal sperm production does not occur until about 75 days , in the rat . here
, we used immature rats ( pd2250 ) and sperm analysis was not appropriate for this age .
although we did not perform sperm analysis , a reduction in spermatogenesis can be assumed .
in addition , it was demonstrated that prenatal caffeine exposure inhibits differentiation of interstitial tissue and leydig cells , leading to a significant reduction in leydig cell number .
like the prenatal effect , we observed histological alteration to the interstitial tissue with loose interstitial tissue and decreased numbers of cells , resulting in significantly increased intertubular distance in the caffeine - fed groups ( figure 4 ) .
consistent with our result , caffeine exposure in adult rat led to degeneration of leydig cells .
this discrepancy might be related to caffeine dose because the latter study used an unusually high dose ( 250 mg / kg / day ) , which exceeds the lethal dose ( 192 mg / kg / day ) in the rat .
in addition , adequate circulating t4 plays an important role in the structural and functional integrity of the reproductive organs .
therefore , the reduction of the testis weight in the caffeine - fed groups is associated with inadequate circulating male hormone ( figure 5 ) . prenatal caffeine exposure caused significantly lower t4 levels in male human offspring and rats .
in agreement with previous work , we found that pubertal caffeine exposure affected t4 levels like prenatal exposure .
another study using immature male rats measured t4 levels after 4 weeks of caffeine exposure ( 5 , 20 , and 100 mg / kg / day ) and reported no clear effect .
furthermore , several clinical and animal studies have reported that chronic caffeine consumption increased serum t4 levels in adult human and adult animals .
the difference may be due to differences in the method of assay , administration protocols , or age of experimental subjects ( young or older rats ) .
however , it is clear that caffeine consumption interferes with serum t4 levels in immature male rats , contributing to the impaired gonadal maturation and function .
on the other hand , decreased t4 may also contribute to the reduction in muscle mass in caffeine - fed animals ( figure 2 ) because it is an important stimulator of skeletal muscle development in men .
the decreased serum t4 level in caffeine - fed animals may be the result of impaired synthesis or enhanced metabolism . as serum t4 is largely produced by the testicular leydig cells , caffeine would be expected to affect androgen - producing cells .
an in vitro study reported that caffeine increased t4 secretion in a cell line and primary rat leydig cells . because in vitro study would not reflect precisely in vivo status , we used cells retrieved from treated animals to measure secretory activity more precisely .
as shown in figure 5 , a blunted response to lh stimulation was noted in leydig cells from the caffeine - fed groups .
it has been reported that prenatal caffeine exposure significantly inhibited the enzyme activity of leydig cells relevant to androgen production .
in addition , an animal study suggested a direct effect of caffeine on the testis instead of an indirect effect via alterations of lh secretion .
therefore , pubertal caffeine exposure may directly affect leydig cells possibly by alteration of enzyme activity and result in a reduction in circulating t4 levels . in conclusion
, caffeine may interfere with testicular steroidogenesis , particularly during puberty and sexual maturation , although the mechanism underlying the effects of caffeine on the leydig cells in immature animals remains to be determined . until now
, there have been no data on the interaction between the leydig cells , t4 , and caffeine exposure during the puberty .
our results demonstrate that caffeine can interfere with the endogenous t4 secretion from the leydig cells , possibly by reducing the sensitivity of the leydig cells to gonadotrophic stimulation .
in addition , we confirmed that pubertal administration of caffeine reduced testis growth and altered testis histomorphology . | prenatal caffeine exposure adversely affects the development of the reproductive organs of male rat offspring .
thus , it is conceivable that peripubertal caffeine exposure would also influence physiologic gonadal changes and function during this critical period for sexual maturation .
this study investigated the impact of high doses of caffeine on the testes of prepubertal male rats .
a total of 45 immature male rats were divided randomly into three groups : a control group and 2 groups fed 120 and 180 mg / kg / day of caffeine , respectively , via the stomach for 4 weeks .
caffeine caused a significant decrease in body weight gain , accompanied by proportional decreases in lean body mass and body fat .
the caffeine - fed animals had smaller and lighter testes than those of the control that were accompanied by negative influences on the histologic parameters of the testes .
in addition , stimulated - testosterone ex vivo production was reduced in leydig cells retrieved from the caffeine - fed animals .
our results demonstrate that peripubertal caffeine consumption can interfere with the maturation and function of the testis , possibly by interrupting endogenous testosterone secretion and reducing the sensitivity of leydig cells to gonadotrophic stimulation .
in addition , we confirmed that pubertal administration of caffeine reduced testis growth and altered testis histomorphology . |
menopause is associated with an increase in intra - abdominal fat , which is considered a major risk factor of atherosclerotic cardiovascular disease ( cvd ) . however
, whether an increased risk of cvd in postmenopausal women is due to altered body composition , changes in reproductive hormones , or some other physiological process associated with menopause has not been clearly established .
it is also uncertain as to what extent androidal fat mass may influence cvd risk factors in healthy nonobese women .
the risk of chronic disease is perceived to be considerably higher in obese compared to normal weight adults
. however , gautier et al . recently reported that the effect of waist circumference , reflecting androidal fat mass , in increasing the risk for diabetes was more profound among men and women with a normal body mass index ( bmi ) of less than 25 kg / m than among those with above normal bmi .
thus , it appears that efforts to reduce abdominal fat accumulation would be beneficial regardless of bmi category .
this study was ancillary to the soy isoflavones for reducing bone loss ( sirbl ) study , a randomized , double - blind , placebo - controlled multicenter ( iowa state university ( isu ) and university of california at davis ( ucd ) ) clinical trial funded by the national institutes of health ( nih ) .
we examined contributors ( androidal fat mass , duration of menopause , isoflavone treatment , family history of cvd , and dietary quality ) to cardiometabolic risk in primarily normal and overweight healthy postmenopausal women .
cardiometabolic outcomes included circulating low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triacylglycerol , glucose , homeostatic model assessment ( homa ) of insulin resistance , uric acid , c - reactive protein ( crp ) , fibrinogen , and homocysteine ( hcy ) .
we hypothesized that consumption of soy isoflavones in tablet form would attenuate the anticipated increase in central fat ( androidal ) mass during the 36 months of treatment , which in turn may favorably modify circulating concentrations of cardiometabolic risk factors .
we also hypothesized that androidal fat mass , other biologic factors , and dietary quality would influence these cardiometabolic risk factors , but that androidal fat mass would predominate .
the parent study examined the effect of two doses ( 80 versus 120 mg / day ) of soy protein - derived isoflavone versus placebo tablets for 36 months on bone loss in healthy postmenopausal women ( 45 to 65 years of age ) who were at risk for osteoporosis .
the parent study included power analysis on the primary outcome ( lumbar spine bone mineral density ) in the methodology and has been previously reported .
many of these cardiometabolic risk factors were tested throughout the study ( ldl - c , hdl - c , tag , glucose , and uric acid ) whereas some were only tested through the 12 month time point ( homa , crp , fibrinogen , and hcy ) .
200210884 - 2 ) approved our study protocol , consent form , and all participant - related materials .
approvals for the dual - energy x - ray absorptiometry ( dxa ) procedures were obtained from each institution 's irb and state department of public health in iowa and california . at prebaseline , each participant received a detailed explanation of the study verbally and in writing before signing an informed consent form .
we recruited ( 2003 to 2005 ) women throughout the state of iowa and in the greater sacramento and bay area regions in northern california primarily through direct mailing lists , stories in local newspapers , and local / regional radio advertisements .
responders ( n = 5,255 ) were initially screened via telephone to identify healthy women ( without diseases or conditions , not taking hormones or medications ) 65 years who had undergone natural menopause ( cessation of menses 1 to 10 years ) , were not experiencing excessive vasomotor symptoms , nonsmokers ( not currently smoking and who had not smoked in the past 6 months ) , and had a bmi between 18.5 through 29.9 ( except for 9 women from ucd who did not meet the range of inclusion criterion : 8 women had bmi values that ranged from 30 to 32.7 and one woman had a bmi of 17.8 , but were enrolled because they were deemed healthy ) . the parent sirbl project established the inclusion / exclusion criteria .
we excluded vegans and high alcohol consumers ( > 7 servings / week ) , as well as those who were diagnosed with chronic disease , had a first - degree relative with breast cancer , or who chronically used medication ( current : cholesterol - lowering and/or antihypertensive ; past 3 months : antibiotics ; past 6 months : estrogen / progestogen creams , calcitonin ; past 12 months : oral hormones / estrogen or selective estrogen receptor modulators ; ever : bisphosphonates ) .
women who met the initial screening criteria ( n = 677 ) were invited to the clinic for further eligibility screening , including bmd assessment using dxa .
the sirbl project focused on disease prevention rather than treatment ; thus , women with bmd lumbar spine ( l1-l4 ) and/or proximal femur t - scores that were low ( > 1.5 sd below young adult mean ) or high ( > 1.0 sd above mean ) or with evidence of previous or existing spinal fractures were excluded .
once each woman qualified based on bmd , fasting blood was drawn for a clinical chemistry profile .
we excluded women with evidence of diabetes mellitus ( fasted glucose 6.93 mmol / l ( 126 mg / dl ) ) , abnormal renal , liver ( elevated enzymes ) , and/or thyroid function , or elevated lipids ( ldl - cholesterol > 4.10 mmol / l ( 160 mg / dl ) ; triacylglycerol > 2.25 mmol /
we excluded 13 women at ucd from this analysis because they did not meet the entry criteria ( 11 had thickened endometrium , 1 had breast cancer , 1 could not provide a baseline blood sample ) .
we excluded an additional 19 women who did not have body composition data at either 12 , 24 , or 36 months because they dropped out of the study , resulting in a sample size of 224 women based upon androidal fat as our primary covariate of interest in this ancillary project . to meet the objectives of the parent project
, participants at each location ( isu , ucd ) were stratified according to baseline proximal femur bmd ( high , medium , and low ) based upon nhanes iii database population values and randomly assigned to one of three treatment groups : placebo control , 80 mg isoflavones , or 120 mg isoflavones .
tablets were provided by the archer daniels midland co. ( decatur , il ) ; tablet composition has been described previously . an independent researcher ( patricia murphy ) at isu
confirmed that the actual isoflavone doses ( mean sd , mg / d ) were similar to those formulated and tested by archer daniels midland , respectively : control = 0 compared with 0.3 0.4 ; 80 mg = 89.5 5.0 compared with 84.3 4.5 ; 120 mg = 124.0 7.7 compared with 122.5 3.4 .
participants in each group were instructed to take three compressed tablets / d . to preserve the double - blind nature of the study , bottles did not indicate treatment assignment . for this study , we used anthropometric and body composition measurements that included weight , height , whole body lean and fat mass , and androidal fat mass , as well as fasting concentrations of lipids / lipoproteins , glucose , and uric acid .
in addition , circulating insulin , homa ( calculated as fasting glucose ( mg / dl ) fasting insulin ( u / ml)/405 ) , crp , fibrinogen , hcy , and red blood cell ( rbc ) folate concentrations were assessed at baseline and 12 months .
standing height was taken twice ( average value recorded ) with a wall - mounted stadiometer ( model s100 ; ayrton corp . ,
prior lake , mn ) and weight was measured at isu using a balance beam scale ( abco health - o - meter ; bridgeview , il ) and at ucd using an electronic scale ( circuits and systems inc ; e. rockaway , ny ) .
women wore hospital scrubs or shorts and a t - shirt , and removed their shoes , belts , watches , and jewelry for the duration of assessment . to ensure standardized data collection ,
body composition measurements were obtained by certified cross - trained dxa operators using matching dxa instruments ( delphi w hologic inc ; bedford , ma ) at each site that were calibrated daily .
to further ensure quality control , one operator assessed overall composition from the whole body dxa scans for both sites . regional adiposity analysis
was performed by one analyzer ( lr ) who sectioned each whole body dxa scan into waist , hip , and thigh regions based on bone landmarks using special software ( discovery version 12.3:7 ) .
the hip region began below the fourth lumbar vertebrae and extended to the tip of the greater trochanter of the femur .
androidal fat mass ( kg ) for each participant was the sum of waist and hip fat mass .
phlebotomists collected fasted ( 9 h ) blood samples between 7:00 and 8:00 am .
we separated serum ( allowed to clot for 30 min prior to centrifugation ) and plasma from whole blood and centrifuged samples for 15 minutes ( 4c ) at 1000 g , storing aliquots at 80c until analyses .
certified clinical laboratories ( labcorp ; kansas city , ks for isu and ucd medical center ; sacramento , ca for ucd ) performed a chemistry panel ( including serum glucose , lipid profile , and uric acid ) on each participant at each time point .
we measured the remaining analytes ( serum insulin , serum crp , plasma fibrinogen , plasma hcy , and rbc folate ) for each participant in batch from isu and ucd samples at baseline and 12 months in duplicate at isu .
we used sufficient in - house sera / plasma as quality - control samples ( frozen at 80c ) to run with each kit to calculate interassay coefficient of variation ( cv ) ; we used duplicate samples to calculate intraassay cv .
the low - to - normal and normal - to - high controls for each kit were well within the acceptable ranges .
serum insulin ( u / ml ) concentration was determined with a radioimmunoassay kit ( linco research , st charles , mo , usa ) using a cobra ii series autogamma counting system ( packard instrument company ; meriden , ct , usa ) .
serum crp ( mg / l ) concentration was determined with a high - sensitivity sandwich enzyme - linked immunosorbent assay kit ( alpco diagnostics ; salem , nh ) and plasma ( heparinized ) fibrinogen ( mg / ml ) concentration was determined with a sandwich enzyme - linked immunosorbent assay kit ( assaypro ; st .
charles , mo ) using a microtiter plate reader ( elx808 ; bio - tek instruments , inc . ,
the intra - assay cvs for crp and fibrinogen were 3.7% and 2.7% , respectively ; the interassay cvs for crp and fibrinogen were 6.0% and 2.3% , respectively .
total hcy ( mol / l ) concentration was determined using a high - performance liquid chromatography ( hplc ) method adapted from araki and sako and ubbink et al . .
the total hcy in plasma consists of free hcy ( i.e. , reduced plus oxidized hcy in the nonprotein fraction of plasma ) and protein - bound hcy .
n - acetylcysteine ( 1 mm ) was added as an internal standard to the plasma samples prior to derivatization .
the fluorescence intensities were measured with excitation at 385 nm and emission at 515 nm , using a jasco fp-1520 fluorescence detector .
intracellular ( rbc ) folate ( ng / ml ) was measured using a radioactive immunoassay kit ( mp biomedicals ; irvine , ca ) . because four hematocrit values were missing , rbc folate could not be calculated for these four samples .
in addition , three baseline samples were incorrectly processed ; thus , rbc folate values have been presented for 217 women .
the intra- and interassay cv for rbc folate were 3.2% and 11.8% , respectively . during the enrollment phase
, trained interviewers administered three questionnaires to participants : a health and medical history [ 1012 ] , a reproductive history , and a nutrition history [ 10 , 11 ] .
we also gathered data on prescription and over - the - counter medications at each time point , as well as previous and/or current use of herbal therapies or dietary supplements ( which they were asked to discontinue prior to baseline testing ) .
we assessed dietary intake at each time point using a semiquantitative food frequency questionnaire from block dietary data systems ( berkeley , ca ) .
a healthy eating index ( hei ) score , with higher scores representing greater adherence to federal dietary guidelines , was calculated for each participant based on this questionnaire and included in the statistical analyses as a covariate .
we performed statistical analyses using version 2.10.1 of the r software , including version 3.1.96 of the nlme package software and considered results statistically significant ( two sided ) at p .05 .
our 3-year longitudinal analyses of ldl - c , hdl - c , triacylglycerol , and glucose included women with complete data at all time points , baseline through 36 months .
our 1-year longitudinal analyses of fibrinogen , insulin , homa , hcy , and crp included women with complete data at baseline and 12 months .
we constructed longitudinal models to identify significant contributors to each cardiometabolic risk factor ( ldl - c , hdl - c , triacylglycerol , uric acid , glucose , insulin , homa , crp , fibrinogen , and hcy ) .
each final longitudinal model included these obligatory variables : treatment ( control versus combined treatment with 80 mg or 120 mg of isoflavones or , in other words , no treatment versus treatment ) , time point ( baseline versus 12 , 24 , or 36 months ) , treatment by time point interaction , and site ( isu versus ucd ) , as well as potential covariates that included androidal fat mass ( kg ) adjusted for height , time since last menstrual period ( tlmp ) ( yr ) ( calculated for each woman : baseline test date date of her last menstrual period ) , family history of cvd coded as a categorical variable ( none versus positive or none versus unknown ) , and hei score .
additionally , the insulin model included glucose as a covariate , and the hcy model included rbc folate as a covariate .
independent variables in modeling the outcomes of interest included those variables that were biologically plausible .
preliminary models also included dietary fat intake ( determined using the block food frequency questionnaire ) and physical activity ( determined using the paffenbarger physical activity recall ) , but those variables did not emerge as remotely significant contributors to any of the cardiometabolic risk factors and thus were not included in the final models .
separate height adjustments of fat and fat - free mass have been suggested for children by wells and cole ; accordingly , we found a significant impact of height on androidal fat mass among the participants in our study based on a log - log regression analysis ( parameter estimate 0.8286 , p = .0058 ) .
this suggested the use of height - adjusted androidal fat mass ( androidal fat mass ( g)/height ( cm ) ) ( exponent of 0.8286 for height changed to 1 for ease of interpretation ) .
in other words , we adjusted androidal fat mass for height because taller women typically had greater waist circumferences due to their larger frame size and thus appeared to be at higher risk for cvd compared with shorter women , whereas this is not necessarily the case .
we performed log transformation of variables with skewed distributions : triacylglycerol , homa , crp , hcy , and rbc folate .
restricted maximum likelihood ( reml ) estimation was used to obtain estimates of variances and correlations between repeated measures .
model selection was guided by a stepwise backwards selection based on model diagnostics , such as akaike 's information criterion and bayes information criterion .
an overall model fit was obtained based on a likelihood ratio test of the ( maximum likelihood fitted ) model at hand and the more parsimonious model of only obligatory covariates .
significance indicates a failure to accept the null hypothesis of covariates contributing to the model in a random fashion .
more specifically , significance indicates that the full model with all of the variables included explains a greater proportion of the variability in the outcome than the parsimonious model .
body composition measurements , including androidal fat mass , did not change significantly during the course of the study .
the isoflavone treatment did not have an effect on any of the body composition outcomes .
the results of longitudinal analysis showed that each cardiometabolic outcome model was highly significant , with overall p - values .0001 for all but the ldl - c model ( p = .002 ) .
androidal fat mass and site ( isu versus ucd ) were consistent predictors of all analytes ( 36 month data ) assessed , including lipids / lipoproteins , glucose , and uric acid .
in general , androidal fat mass was typically the strongest covariate ( positive ) in each model ( except the model with hdl - c as the outcome , where it was significantly and negatively associated ) .
time point also emerged as a significant covariate in the hdl - c and glucose models , indicating that the median concentrations of these analytes increased with time ; however , the analyte concentrations at subsequent time points were not significantly different compared to baseline .
the median glucose concentration was 85 mg / dl at baseline and 88 mg / dl at 36 months .
the median hdl - c concentration was 64 mg / dl at baseline and 65 mg / dl at 36 months .
family history of cvd had a significant association with glucose : women who were unaware of their family history ( do not know , n = 9 ) , taking into account key factors in the model , had on average a 6.8 mg / dl higher glucose concentration compared with women who indicated that they had a positive family history .
isoflavone treatment , tlmp , or hei did not influence any of the 36 month analytes .
similar to the 36 month outcomes , each 12 month outcome model was highly significant ( overall model p - values
.0001 ) , except for hcy ( p = .23 , data not shown ) .
androidal fat mass was positively associated with each outcome ( table 3 ) and was the strongest covariate ( positive ) in each model .
the parameter estimate for site was negative , indicating that the women from ucd had lower values than the women at isu .
in addition , tlmp ( p = .0020 ) and tlmp - by - site interaction ( p = .0021 ) contributed significantly , and a positive family history of cvd contributed marginally ( p = .070 ) to fibrinogen concentration .
the unadjusted mean insulin concentration was significantly greater ( 2.86 u / ml ; p .0001 ) at 12 months compared with baseline .
isoflavone treatment or hei did not emerge as significant predictors in any of the 12 month models .
the main objective of this study was to identify significant contributors to cardiometabolic risk in primarily normal and overweight ( bmi < 30 ) healthy postmenopausal women . in agreement with our hypothesis ,
androidal fat mass was the strongest and most consistent predictor of all cardiometabolic outcomes ( except for hcy ) examined in this study .
our participants , as a group , showed no significant changes in body composition measurements , including androidal fat mass , during the course of the study .
it should be noted , however , that the women were instructed to maintain their weight throughout the course of the study by following their usual diet and physical activity patterns , and hence , we did not expect to document overall or androidal fat mass change from baseline to 36 months . similarly , with the exception of insulin , none of the cardiometabolic risk factors changed significantly between baseline and the time of final assessment ( either 12 mo or 36 mo ) .
nevertheless , strong associations between androidal fat and cardiometabolic risk factors suggested that even a small increase in androidal fat mass may have considerable health consequences .
indeed , biggs et al . reported that the incidence of type 2 diabetes was 70% higher ( a hazard ratio of 1.7 ) in adults 65 years of age and older who gained at least 10 cm in waist circumference over several years compared with those who remained within 2 cm of their baseline waist circumference .
although the biggs 's study did not specifically target postmenopausal women , it provided some insight into the magnitude of the impact of central adiposity on the incidence of type 2 diabetes .
certain factors such as duration of menopause , isoflavone treatment , and diet quality that are recognized in the literature as influential with respect to cardiovascular health did not emerge as significant predictors of the cardiometabolic outcomes examined in this study
for instance , denino et al . found that the relationship between age and blood lipids in nonobese women was abolished after controlling for visceral fat .
similarly , in our study , tlmp did not emerge in any model ( except for fibrinogen ) as a significant covariate , because time point took precedence over tlmp .
results also suggested that androidal fat mass in combination with other factors predominated in these cardiometabolic risk models . on the other hand , our findings for isoflavone treatment and dietary quality conflict with some of the previously published reports .
the lowering effect of soy protein rich in isoflavones on blood lipids / lipoproteins , albeit modest ( 6% reduction ) , has been documented [ 18 , 19 ] .
similarly , the effect of diet quality on cvd outcomes is well known : a healthier diet ( high in fiber and low in fat and sodium ) is associated with a lower risk of cvd [ 20 , 21 ] .
we included both a hei score as a measure of diet quality and isoflavone treatment in our analysis .
the lack of association between hei and cardiometabolic outcomes in our study could be in part explained by a majority of women who had relatively healthy diets ( median hei of 67 out of 100 ) .
very few studies have examined the association between hei and cardiometabolic outcomes with mixed results .
for instance , kant and graubard reported that hei emerged as a negative predictor of serum hcy , crp and plasma glucose ( p < .05 ) , whereas fung and colleagues did not find an association between hei and crp .
in a recent study of 125 multiethnic overweight and obese women in early postpartum , the hei scores were negatively associated with ldl - c and total cholesterol and positively related to hdl - c after adjustment for energy intake , body weight , and lactation status .
based on the results of our study , hei does not appear to be a predictor of cardiometabolic risk factors . on the other hand
, the effects of dietary factors as well as isoflavone treatment on cardiometabolic outcomes may be mediated by androidal fat mass .
we previously reported that soy isoflavone treatment for 12 months did not exert an effect on body composition , including androidal fat , in our sample of women .
with respect to diet , fox et al . determined that premenopausal women who participated in a 24 week diet and/or exercise program showed reductions in weight ( 7 kg ) and total percentage body fat ( although it remained greater than 35% for all groups ) , but no significant improvements in blood lipids , glucose , or insulin concentrations .
further analysis revealed a lack of change in the waist - to - hip ratio , which in turn indicated that body fat distribution was not influenced by the intervention . to summarize , dietary interventions that are not sufficiently potent to reduce androidal fat mass are not likely to produce changes in cardiometabolic outcomes .
in addition to androidal fat mass , the other most common independent predictors of cardiometabolic outcomes in our study included time and site .
although concentrations of hdl - c and glucose did not change significantly during the study , both analytes showed an upward trend from baseline to 36 mo .
the effect of time could be related to biological and/or behavioral factors that were not included in our models .
in fact , tlmp was no longer significant when time was included , indicating that time was more important than tlmp .
the site variable also emerged as a significant covariate in all models : compared with women at isu , women at ucd had lower concentrations of triacylglycerol , hdl - c , glucose , insulin , homa , crp , and fibrinogen and higher concentrations of ldl - c and uric acid .
the site effect may be in part explained by baseline characteristics : isu women were slightly ( albeit not significantly ) heavier and younger compared with ucd women .
it is also quite possible that the site effect was due to some inherent differences between the two geographic locations .
we did not document a relationship between androidal fat mass , as well as other factors , and hcy likely because the women in our study were well below the clinical cut - off of 15 , and hcy values did not indicate great variability .
some of the strengths of our study were that we followed a relatively large number of women and monitored longitudinal changes ( baseline to 12 or 36 mo ) in cardiometabolic risk factors .
limitations were that these women were free - living ( both a strength and limitation ) , lacked ethnic diversity , and underwent infrequent measurements ( yearly ) .
thus , some of our women may have been identified as obese using other criteria , such as percentage body fat ( 3540% = overweight , >
40% = obese ) . in conclusion , although cardiometabolic outcomes examined in this study were not affected by isoflavone treatment , each outcome ( except for hcy ) had a strong , significant association with androidal fat mass .
thus , even small changes in androidal fat are likely to alter the cardiometabolic profile in healthy postmenopausal women .
the sirbl clinical trial was supported mainly by a grant ( ro1 ar046922 ) from the national institute of arthritis and musculoskeletal and skin diseases ( niams ) .
the project was also supported by the nutrition and wellness research center , iowa state university ; usda / ars , western human nutrition research center , clinical and translational science center , clinical research center at the university of california ( 1m01rr19975 - 01 ) , and national center for medical research ( ul1 rr024146 ) .
archer daniels midland company ( decatur , il ) donated soy isoflavone tablets ( novasoy ) and glaxosmithkline ( moon township , pa ) donated calcium and vitamin d supplements ( os - cal ) . | we hypothesized that soy isoflavones would attenuate the anticipated increase in androidal fat mass in postmenopausal women during the 36-month treatment , and thereby favorably modify the circulating cardiometabolic risk factors : triacylglycerol , ldl - c , hdl - c , glucose , insulin , uric acid , c - reactive protein , fibrinogen , and homocysteine .
we collected data on 224 healthy postmenopausal women at risk for osteoporosis ( 45.865 y , median bmi 24.5 ) who consumed placebo or soy isoflavones ( 80 or 120 mg / d ) for 36 months and used longitudinal analysis to examine the contribution of isoflavone treatment , androidal fat mass , other biologic factors , and dietary quality to cardiometabolic outcomes . except for homocysteine , each cardiometabolic outcome model was significant ( overall p - values from .0001 to .0028 ) .
androidal fat mass was typically the strongest covariate in each model .
isoflavone treatment did not influence any of the outcomes .
thus , androidal fat mass , but not isoflavonetreatment , is likely to alter the cardiometabolic profile in healthy postmenopausal women . |
polyneuropathy , organomegaly , endocrinopathy , monoclonal gammopathy , skin changes ( poems ) syndrome causes the following clinical manifestations : ( the initial letters of which form the word poems ) as well as extravascular volume overload , erythrocytosis / thrombocytosis , osteosclerotic lesions , and elevated vascular endothelial growth factor ( vegf ) levels 1 .
the condition is diagnosed based on the presence of both polyradiculoneuropathy and a monoclonal plasma cell disorder , as well as at least one of the other three major criteria and at least one minor criterion .
in addition , it has a rapidly progressive course ; therefore , early diagnosis is important .
pleural effusion , ascites , and/or cardiac effusion are the first symptoms to appear in more than half of patients with poems syndrome , and these conditions can become lifethreatening in some cases 2 . the criteria for poems syndrome vegf : vascular epithelial growth factor . due to the abovementioned unique manifestations of poems syndrome
, it can be difficult to achieve an early or accurate diagnosis in cases in which a patient with undiagnosed poems syndrome develops cancer .
in other words , pleural effusion , ascites , lymphadenopathy , and bone lesions caused by poems syndrome might be misdiagnosed as cancer metastases if the preexisting poems syndrome is not detected .
herein , we present the first case report of poems syndrome with synchronous breast cancer and describe potential diagnostic problems associated with such cases .
a 65yearold female with a chief complaint of coxalgia , which had lasted for 2 years , presented to a nearby hospital .
a computed tomography ( ct ) scan showed multiple sclerotic bone lesions , which were suggestive of an occult primary tumor and multiple bone metastases . despite extensive testing for 3 months
, no final diagnosis was made so the patient was referred to our hospital for a second opinion . by that time , she had developed further symptoms ; that is , a prolonged bloating sensation and visible abdominal distension , which had lasted for a month .
she received a thorough examination at our hospital 's internal medicine department , including various blood tests ; imaging examinations , such as ct and mammography ; upper and lower endoscopy ; and cytoscreening ( a biochemical examination ) of her ascites .
the differential diagnosis process particularly focused on autoimmune connective tissue disorders in addition to a further examination for occult primary cancer .
the patient 's laboratory data were indicative of anemia , thrombocytopenia , low serum albumin levels , kidney dysfunction , and subclinical hypothyroidism , but no autoantibodies associated with autoimmune connective tissue disorders were detected ( table 2a b ) .
a ct scan showed a small to moderate amount of pleural effusion , a large amount of ascites , enlargement of both axillary lymph nodes , and multiple sclerotic bone lesions , but did not detect any tumors of the parenchymal organs or lymphadenopathy ( fig .
c ) . diagnostic aspiration indicated that the patient 's ascites ( table 3 ) was transudative , but not malignant .
a breast ultrasound scan detected a hypoechoic solid tumor ( size : ~1 cm ) containing internal echogenic spots with interruption of both the anterior and posterior borders of the mammary gland ( fig .
therefore , an ultrasoundguided needle biopsy was performed , leading to a histopathological diagnosis of hormone receptorpositive invasive ductal carcinoma without human epidermal growth factor receptor type 2 ( her2 ) protein overexpression ( fig .
although the diagnostic aspiration did not provide conclusive evidence of malignant effusion , after reflecting on the patient 's pathological characteristics , the physicians made a final diagnosis of stage iv / metastatic breast cancer with metastases involving the pleural cavity , peritoneal cavity , lymph nodes , and bone .
the patient began receiving tamoxifenbased hormonal therapy because chemotherapy was deemed inappropriate due to coexisting renal impairment . 1 month later , it became difficult for the patient to live at home because her massive ascites induced a bloating sensation accompanied by pain , shortness of breath , a diminished appetite , nausea , severe fatigue , and edema of the lower extremities .
therefore , she was admitted to our surgical ward in order to undergo peritoneal portcatheter placement to manage her refractory ascites .
laboratory findings.(a ) abnormal laboratory data obtained on admission and the changes in these parameters after chemotherapy treatment .
( b ) a list of the autoantibodies associated with autoimmune connective tissue disorders that were tested for on admission bun : blood urea nitrogen , tsh : thyroidstimulating hormone , ft3 : free triiodothyronine , ft4 : free thyroxine , rnp : ribonucleoprotein , ssa : sjgren 's syndromerelated antigen a , anca : antineutrophil cytoplasmic antibodies , tpo : thyroid peroxidase .
[ correction added on 21 march 2016 after first online publication : the units in tables 2 and 3 were incorrect and have been updated in this version ]
ct scan obtained on admission .
bilateral axillary lymph node enlargement ( white arrows ) , ( b ) . a large amount of ascites , and ( c ) . a bone sclerotic lesion in the pubic symphysis ( white arrow ) .
the results indicated that the ascites was transudative , but not malignant [ correction added on 21 march 2016 after first online publication : the units in tables 2 and 3 were incorrect and have been updated in this version ] imaging studies of the right breast .
a breast ultrasound detected a hypoechoic solid tumor ( size : ~1 cm ) containing internal echogenic spots with interruption of both anterior and posterior border of the mammary gland ( white arrows ) .
histopathological examinations of the right breast an ultrasoundguided needle biopsy detected hormone receptorpositive invasive ductal carcinoma without human epidermal growth factor receptor type 2 ( her2 ) protein overexpression .
the surgeon who performed the physical examination on admission ( the lead author of this article ) noted that the patient had several characteristic physical findings : clubbing and hyperpigmentation of the fingers ( fig .
4b ) ; and polyneuropathy , for example , numbness in both fingers , an inability to sense vibrations in both upper extremities , muscle weakness , and diminished deep tendon reflexes .
the detection of these characteristic physical findings triggered a fundamental review of the previous diagnosis , which required us to reexamine whether the patient 's breast cancer had played a causal role in the development of her pleural effusion , ascites , axillary lymphadenopathy , and sclerotic bone lesions .
when we considered all of the patient 's findings ( other than the breast cancer ) in a comprehensive manner , the differential diagnoses were divided into the following three categories : 1 hematological disorders , such as multiple myeloma , poems syndrome , monoclonal gammopathy of undetermined significance , waldenstrom 's macroglobulinemia , cryoglobulinemia , and amyloidosis ; 2 immunemediated peripheral neuropathies , such as chronic inflammatory demyelinating polyneuropathy ; and 3 autoimmune connective tissue disorders , such as scleroderma .
however , autoimmune connective tissue disorders had already been ruled out , as mentioned above . on the other hand , the patient 's physical findings ; that is , the skin and neurological manifestations , represented valuable clues and led to an indepth examination for poems syndrome . skin changes ( a ) .
finger clubbing with hyperpigmentation ( b ) hyperpigmentation of the back with angiomas to examine whether the patient met the major criteria for poems syndrome ( table 1 ) , we performed an iliac crest bone marrow biopsy , a test for mprotein , and a nerve conduction velocity ( ncv ) test .
the bone marrow biopsy showed normal cellularity and did not detect any bone marrow plasma cell involvement , which excluded multiple myeloma .
5 ) , and the ncv test demonstrated both demyelination and axonopathy in the right upper extremities ( table 4 ) , which were considered to be comorbidities of the patient 's peripheral polyneuropathy .
thereby , the diagnostic criteria for poems syndrome were fulfilled because one element of the other three major criteria ( sclerotic bone lesions ) and three of the minor criteria ( lymphadenopathy , extravascular volume overloading , and skin changes ) had already been met .
in addition , we obtained further strong evidence to support this diagnosis ; that is , a markedly raised vegf level ; the patient 's plasma and serum vegf levels were 555 pg / ml and 1530 pg / ml , respectively .
it has been reported that a plasma vegf level of 200 pg / ml exhibits 95% specificity and 68% sensitivity for diagnosing poems syndrome 3 . according to the japanese diagnostic criteria for poems syndrome issued by the japan intractable diseases information center
, a significantly elevated vegf level is defined as a serum level of > 1000 ng / ml 4 .
therefore , we were able to make a definitive diagnosis of poems syndrome in this rare case , which also involved synchronous breast cancer
a ncv study of the patient 's right side ncv , nerve conduction velocity . as a reference ,
22 this test detected mixed neuropathy in the median motor nerve and ulnar motor nerve ( a long latency , low amplitude , and low ncv ) , and axonal neuropathy in the right ulnar sensory nerve ( a low ncv ) .
the ncv of the median sensory nerve , tibial motor nerve , peroneal motor nerve , and sural sensory nerve were unmeasurable . immediately after the final diagnosis ,
the patient began receiving chemotherapy based on a regimen for multiple myeloma ( 1.3 mg / m bortezomib , sc , on days 1 , 8 , 22 , and 29 of each cycle and 20 mg dexamethasone , po , on days 1 , 2 , 8 , 9 , 22 , 23 , 29 , and 30 of each cycle ; each cycle lasted 6 weeks ) 5 as her condition was becoming increasingly lifethreatening .
after two courses of chemotherapy , the patient 's pleural effusion and ascites had almost disappeared , and her plasma and serum vegf levels fell significantly to 47 pg / ml and 266 pg / ml , respectively ; that is , below the diagnostic cutoff values of 200 pg / ml and 1000 pg / ml , respectively , indicating that these symptoms had been caused not by cancerous dissemination , but by poems syndrome . with the decreasing extravascular volume overload , her circulating plasma volume increased , leading to an improvement in her kidney function ( see table 2a ) , for example , her estimated glomerular filtration rate improved from 18 ml / min to 28 ml / min .
thus , the patient 's condition had improved markedly , and she was well enough to undergo a radical operation for breast cancer under general anesthesia .
a histopathological examination of the resected specimens detected a 10 5 mmsized invasive ductal carcinoma with no lymph node involvement ( 0/19 ) . when investigating the dissected axillary lymph nodes for poems syndrome ,
none of the following histopathological features of castleman 's disease were detected : hyaline vascular , plasmacytic , or mixed lymph node features .
, adjuvant endocrine therapy ( 5 years ' tamoxifen treatment ) was considered sufficient . after receiving several more cycles of the same chemotherapy regimen ,
the patient is scheduled to receive an autologous stem cell transplant in the near future , which is expected to result in a substantial improvement in her neuropathy 7
8 .
poems syndrome was first reported by crow in 1956 9 and was subsequently described by fukase in 1969 10 .
the acronym poems was coined by bardwick in 1980 11 and stands for the five main features of the disease ; that is , polyneuropathy , organomegaly , endocrinopathy , monoclonal gammopathy , and skin changes .
the syndrome was also referred to as crow fukase syndrome by nakanishi in 1984 based on a japanese study of 102 cases 12 , and this name is more widely known in japan than poems syndrome .
although the pathophysiology of poems syndrome is poorly understood , the production of various inflammatory cytokines , such as interleukins ( il1 and il6 ) and vegf , by abnormal plasma cells is postulated to play a direct causal role in various symptoms of the condition 13 , 14 . in particular ,
vegf levels exhibit the strongest correlations with disease activity in poems syndrome 3 , 15 .
plasma and serum vegf levels of 200 pg / ml and 1,000 pg / ml , respectively , are considered to be diagnostic cutoff values for poems syndrome .
elevated vegf levels are postulated to be involved in the pathogenesis of enhanced vascular permeability , increased endoneurial pressure , and the deposition of plasma cellderived material 14 , 16 , 17 , which eventually cause a variety of symptoms , including extravascular volume overloading ( edema , pleural effusion , and/or ascites ) , polyneuropathy ( demyelination ) , sclerotic bone lesions , and skin changes 18 , 19 .
however , the mixed results seen with antivegf therapy indicate that vegf might not be the driver of the clinical manifestations of poems syndrome 20 .
further studies of this issue are required . to the best of our knowledge ( based on a search of the pubmed database ) , this is the first case report about a case of poems syndrome with synchronous breast cancer . using
poems syndrome and synchronous cancer as search keywords , we did not find any relevant articles ( accessed oct 19 , 2015 ) . at present
, there is no evidence that certain types of cancer are strongly associated with poems syndrome .
thus , it is likely that the current patient developed poems syndrome and breast cancer synchronously by chance .
the following three major factors might help to explain the diagnostic difficulties encountered in the present case .
first , poems syndrome is a rare disease ; its prevalence rate was reported to be 0.3 per 100,000 in japan 17 . however , there is another fact that some patients with poems syndrome are first diagnosed at autopsy or go undiagnosed unless properly autopsied because of diagnostic challenges specific to the syndrome 21 , which makes the actual prevalence rate unclear .
second , many of the signs and symptoms of poems syndrome mimic those of other disorders , leading to confusion and difficulties during the diagnostic process .
especially when being diagnosed with a certain type of cancer before the detection of preexisting poems syndrome , pleural effusion , ascites , lymphadenopathy , and bone lesions might be considered to be carcinomatous in origin .
the risk of such mistakes is probably higher in cases involving a metastasisprone cancer , for example , breast cancer .
third , clinicians have recently appeared to rely excessively on laboratory findings and/or imaging studies during the diagnostic process .
the present case should act as a warning against this unfavorable trend and could yield a bitter but helpful lesson that , the more complex a disease is , the more important a physical examination tends to be .
| key clinical messagepolyneuropathy , organomegaly , endocrinopathy , monoclonal gammopathy , skin changes ( poems ) syndrome is a rare plasma cell disorder that causes a paraneoplastic syndrome .
we report the first case of poems syndrome with synchronous breast cancer .
the patient was at risk of being misdiagnosed with metastatic cancer , and it is important to emphasize that physical examinations provided vital diagnostic clues . |
serotonin transporter gene ( slc6a4 , solute carrier family 6 , member 4 ) encodes an integral plasma membrane protein that plays a major role in the regulation of serotonin neurotransmission ( 1 ) .
it has been widely recognized as the target sites of selective serotonin reuptake inhibitor ( ssris ) , which have been recognized as both anti - depressant and anti - anxiety drugs .
two common polymorphisms in the promoter region of the serotonin transporter gene , i.e. 5-httlpr ( polymorphisms in the promoter region of the serotonin transporter gene ) have been reported , together with their functional properties .
( " l " ) and short ( " s " ) alleles confirmed by conventional polymerase chain reaction and electrophoresis , were involved in the transcriptional regulation of the serotonin transporter gene ( 2 , 3 ) .
the short allele has been suggested to decrease transcriptional activities of serotonin transporter gene , although it is uncertain whether they act in a dominant fashion ( 4 ) or a recessive fashion ( 5 - 7 ) . studies have shown that reliably measured personality dimensions are substantially influenced by genetic factors ( 8) . among them , anxiety - related traits such as harm avoidance or neuroticism appears to be one of the most fundamental , enduring , and continuously distributed personality dimensions , which are associated with 5-httlpr ( 2 , 9 , 10 ) .
these associations , however , remain controversial , because numerous other studies yielding negative results ( 11 - 15 ) .
these inconsistencies could be accounted for by various factors including different measures applied , confounding effects of age and gender , and inadequate sample size .
perhaps more importantly , ethnic differences in allele frequencies and population substructure may underlie those inconsistent results . in the present study
, we examined whether association between 5-httlpr and harm avoidance of korean version of the temperament and character inventory ( k - tci ) ( 16 ) should be replicated in korean population .
we also examined other personality traits in k - tci since other personality dimensions of novelty seeking , reward dependence , and persistence are reported to be independently heritable ( 18 ) .
we confined our sample to one college students , which was presumed to be relatively homogenous population regarding age and sociocultural backgrounds .
there were 69 males and 89 females , average age of 23.83.1 ( means.d . )
a 10 ml venous blood sample was obtained , and genomic dna was isolated from peripheral blood leukocytes according to standard procedures .
dna fragments were amplified by polymerase chain reaction using 5'-ggcgttgccgctctgaattgc ( -1,416 ->-1,397 ) and 5'-gagggactgagctggacaacccac ( -910 ->-889 ) primers ( 3 ) .
the pcr reaction mixture contained a total volume of 20 l with the following composition : 12 ng genomic dna , 4 pmoles of the each primers , 2.5 mm dntps , 5 mm of deaza dgtp , and 1 u ampli taq with the appropriate buffer .
after an initial 5 min denaturation step at 95 , 5 cycles were performed consisting of 40 sec at 95 , 40 sec at 58 , and 60 sec at 72 , followed by an additional 35 cycles of 40 sec at 95 , 40 sec at 61 , and 60 sec at 72. the reaction was ended by incubation at 72 for 7 min .
pcr products were separated by the long ( 524 bp ) and short ( 484 bp ) variants on 3% agarose gels with ethium bromide .
we compared harm avoidance scores across the different genotypes ( " ss " , " sl " and " ll " ) and sex .
then , we compared the scores between " ss " group and " sl"+"ll " group , since " s " allele might act as recessive fashion ( 5 , 19 ) . additionally , we compared another 3 dimensional scores of novelty seeking , reward dependence , and persistence .
statistical package for the social sciences ( spss , version 10.0 , spss inc , chicago , il . , u.s.a . , 2000 ) was used .
the genotypes of 5-httlpr were distributed according to the hardy - weinberg equilibrium ( =0.13 , p=0.94 ) .
ninety - five subjects ( 60.1% ) were " ss " genotype , and subjects with " sl " and " ll " were 54 ( 34.2% ) and 9 ( 5.7% ) , respectively .
there were no significant differences in the scores of harm avoidance ( f=0.38 , p=0.69 ) , novelty seeking ( f=0.07 , p=0.93 ) , reward dependence ( f=0.16 , p=0.86 ) and persistence ( f=0.24 , p=0.79 ) using genotype and sex as independent variables ( table 1 ) .
when dividing the subjects into 2 groups of " ss " ( 60.1% ) and " sl"+"ll " ( 39.9% ) , we could not find associations between the two genotype group and personality traits , either ( table 1 ) . while the 5-httlpr genotypes frequencies ( =111.04 , p<0.001 ) and the allele frequencies ( =110.21 , p<0.001 ) in our sample were significantly different from those of lesch et al .
( 2 ) , those frequencies are quite similar to other studies of korean ( 20 , 21 ) , japanese ( 6 , 12 , 13 ) , and chinese ( 22 ) .
in the present study , we could not find evidence for an association between 5-httlpr and harm avoidance measured by k - tci in healthy korean subjects .
it is contrary to two recent meta - analyses including more than 23 studies which have shown the association between the polymorphism and trait anxiety , despite its weak effects ( 14 , 15 ) .
this relationship was not reported in previous two studies using korean samples ( 20 , 21 ) . even though the association between 5-httlpr and harm avoidance is not confined to one ethnic group ( 10 ) , majority of studies in east asian populations did not show the association ( 20 - 23 ) , with two possible exceptional studies in japan ( 6 , 12 ) .
this raises the possibility that the reported associations between the 5-httlpr and harm avoidance do not exist in east asian population .
we found that allele frequencies in our subjects were similar to those in other east asian subjects ( 6 , 12 , 13 , 20 , 21 ) , but they were significantly different from those in caucasian subjects ( 2 , 5 , 24 ) .
it is well known that allele frequencies distribution has large effects on the power of genetic association studies ( 25 ) .
together with small effect size of harm avoidance ( 14 , 15 ) , it is certainly possible that genotype distribution of east asian samples , especially marked low frequencies of long alleles , could reduce the power of the tests , which might lead to negative results .
there have been many reports suggesting dominance of long allele ( 5 , 19 ) .
there are also reports that " ss " genotype acts in recessive fashion in east asian populations ( 6 , 7 , 26 ) . in accordance with these , we divided the subjects into " ss " genotype group and " sl"+"ll " genotype group .
there were no significant differences between the two genotype groups and 4 dimensional scores , either .
it has been suggested that high frequency of short allele in the japanese population may be responsible for the interpersonal sensitivity and emotional restraints that is generally regarded as characteristic of japanese culture ( 6 , 23 ) .
the ethnic differences of allele frequencies between asians and caucasians might partially explain personality tendencies towards greater or less cautiousness and shyness from a genetic perspective .
however , in our sample , we could not find the correlation between the high scores of harm avoidance and short alleles of 5-httlpr . in terms of gender differences
, we could speculate that genetic factors have different effects on personality differences between sex ( 27 ) .
however , we could not find previously reported gender differences ( 5 , 11 ) in this study . in another korean study exclusively including female subjects , they could not find association between harm avoidance and 5-httlpr ( 28 ) . though tci has been known to reflect the genetic components of personality dimensions including harm avoidance
, it also includes non - genetic , environmental components ( 29 ) . considering that age
, sex and culture could affect on personality dimensions ( 30 ) , our study has some strength in that the study subjects could be relatively homogenous regarding ethnicity , age , cultural and socioeconomic status , because we confined our sample to the students of one university in seoul , korea .
the average age of the participants were 23.83.1 , minimizing previously reported age effect on the tci dimensional scores ( 31 ) .
we also found no correlations between age and personality traits scores in our sample ( result not shown ) . though college students do not represent entire population , we could reduce potential confounding effects of age and cultural factors on the personality traits .
overall , we could not find the association between 5-httlpr and harm avoidance and other tci measures . while the allele frequencies of our sample are similar to those of other studies in east asia including korea , they were significantly different from those of lesch et al .
( 2 ) , which support further cross - cultural replication studies with a lager sample . | there have been numerous studies on the association between 5-httlpr ( polymorphisms in the promoter region of the serotonin transporter gene ) and anxiety - related personality traits , with conflicting results . in this study , we administered korean version of the temperament and character inventory ( k - tci ) to a sample of 158 korean college students and genotyped for the 5-httlpr in order to compare the tci dimensional scores including harm avoidance according to the 5-httlpr genotype and sex .
we could not find the association between 5-httlpr and harm avoidance and other tci measures . considering known allele frequencies differences of 5-httlpr among different ethnic groups , further cross - cultural studies with a larger sample would be needed . |
spinal cord neoplasms are rare in the pediatric and adult patient population ; however , spinal neoplasms are less common in children compared with adults .
only 0.51% of the central nervous system ( cns ) neoplasms are located within the spinal cord . the vast majority ( 90% ) of spinal cord neoplasms
the rarity of these lesions is also demonstrated by the fact that of all cns glial tumors , only 24% are located in the spinal cord .
all age groups may be affected , but spinal cord neoplasms are more frequent towards the end of the first decade and the beginning of the second decade of life .
many children present with a history of non - specific findings which progress slowly over time .
in addition , children frequently have a long history of exacerbation and remission of symptoms , which is believed to result from fluctuant degrees of peritumoral spinal cord edema .
so - called key clinical / neurological features include ( a ) progressive motor weakness , ( b ) progressive scoliosis , ( c ) gait disturbance , and ( d ) muscle rigidity with paraspinal muscle spasm .
. back pain may be a leading clinical complaint ; 2530% of children with a spinal cord tumor suffer from recurrent episodes of back pain .
back pain can be classified as spinal pain ( 70% ) , root pain and tract pain .
spinal pain is characterized as a dull and aching pain , localized to the bone segments adjacent to the tumor .
spinal pain is also believed to result from focal distension of the dural sack by the tumor .
tract pain is typically described as a vague , burning pain with ill - defined paresthesias .
tract pain likely results from a direct infiltration and compression of the spino - thalamic tracts within the spinal cord .
children with back pain should be taken seriously , especially because back pain in healthy children without a history of recent trauma is very rare .
frequently , back pain in children is initially interpreted as so - called growth pain .
many of these children are in a phase of rapid growth and are physically highly active . because most symptoms are non - specific and symptoms may wax and wane , diagnosis may be delayed .
physicians frequently do an array of diagnostic tests to rule out common causes for back pain , such as renal ultrasound for kidney stones , chest radiography for pneumonia and even spinal radiographic survey or bone scintigraphy for spondylodiscitis .
when tests are negative , physicians may feel comfortable starting conservative treatments , which may result in a significant delay in diagnosis . in rare cases ,
a spinal cord tumor can be missed because a limited magnetic resonance imaging ( mri ) examination of the spine was requested , e.g. a lumbar spine mri .
however , due to the complex segmentational development of the spinal cord and adjacent musculoskeletal derivates , a tumor may be located significantly higher than the symptoms suggest .
. a smaller percentage of children with spinal cord neoplasms may present with symptoms of increased intracranial pressure .
several explanations are discussed : ( a ) cerebrospinal fluid ( csf ) resorption may be impaired by a tumor - related elevation of the csf proteins ; ( b ) blockage of the foramen of magnum by a high cervical cord tumor ; ( c ) by an intraspinal , subarachnoidal tumor hemorrhage ; and ( d ) by subarachnoidal tumor seeding . consequently ,
if an unexplained , new hydrocephalus is diagnosed , mri of the spine to rule out spinal cord tumors should be considered . finally , diagnosis of spinal cord tumors may be delayed because these tumors are so rare that many physicians just do not think about it .
back pain , mild motor weakness or a mild gait disorder may be signs of a spinal cord tumor
. the goal should be : diagnose aggressively ( mri ) and treat as gently as possible as early as possible .
the high spatial resolution , the different image contrasts that can be generated , the combination of anatomical and functional data that can be collected and the lack of ionizing radiation make mri the primary imaging modality for examining spinal cord lesions .
computed tomography ( ct ) is contraindicated because of the high radiation dose if the entire spine is examined .
nuclear medicine studies are usually considered if an infectious process such as spondylodiscitis or an abscess is suspected .
mri should always include the entire spine and if a focal spinal cord neoplasm is identified , the brain should be included to exclude additional or metastatic lesions .
mri protocols should include multiple planes taking advantage of different imaging contrasts like t1- and t2-weighted images .
finally , functional sequences including diffusion tensor imaging ( dti ) and h magnetic resonance spectroscopy ( mrs ) may be helpful .
frequently , spinal cord neoplasms are treated by a combination of chemotherapy , radiotherapy and neurosurgical resection . for resection ,
this hardware , even when it is of non - ferromagnetic material , may significantly limit the diagnostic sensitivity of mri .
imaging sequences should be carefully chosen to limit artifacts that can obscure residual or recurrent tumor .
imaging features suggestive of a spinal cord neoplasm include ( a ) focal signal alteration with cord expansion , ( b ) corresponding contrast enhancement and ( c ) tumoral and non - tumoral cysts . a focal t1- or t2-weighted signal alteration in combination with a cord expansion is the leading criterion for a spinal neoplasm .
if a significant focal mass effect is lacking other causes for the signal alteration should be considered , such as transverse myelitis , demyelinating processes like multiple sclerosis and acute disseminated encephalomyelitis , sarcoidosis , arteriovenous fistula with intramedullary edema , or focal ischemia .
non - tumoral cysts are frequently seen at the poles of the tumor ; they may be a reactive dilatation of the central canal or represent fluid produced by the tumor itself .
tumoral cysts have to be resected because the cyst wall is likely invaded by tumor cells .
mri usually allows differentiating neoplastic from non - neoplastic spinal cord lesions ; however mri is limited in defining tumor histology .
compared to the adult patient population , the same kind of spinal neoplasms are seen in children
the most frequent are the pilocytic and anaplastic astrocytomas ( 60% ) and ependymomas ( 30% ) . in children a benign subgroup of the ependymomas
non - glial tumors are much less frequent and include a variety of histologic subtypes such as hemangioblastoma , subependymoma , ganglioglioma , paraganglioma , lymphoma , primitive neuroectodermal tumor ( pnet ) neurocytoma , oligodendroglioma and metastases . as rule of thumb
, it can be said that in children , astrocytomas are more frequent than ependymomas ; in adults ependymomas are more frequent than astrocytomas .
some additional rules mention that ependymomas are more frequently seen in neurofibromatosis ( nf ) ii patients while astrocytomas are more frequently seen in nf i patients .
finally , the higher the tumor location ( cervical versus thoracic or lumbar ) , the more likely a syrinx will develop .
their primary location is more rostral than in adults ; 50% are in the cervico - thoracic region and usually affect a small number of segments .
astrocytomas most frequently present with pain and motor dysfunction , followed by gait disturbance , torticollis and scoliosis . bowel and bladder dysfunction is uncommon due to their predominant cervical location .
the majority are of low grade , who grade i and ii ( 7580% ) including pilocytic and fibrillary type astrocytomas .
anaplastic astrocytoma ( who iii ) and glioblastoma multiforme ( who iv ) are much rarer ( 2025% , 0.21.5% ) .
spinal astrocytomas rarely present with a clear cleavage plane between tumor and normal spinal cord because of the infiltrative characteristics ; tumor cells extend along the scaffold of normal astrocytes .
tumor cysts , both polar and intratumoral , are seen in 2040% of children , frequently accompanied by a rostral and/or caudal syrinx .
are frequently eccentric within the spinal cord and may show an asymmetric spinal cord expansion .
lesions are typically t2-hyperintense , t1-iso- or hypointense and may show a mild to moderate contrast enhancement . rarely , exophytic tumor components may be observed . on follow - up imaging ,
low grade spinal cord astrocytomas are typically stable or may show a slow progression . on dti ,
careful analysis of the adjacent soft tissues as well as the brain is essential to rule out additional lesions that may suggest nf1 .
imaging features are very similar to the classical astrocytomas , however they frequently present early with diffuse subarachnoidal dissemination .
gangliogliomas resemble the signal characteristics of astrocytomas on imaging and are also frequently eccentric in location .
this also explains the more frequently observed sensory symptoms due to their proximity to the spino - thalamic tracts .
classical spinal ependymomas are usually low grade ( who i , ii ) and are consequently slow growing .
ependymomas are less infiltrative than astrocytomas , they tend to compress and displace the adjacent spinal cord tissue . on dti ,
many ependymomas reveal a high vascularity with multiple small feedings vessels and a strong contrast enhancement .
a rim of t2-hypointense hemosiderin may be observed along the pole of the tumor , also known as
cap sign. myxopapillary ependymoma is a special variant that differs by age of presentation and location from the classic ependymomas .
myxopapillary ependymomas encompass about 13% of all spinal ependymomas and are more common in male children .
they typically arise from the ependymal glia of the filum terminal and are consequently seen predominantly in the area of the conus medullare and filum terminale .
these tumors are frequently polylobulated , fill the spinal canal , may scallop the adjacent vertebral bodies and can produce mucin .
clinically , due to their low location , they present with lower back pain , leg weakness and sphincter dysfunction .
these tumors are much less frequent and are usually diagnosed after biopsy or if a matching clinical history suggest another histologic subtype than astrocytoma or ependymoma , e.g. lymphomas , pnets or oligodendrogliomas . hemangioblastomas and metastatic disease deserve a separate discussion .
in addition , hemangioblastomas should be ruled out in children with von hippel lindau ( vhl ) syndrome .
vhl syndrome is an inherited multi - system disorder characterized by abnormal growth of blood vessels with an increased risk for hemangioblastomas .
intramedullary metastases are rare and may result from hematogenous spread or from direct extension from the leptomeninges .
children with intramedullary metastatic disease usually have a poor prognosis with rapid progression of clinical symptoms .
extramedullary , intradural metastasis are much more frequent and usually result from csf seeding from primary cns neoplasms which are in close proximity or within the csf compartments . typically csf seeding is seen in , e.g. medulloblastomas , ependymomas , high grade astrocytomas , germinomas , or choroid plexus tumors .
the lumbosacral region is most frequently affected with a nodular and irregular , contrast - enhancing thickening of the thecal sac and nerve roots .
the surface of the spinal cord may be coated , also known as sugar coating. contrast - enhanced t1-weighted sequences are mandatory .
finally , paraspinal neoplasms may extend intraspinal , e.g. paraspinal rhabdomyosarcomas or extraosseous ewing sarcomas .
correlation with the clinical history and dedicated anatomical and functional mri usually solves this issue .
their primary location is more rostral than in adults ; 50% are in the cervico - thoracic region and usually affect a small number of segments .
astrocytomas most frequently present with pain and motor dysfunction , followed by gait disturbance , torticollis and scoliosis . bowel and bladder dysfunction is uncommon due to their predominant cervical location .
the majority are of low grade , who grade i and ii ( 7580% ) including pilocytic and fibrillary type astrocytomas .
anaplastic astrocytoma ( who iii ) and glioblastoma multiforme ( who iv ) are much rarer ( 2025% , 0.21.5% )
. spinal astrocytomas rarely present with a clear cleavage plane between tumor and normal spinal cord because of the infiltrative characteristics ; tumor cells extend along the scaffold of normal astrocytes .
tumor cysts , both polar and intratumoral , are seen in 2040% of children , frequently accompanied by a rostral and/or caudal syrinx .
are frequently eccentric within the spinal cord and may show an asymmetric spinal cord expansion .
lesions are typically t2-hyperintense , t1-iso- or hypointense and may show a mild to moderate contrast enhancement . rarely , exophytic tumor components may be observed . on follow - up imaging ,
low grade spinal cord astrocytomas are typically stable or may show a slow progression . on dti ,
careful analysis of the adjacent soft tissues as well as the brain is essential to rule out additional lesions that may suggest nf1 .
imaging features are very similar to the classical astrocytomas , however they frequently present early with diffuse subarachnoidal dissemination .
gangliogliomas resemble the signal characteristics of astrocytomas on imaging and are also frequently eccentric in location .
this also explains the more frequently observed sensory symptoms due to their proximity to the spino - thalamic tracts .
classical spinal ependymomas are usually low grade ( who i , ii ) and are consequently slow growing .
ependymomas are less infiltrative than astrocytomas , they tend to compress and displace the adjacent spinal cord tissue . on dti ,
many ependymomas reveal a high vascularity with multiple small feedings vessels and a strong contrast enhancement .
a rim of t2-hypointense hemosiderin may be observed along the pole of the tumor , also known as
cap sign. myxopapillary ependymoma is a special variant that differs by age of presentation and location from the classic ependymomas .
myxopapillary ependymomas encompass about 13% of all spinal ependymomas and are more common in male children .
they typically arise from the ependymal glia of the filum terminal and are consequently seen predominantly in the area of the conus medullare and filum terminale .
these tumors are frequently polylobulated , fill the spinal canal , may scallop the adjacent vertebral bodies and can produce mucin .
clinically , due to their low location , they present with lower back pain , leg weakness and sphincter dysfunction .
these tumors are much less frequent and are usually diagnosed after biopsy or if a matching clinical history suggest another histologic subtype than astrocytoma or ependymoma , e.g. lymphomas , pnets or oligodendrogliomas . hemangioblastomas and metastatic disease deserve a separate discussion .
in addition , hemangioblastomas should be ruled out in children with von hippel lindau ( vhl ) syndrome .
vhl syndrome is an inherited multi - system disorder characterized by abnormal growth of blood vessels with an increased risk for hemangioblastomas .
intramedullary metastases are rare and may result from hematogenous spread or from direct extension from the leptomeninges .
children with intramedullary metastatic disease usually have a poor prognosis with rapid progression of clinical symptoms .
extramedullary , intradural metastasis are much more frequent and usually result from csf seeding from primary cns neoplasms which are in close proximity or within the csf compartments . typically csf seeding is seen in , e.g. medulloblastomas , ependymomas , high grade astrocytomas , germinomas , or choroid plexus tumors .
the lumbosacral region is most frequently affected with a nodular and irregular , contrast - enhancing thickening of the thecal sac and nerve roots .
sugar coating. contrast - enhanced t1-weighted sequences are mandatory . , to avoid misinterpretation imaging should be performed prior to surgery .
finally , paraspinal neoplasms may extend intraspinal , e.g. paraspinal rhabdomyosarcomas or extraosseous ewing sarcomas .
correlation with the clinical history and dedicated anatomical and functional mri usually solves this issue . | abstractspinal cord neoplasms are rare lesions in children . most of the spinal cord neoplasms are astrocytomas . frequently the clinical symptoms are non - specific .
back pain is often the leading symptom .
diagnosis is often delayed .
magnetic resonance imaging is the imaging modality of choice .
preoperative brain and spine imaging covering the entire spine is mandatory to exclude cerebrospinal fluid seeding . |
chronic obstructive pulmonary disease ( copd ) is characterized by progressive and partially reversible airflow limitation , and it is among the leading causes of mortality worldwide.1 copd is a multicomponent disease , and patients present a range of comorbidities that have an impact on prognosis and may increase the risk of mortality.2 the effects of copd on respiratory and physical function have been well studied ; moreover , due to the heavy burden of psychological disturbance , psychiatric morbidity , and disability in daily life , the mental health of copd patients has received growing attention in recent years.3,4 the prevalence of cognitive impairment in patients with copd ranges from 12% to 88%5 and is associated with depression , poor quality of life , which may affect patients ability to manage their disease , and reduced compliance with medication and oxygen therapy,6,7 leading to adverse clinical outcomes.811 therefore , cognitive function as well as emotional function are important aspects of the overall clinical care of patients with copd .
it has been suggested that a multidimensional assessment and personalized disease management approach could be an optimal strategy for addressing comorbidities , self - management education , and risk factor modification in copd patients.7 the early identification of cognitive dysfunction is critical if outcomes are to be improved in this population , and an understanding of the characteristics associated with a higher risk of cognitive impairment may assist health care professionals to address this challenge .
the primary aim of this study was to examine the cross - sectional prevalence of cognitive impairment in an unselected population of copd patients ; the secondary objective was to determine the relationships between cognitive impairment and major demographic and clinical variables , such as lung function , depression , and quality of life .
the present study is a post hoc analysis of a cross - sectional , observational study conducted in respiratory medicine departments and primary care centers in spain.11 this study was aimed at examining the prevalence of depressive symptoms and moderate - to - severe depression in copd and included ambulatory patients who were 40 years of age or older , with stable copd ( confirmed by postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 70% and absence of exacerbations in the previous 3 months ) .
all patients that completed the mini - mental state examination ( mmse ) were included in this post hoc analysis .
the study was approved by the institutional ethics committee of the hospital clinic ( barcelona , spain ) and was conducted in accordance with the principles of the declaration of helsinki .
prior to participation , all patients signed written documentation that the informed consent process was completed .
investigators recorded patients sociodemographic data and clinical information on copd severity using the modified medical research council dyspnea scale12 and the body mass index , airflow obstruction , dyspnea and exacerbations ( bodex ) index,13 comorbidity according to the charlson index,14 exacerbations in the previous year , and treatment .
this instrument explores spatial and temporal orientation , registration , attention , and calculation , recall , language , and visual construction in 12 items and 30 questions . a correct answer to one question
a score of less than 27 indicated cognitive impairment.17 depressive symptoms were measured using the short beck depression inventory questionnaire.18,19 this tool is a 13-item self - administered inventory that assesses affective , cognitive , motivational , and vegetative symptoms of depression .
items use a 4-point scale that ranges from 0 to 3 and a total score is calculated by adding up the item responses .
health - related quality of life was assessed by the generic euroqol-5 dimensions ( eq-5d ) questionnaire and the specific copd assessment test ( cat ) .
the eq-5d consists of a 5-item descriptive system ( including mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) , with each item rated as no problems , some problems , or severe problems , and an overall health state score for the day of the assessment , measured on a visual analog scale ( 0100 ; 100 represents best overall health).20 the cat is a short , specific quality of life questionnaire for measuring the impact of copd on the patient s well - being and daily life .
it consists of eight items , each presented as a 6-point semantic differential scale , providing a score out of 40 , indicating the impact of the disease.21 we used the validated spanish version of cat.22 physical activity was measured by asking patients how many minutes , on average , they walked every weekday , as previously described.23,24 depending on their daily walking activity , patients were classified into three groups : patients who walked < 30 minutes , patients who walked between 30 and 60 minutes , and patients who walked 60 minutes .
fisher s exact test was used for comparisons of qualitative variables , with the bonferroni correction for all pairwise comparisons .
student s t - test was used to determine the relationship between qualitative variables by group .
odds ratio ( or ) univariates were calculated by logistic regression to evaluate the different risks contemplated in the study , including all demographic , clinical , and questionnaires variables .
the first model was developed with cognitive status as a dependent variable and all variables that showed a significant association with cognition in univariate analysis as independent variables ( model 1 ) . a second model ( model 2 ) excluded any variables derived from the use of the questionnaires from the independent variables .
the objective of model 2 was to identify the factors associated with cognitive impairment that could be identified in routine clinical practice without the administration of questionnaires .
statistical analyses were performed using sas version 9.1.3 service pack 3 software ( sas institute inc . , cary , nc , usa ) .
the present study is a post hoc analysis of a cross - sectional , observational study conducted in respiratory medicine departments and primary care centers in spain.11 this study was aimed at examining the prevalence of depressive symptoms and moderate - to - severe depression in copd and included ambulatory patients who were 40 years of age or older , with stable copd ( confirmed by postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 70% and absence of exacerbations in the previous 3 months ) .
all patients that completed the mini - mental state examination ( mmse ) were included in this post hoc analysis .
the study was approved by the institutional ethics committee of the hospital clinic ( barcelona , spain ) and was conducted in accordance with the principles of the declaration of helsinki .
prior to participation , all patients signed written documentation that the informed consent process was completed .
investigators recorded patients sociodemographic data and clinical information on copd severity using the modified medical research council dyspnea scale12 and the body mass index , airflow obstruction , dyspnea and exacerbations ( bodex ) index,13 comorbidity according to the charlson index,14 exacerbations in the previous year , and treatment .
this instrument explores spatial and temporal orientation , registration , attention , and calculation , recall , language , and visual construction in 12 items and 30 questions . a correct answer to one question
a score of less than 27 indicated cognitive impairment.17 depressive symptoms were measured using the short beck depression inventory questionnaire.18,19 this tool is a 13-item self - administered inventory that assesses affective , cognitive , motivational , and vegetative symptoms of depression .
items use a 4-point scale that ranges from 0 to 3 and a total score is calculated by adding up the item responses .
health - related quality of life was assessed by the generic euroqol-5 dimensions ( eq-5d ) questionnaire and the specific copd assessment test ( cat ) .
the eq-5d consists of a 5-item descriptive system ( including mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) , with each item rated as no problems , some problems , or severe problems , and an overall health state score for the day of the assessment , measured on a visual analog scale ( 0100 ; 100 represents best overall health).20 the cat is a short , specific quality of life questionnaire for measuring the impact of copd on the patient s well - being and daily life .
it consists of eight items , each presented as a 6-point semantic differential scale , providing a score out of 40 , indicating the impact of the disease.21 we used the validated spanish version of cat.22 physical activity was measured by asking patients how many minutes , on average , they walked every weekday , as previously described.23,24 depending on their daily walking activity , patients were classified into three groups : patients who walked < 30 minutes , patients who walked between 30 and 60 minutes , and patients who walked 60 minutes .
fisher s exact test was used for comparisons of qualitative variables , with the bonferroni correction for all pairwise comparisons .
student s t - test was used to determine the relationship between qualitative variables by group .
odds ratio ( or ) univariates were calculated by logistic regression to evaluate the different risks contemplated in the study , including all demographic , clinical , and questionnaires variables .
the first model was developed with cognitive status as a dependent variable and all variables that showed a significant association with cognition in univariate analysis as independent variables ( model 1 ) . a second model ( model 2 ) excluded any variables derived from the use of the questionnaires from the independent variables . the objective of model 2 was to identify the factors associated with cognitive impairment that could be identified in routine clinical practice without the administration of questionnaires
statistical analyses were performed using sas version 9.1.3 service pack 3 software ( sas institute inc . , cary , nc , usa ) .
of 1,273 screened patients , 333 had not had the mmse evaluation and were excluded from the analysis . both populations ( excluded and included ones )
included patients were predominantly male , with an age ranging from 40 to 90 years .
a total of 370 patients ( 39.4% ) scored below the threshold of 27 for suspicion of mild cognitive impairment ( mci ; figure 1 ) .
patients with mmse scores < 27 were older , had a lower level of formal education , had heavier smoking exposure , presented more respiratory symptoms , had more frequently domiciliary oxygen , suffered a higher number of and more severe exacerbations during the previous year , and had a greater comorbidity burden .
in addition , higher levels of depression , more suicidal ideation , poorer quality of life , and less physical activity were more frequent in cognitively impaired patients . in the univariate analysis ,
cognitive impairment was associated with higher age , lower educational level , worse lung function and more severe copd , more frequent respiratory symptoms and exacerbations , and oxygen therapy requirement .
higher comorbidity burden , more depressive symptoms , and more impaired health status ( eq-5d and cat ) were also associated with cognitive impairment .
higher levels of physical activity were associated with better cognitive function ( table 2 ) . in the multivariate analysis , only educational level and the eq-5d social tariff were independently and significantly associated with cognitive impairment ( table 2 ) . when the analysis was limited to the usual demographic and clinical variables , and the questionnaires
were excluded from the model , the significant factors in the multivariate analysis were educational level , history of exacerbations , the bodex index , and the charlson comorbidity index ( table 3 ) .
of 1,273 screened patients , 333 had not had the mmse evaluation and were excluded from the analysis . both populations ( excluded and included ones )
included patients were predominantly male , with an age ranging from 40 to 90 years .
a total of 370 patients ( 39.4% ) scored below the threshold of 27 for suspicion of mild cognitive impairment ( mci ; figure 1 ) .
patients with mmse scores < 27 were older , had a lower level of formal education , had heavier smoking exposure , presented more respiratory symptoms , had more frequently domiciliary oxygen , suffered a higher number of and more severe exacerbations during the previous year , and had a greater comorbidity burden .
in addition , higher levels of depression , more suicidal ideation , poorer quality of life , and less physical activity were more frequent in cognitively impaired patients .
in the univariate analysis , cognitive impairment was associated with higher age , lower educational level , worse lung function and more severe copd , more frequent respiratory symptoms and exacerbations , and oxygen therapy requirement .
higher comorbidity burden , more depressive symptoms , and more impaired health status ( eq-5d and cat ) were also associated with cognitive impairment .
higher levels of physical activity were associated with better cognitive function ( table 2 ) . in the multivariate analysis , only educational level and the eq-5d social tariff were independently and significantly associated with cognitive impairment ( table 2 ) . when the analysis was limited to the usual demographic and clinical variables , and the questionnaires
were excluded from the model , the significant factors in the multivariate analysis were educational level , history of exacerbations , the bodex index , and the charlson comorbidity index ( table 3 ) .
copd is a multicomponent inflammatory disease that affects physical and nonphysical functions , one of which is cognition . in our study , we found some degree of cognitive impairment in almost 40% of unselected copd patients , as determined by a mmse score < 27 .
previous estimates of cognitive dysfunction in copd patients ranged from 12% to 88% , depending on the study population and the tools used for neuropsychological assessment.5,9 a recent study in a very similar population ( including not only elderly patients , but also young patients ) to ours17 found mci in 36% of copd patients after comprehensive neuropsychological testing .
the authors reported that a mmse score of 27 provided optimal maximum accuracy and a diagnostic cutoff ( < 27 indicated impairment ) , with 97% specificity and 73% of patients correctly classified .
however , they also found that the montreal cognitive assessment performed better as a screening test for detecting mci in patients with copd .
so , this threshold for mild impairment is not necessarily indicative of a clinically significant cognitive decline associated with functional impairment , but could serve as an early identification of an affected cognitive function .
cognitive symptoms are core symptoms in many mental disorders;25 they impact on cognitive functioning , which deteriorates as patients get older , particularly after the age of 60.26 copd patients score lower on standard cognitive performance tests over time , compared with individuals of the same age without the disease.27 one hypothesis for this accelerated decline in copd patients is altered brain perfusion.28 changes in brain perfusion due to hypoxemia in subjects with severe copd may increase cognitive impairment.29 in our population , more cognitively affected patients were receiving domiciliary oxygen that could be a correlate of higher disease severity and sustained periods of hypoxemia .
in contrast , a recent study indicated that long - term home oxygen therapy allowed to preserve cognitive functions from the copd - induced deterioration,30 so the role of supplemental oxygen in preventing copd - induced cognitive deterioration is still controversial . indeed , other factors , such as hypercapnia or oxidative stress , may also be involved.31 other authors have suggested that intermittent and continuous hypoxia resulting from poor lung function may lead to transient deficits in neurotransmitter metabolism in the central nervous system.3234 several mechanisms , then , appear to be involved in cognitive decline in this population .
prospective neuroimaging studies are required to characterize brain changes and corresponding disturbances in cognitive function in these patients over time .
cognitive impairment represents a critical health care burden in terms of costs.35 individuals with mci have a higher risk of developing dementia than the general population.36 moreover , cognitive dysfunction in copd has been associated with poorer outcomes and even with an increase in disability and mortality.8,37,38 thus , it is essential that this condition is identified early in copd patients , in order to prevent or delay progression to clinical dementia or increased morbidity .
if the factors affecting cognition are recognized , cognitive impairment may be detected earlier and copd patients at higher risk may be identified . in our study , the multivariate analysis showed that educational level and quality of life assessed by eq-5d were significantly and independently associated with the presence of cognitive impairment .
however , the use of health - related quality of life or other questionnaires is not a routine practice in most primary care offices , so we ran a multivariate model , discarding the scores of these questionnaires and including only demographic and clinical variables . in this model ,
cognitive impairment was associated with exacerbations in the previous year , the severity of copd measured by the bodex index , and a higher comorbidity burden .
they have been associated with increased health care costs,39 a significant decline in health status,40 and substantial mortality.41 our results suggest that frequent exacerbations also have negative consequences on cognitive function .
this is in line with a previous study that investigated the cognitive function of copd patients who were hospitalized following an acute exacerbation .
these patients had significantly poorer cognitive function compared with control participants 3 months after discharge from the hospital.10 other studies have shown that cognitive impairment during the exacerbation period resolves during periods of stability.4244 more exacerbations and increased copd severity could reflect the poorer compliance with medication associated with cognitive impairment.6,7 the systemic inflammation seen in severe copd and during acute exacerbations45 may participate in neurocognitive impairment via a direct neurotoxic effect or by affecting cerebral atherosclerosis.9 a high comorbidity burden may also contribute to persistent brain injury : multiple concomitant diseases , such as cerebrovascular disease and related mechanisms , including endothelial dysfunction and oxidation , may lead to neuronal death , synaptic dysfunction , and cognitive impairment.46 all these data confirm that the cognitive impairment that occurs in copd patients is associated with disease severity .
neuropsychiatric assessment should become a routine part of the diagnostic procedure for these patients , to help physicians grade the overall impact of copd and determine the most effective treatment and strategies .
the cross - sectional design of our study limits any type of causal inferences , and the directionality between copd and cognition remains unclear . despite the frequent use of the mmse in clinical research and practice ,
this tool for cognitive impairment detection could have missed key domains of cognition often affected in copd ( ie , executive functioning ) .
an additional limitation of our study is the lack of data on our patients medical treatment . as treatment
may have great impact on the symptom burden and mental health of the patients , such information should be collected in future studies . in particular , prospective studies
are urgently needed to determine the most effective behavioral and medical interventions for reducing the risk of poor neurocognitive outcomes in patients with copd .
early detection of cognitive decline is crucial , in view of its association with poorer copd outcomes , including increased mortality , and patients with frequent exacerbations and or when the bodex index appears concerning the clinician may want to ask the patient and/or caregivers about cognitive function .
these patients may need more individualized educational and care interventions to help them manage their daily lives .
clinicians must involve family caregivers in the care plan of patients with severe copd and cognitive deficits . | purposewe investigated the association between cognitive impairment and chronic obstructive pulmonary disease ( copd ) , taking into account demographic and clinical variables evaluated during routine practice.patients and methodswe performed a post hoc analysis of a cross - sectional study that included subjects with stable copd .
sociodemographic and clinical information was recorded using the body mass index , airflow obstruction , dyspnea and exacerbations index and the charlson comorbidity index .
cognitive performance was studied by the mini - mental state examination , with a score less than 27 indicating clinical impairment .
depressive symptoms , physical activity , and quality of life ( euroqol-5 dimensions and copd assessment test ) were also evaluated.resultsthe analysis included 940 subjects .
the prevalence of cognitive impairment was 39.4% .
multivariate logistic regression models revealed that cognitive impairment was associated with educational level ( odds ratio [ or ] = 0.096 , 95% confidence interval [ ci ] = 0.0110.447 ) and poorer quality of life measured by the euroqol-5 dimensions social tariff ( or = 0.967 , 95% ci = 0.9500.983 ) . when questionnaires were not included in the analysis ,
cognitive impairment was associated with educational level ( or = 0.063 , 95% ci = 0.0100.934 ) , number of exacerbations ( or = 11.070 , 95% ci = 1.45084.534 ) , body mass index , airflow obstruction , dyspnea and exacerbations index score ( or = 1.261 , 95% ci = 1.0491.515 ) , and the charlson comorbidity index ( or = 1.412 , 95% ci = 1.1181.783).conclusioncognitive impairment is common in copd and is associated with low educational level , higher disease severity , and increased comorbidity .
this could have therapeutic implications for this population . |
sepsis is a clinical syndrome that results from complex interaction between host and infectious agents and is characterized by systemic activation of multiple inflammatory pathways .
the pathophysiology of sepsis could be initiated by the outer membrane components of gram - negative and gram - positive organisms .
signaling by these mediators occurs via a family of transmembrane receptors known as toll like receptors . within the monocyte , nuclear factor kappa b ( nfkp ) and mitogen activated protein kinase ( mapk )
it is thought that edaravone has antioxidant action , inhibits lipid peroxidation and causes quenching of active oxygen leading to protection of various cells against damage by reactive oxygen species .
some studies have shown beneficial effect of edaravone on postischemic reperfusion injury in cardiac , cerebral and hepatic tissues .
so , in this work we attempt to evaluate its role in cases of septic peritonitis ( sp ) .
this is a prospective observer - blinded study carried out in surgical intensive care unit ( icu ) during the period from march 2014 to april 2015 in 60 patients with sp after approval by hospital ethical committee .
inclusion criteria included american society of anesthesiologists physical status i or ii having sp after gastrointestinal perforation ( perforated peptic ulcer , traumatic intestinal perforation or postcorrection of intestinal anastomosis leakage ) .
exclusion criteria included patients in septic shock , on vasopressor or with one or more organ dysfunction .
criteria of sepsis according to american college of chest physician / society of critical care medicine definitions which include strongly suspected infection or positive blood culture plus two or more of the following : temperature > 38c or < 36 , heart rate ( hr ) > 90 b / min , respiratory rate > 20 c / min , pco2<32 mmhg and white blood cells > 12000 mm or < 4000 mm . all patients were treated with antibiotics according to culture and sensitivity test and received intravenous ( iv ) fluids to keep central venous pressure ( cvp ) 12 cm h2o and mean arterial pressure 65 mmhg .
randomization was performed using a random number list generated by means of the quickcalcs ( graphpad software inc . , la jolla , ca , usa ) .
the group assignment numbers were sealed in an envelope and kept by the study supervisor .
patients were randomly divided into two groups of thirty patients each - group ( c ) : control group managed according to the routine protocol of sepsis and group ( e ) : edaravone treated sp managed according to the routine protocol of sepsis + edaravone at dose of 30 mg/12 h iv infusion for 2 weeks .
edaravone was supplied from sigma company ( license from mitsubishi pharmaceutical , inc . ) .
all patients were monitored for invasive blood pressure , hr , cvp , temperature , urine output , total fluid balance and routine investigations ( complete blood cell count , arterial blood gases , serum electrolytes , liver , and kidney function tests ) .
blood sample were taken weekly for 2 weeks to measure the following parameters :
nuclear transcription factor kappa b activity ( nfkb ) was measured in activated monocyte macrophage by elisa based kits assay against p50 and p65 ( trans am .
nfkb p56 from active motif rixensart belgium).mapk was measured in activated monocyte / macrophage by nonradioactive assay kits ( western blot analysis kits ) from cell signaling technology inc .
, u.s.a).heat shock protein 72 ( hsp 72 ) , an indicator for intracellular response to stress , was measured by immunoblotting.total antioxidant capacity ( tac ) was measured by ferric reducing ability of plasma method , comparing absorbance changes at 593 nm which will be linear over a wide concentration range of antioxidant mixtures
.
nuclear transcription factor kappa b activity ( nfkb ) was measured in activated monocyte macrophage by elisa based kits assay against p50 and p65 ( trans am .
mapk was measured in activated monocyte / macrophage by nonradioactive assay kits ( western blot analysis kits ) from cell signaling technology inc . , u.s.a )
heat shock protein 72 ( hsp 72 ) , an indicator for intracellular response to stress , was measured by immunoblotting .
total antioxidant capacity ( tac ) was measured by ferric reducing ability of plasma method , comparing absorbance changes at 593 nm which will be linear over a wide concentration range of antioxidant mixtures .
sample size of thirty patients was calculated for 90% power , = 0.05 , = 0.1 , and anticipated effect size = 0.40 using sample size software ( g*power version 3.00.10 , germany ) .
analytic statistics was performed on ibm compatible computer using spss 11.5 software ( spss inc . ,
data was compared using unpaired student 's t - test ; p < 0.05 was considered statistically significant .
sample size of thirty patients was calculated for 90% power , = 0.05 , = 0.1 , and anticipated effect size = 0.40 using sample size software ( g*power version 3.00.10 , germany ) .
analytic statistics was performed on ibm compatible computer using spss 11.5 software ( spss inc . ,
data was compared using unpaired student 's t - test ; p < 0.05 was considered statistically significant .
there was significant decrease ( p < 0.05 ) in serum level of nfkb , mapk in group e in comparison with group c. while serum level of hsp 72 and tac showed significant increase ( p < 0.05 ) in group e in comparison with group c. mortality rate was lower in group e than in group c. the number of patients developed septic shock was lower in group e than in group c [ tables 1 and 2 ] .
the randomized trial flow diagram , including enrollment , intervention allocation , and analysis patients demographic data and outcome effect of edaravone on blood level of nuclear factor kappa b , p38 mitogen activated protein kinase , heat shock protein 72 and total antioxidant capacity
this work demonstrated that the edaravone treated sp group recorded significant improvement in the inflammatory and oxidative processes in comparison with control group manifested by a significant decrease in serum level of p38 mapk and nfkb .
septic shock can initiate an inflammatory response which is characterized by up regulation of cytokine expression and neutrophil accumulation in a variety of tissues .
the cytokine expression is mediated by two separate signal pathways : p38 mapk and nfkb in activated monocyte / macrophages which are activated by several incoming signals from cell surface including the reactive oxidative intermediates and lipopolysaccharides of most clinically relevant gram - negative organisms found in septic infection .
the expression of several inflammatory cytokines as tumor necrosis factor alpha ( tnf- ) and interleukin 6 bind to their receptors in various cell types resulting in functional impairment .
edaravone is a free radical scavenger quenching of reactive oxygen and hence attenuating the oxidative process and inhibiting subsequent injurious inflammation by reducing expression of inflammatory cytokines and adhesion molecules .
, 2008 stated that edaravone has an anti - inflammatory effect by inhibiting induction of inducible nitric oxide synthase ( inos ) as well as pro - inflammatory cytokines tnf- and this is mediated by the inhibiting activity of transcription factor nfk - b which is an essential signal for inos production .
also , edaravone could decrease the stability of inos mrna and hence decreasing expression of inos .
the inhibition of no production will subsequently inhibit production of perioxynitrite and hence attenuat lipid peroxidation .
higashi et al . , stated that edaravone could inhibit lipoxygenase metabolism of arachidonic acid by trapping hydroxyl radicals .
also it could inhibit alloxan induced lipid peroxidation and hence prevent cellular damage . moreover , suzuki et al .
demonstrated that the antioxidant action of edaravone could be attributed partly to the enhancement of prostacyclin production leading to protection of various cells .
in addition , this work demonstrated a significant increase in serum level of cytoprotective hsp 72 in edaravone treated sp group in comparison with control one .
although the mechanism of hsp production is still unclear , it may be attributed to activation of adenosine monophosphate - activated protein kinase and mapks .
activation of these intracellular signaling pathways cause quiescent cells to enter the cell cycle and initiate regenerative response inducing hsp as sort of stress adaptation .
heat shock 72 are intracellular proteins ( named according to their molecular weight ) which present in small amount in normal cells and a larger amount in cells subjected to a variety of stressful stimuli as a sort of cellular defense mechanism protecting cells from excessive stress damage . also , they play a role in protein - protein interaction stabilizing the protein conformation and preventing the unwanted protein aggregation
this study elucidated that edaravone induced a significant increase in tac of plasma in comparison with a control group which could be attributed partly to the significant increase in serum level of hsp 72 induced by edaravone .
zhi et al . , stated that hsp 72 could increase the antioxidant capacity of cells .
, elucidated that edaravone could reduce the oxidative stress by enhancing the production of hsp , which could limit cellular injury by refolding the disrupted protein stabilizing cell membrane and hence attenuating neutrophil infiltration .
in cases of sp treatment with edaravone could significantly improve the inflammatory and oxidative states with better patient outcomes .
| background and aims : sepsis is a complex rapidly progressive infectious disease that remain a major cause of morbidity and mortality in surgical patients and trauma victims .
edaravone a novel free radical scavenger was approved in 2001 in japan for treatment of acute cerebral and myocardial infarction . hence , in this work we attempt to evaluate its role in cases of septic peritonitis ( sp).material and methods : this is a prospective randomized observer - blinded study carried out in surgical intensive care unit ( icu ) after approval by hospital ethical committee .
after admission to icu patients were randomly divided into two groups of thirty patients each - group ( c ) : control group managed according to the routine protocol of sepsis and group ( e ) : edaravone treated sp managed according to the routine protocol of sepsis + edaravone at dose of 30 mg/12 h intravenous infusion for 2 weeks .
all patients were monitored for invasive blood pressure , central venous pressure , heart rate , temperature , urine output , total fluid balance , and routine investigation .
blood sample was taken weekly for 2 weeks to measure the following parameters : nuclear transcription factor kappa b activity ( nfkb ) , mitogen - activated protein kinase ( mapk ) , heat shock protein 72 ( hsp 72 ) and total antioxidant capacity ( tac).results : there was significant decrease ( p < 0.05 ) in serum level of nfkb , mapk in group e in comparison with group c. while serum level of hsp 72 and tac showed significant increase ( p < 0.05 ) in group e compared with group c with better outcome.conclusion:sp treatment with edaravone could significantly improve the inflammatory and oxidative states with better patient outcomes . |
the major objectives of root canal therapy are removal of the pathologic pulp , shaping and cleaning of the root canal system , disinfection of the contaminated root canals and three dimensional obturation to prevent reinfection.1 the success of endodontic therapy depends on adequate access , thorough biomechanical preparation and proper obturation of the root canal system .
endodontically treated teeth are widely considered to be more susceptible to fracture than vital teeth .
the reasons most often reported have been the removal of tooth structure during endodontic therapy , dehydration of dentin after endodontic therapy and excessive pressure during obturation.2,3 the strength of the endodontically treated teeth is directly related to the method of canal preparation and the amount of remaining sound tooth structure .
it is observed that the greatest incidence of vertical root fracture occurs in teeth that have undergone endodontic therapy.4 vertical root fracture is a longitudinal fracture of the root , extending throughout the entire thickness of dentin from the root canal to the periodontium .
it is of serious clinical concern and has an unfavorable prognosis resulting in extraction of the tooth or resection of the affected root.5,6 although obturation may not necessarily be the most critical stage in root canal therapy , it should still be performed according to the highest clinical standards .
gutta - percha has widely been accepted for years as the gold standard filling material to obturate root canals .
furthermore , the adhesive strength between the root canal walls , endodontic sealers , and gutta - percha was found to be very weak.7 although very few materials have seriously challenged gutta - percha and sealer in majority of filling situations , research continues to find alternatives that may seal better and also mechanically reinforce compromised roots.7 the bonding concept of the root filling material is hampered by the lack of a chemical union between the polyisoprene component of gutta - percha and methacrylate - based resin sealers.8 recently , two challenging strategies have been employed to effectively reinforce the endodontically treated tooth structure .
a new material resilon ( pentron clinical technologies , wallington , ct ) was introduced as a better alternative to gutta - percha .
this synthetic polymer claimed not only to provide a better seal but also reinforces the tooth structure through a combination of primer , dual cure sealer and resin obturating material .
the polyester chemistry containing bioactive and radiopaque fillers made it to possess better handling characteristics and look alike gutta - percha .
in addition , when used in conjunction with a resin - based sealant or bonding agent it forms a monoblock within the canals that bonds to the dentinal walls , it appears logical that they have the potential to strengthen the walls against fracture.1,8 a second strategy has been employed in which an unusual resin is created by first reacting one of the isocyanato groups of a diisocyanate with the hydroxyl group of a hydroxyl terminated polybutadiene as the latter is bondable to hydrophobic polyisoprene .
this is followed by grafting of a hydrophilic methacrylate functional group to the other isocyanato group of the diisocyanate producing a gutta - percha resin coating that is bondable to a methacrylate - based resin sealer .
the resin coated gutta - percha is recommended to be used with a recently modified patented version of a hydrophilic methacrylate - based dual - cured resin sealer ( endorez).9 in the light of incidence of vertical root fractures associated with gutta - percha filling techniques , this study was undertaken to compare the fracture resistance of roots endodontically treated with different resin - based adhesive sealers with conventional lateral condensation technique .
a total of 60 single canalled extracted maxillary anterior teeth , caries free and periodontally compromised , which was confirmed by the department of periodontology , km shah dental college , were collected and cleaned of soft tissue debris and calculus and stored in phosphate buffered saline ( pbs , ph 7.2 ) containing 0.1% sodium azide to inhibit bacterial growth for a maximum of 5 days .
caries free teeth , with a single root and a single , straight canal confirmed radiographically , completely formed apices with root lengths ranging from 14 to 16 mm and bucco lingual diameter ranging from 6 to 8 mm were included for the present study .
all the teeth were carefully examined under 16 magnifications with the help of digital operating microscope ( seiler dental microscope , seiler instruments inc . , st .
louis , usa ) to rule out preexisting fractures and all unacceptable ones were discarded .
the selected teeth were sectioned at the cement - enamel junction with a diamond disk operated on a slow speed micromotor hand piece under a constant water coolant flow .
the first group served as a negative control group containing the roots neither instrumented nor obturated . in the remaining four groups ,
the working length was determined by introducing a size 10 k file into the canal until it exited from the apex and the final working length was set 1 mm short of that length .
after the introduction of hand files and establishment of a glide path , an automated torque control endomotor with an attached reduction gear hand piece was used with profile rotary series to clean and shape the root canals using glyde as a lubricant and a chelator ( figure 1 ) .
instrumentation of the root canals with profile rotary series instruments and torque control endomotor with a mounted reduction gear hand piece .
profile orifice shapers ( o.s ) , profile 0.06 and profile 0.04 tapers , were the three types of instruments used in the shaping of root canals .
instrumentation was initiated by the introduction of profile o.s 4 ( # 0.07/50 ) and profile o.s 3 ( # 0.06/40 ) files until the point of resistance without exerting any pressure .
roots were then submitted to chemomechanical preparation with the following sequence : # 30/.06 , # 25/.06 , # 20/.06 , # 30/.04 , # 25/.04 , # 20/.04 , and finally # 30/.06 . in between each preparation 2 ml
after completion of instrumentation , all specimens received a final flush of 5 ml 17% of edta irrigating solution following manufacturer s instructions followed by 5 ml of 2.5% naocl solution in order to remove the smear layer .
then the specimens were randomly assigned into five experimental groups ( n = 12 ) per group as follows .
neither instrumentation nor obturation was done in the samples in this group ; the root canal openings were sealed with cavit .
these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) .
the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer .
the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) .
after checking the snug fit of the master resilon cone , the realseal primer was dispensed onto a microbrush available in the kit ( figure 3 ) , the excess removed by using paper points .
the apical third of the master cone was coated with the sealer and placed into canal and then a size 20 finger spreader was inserted , rotated and withdrawn .
an accessory resilon cone coated with a thin layer of sealer was placed into the space created by the spreader and process repeated until the canal was completely obturated .
the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument .
realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) .
the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points .
the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) .
after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) .
the former was inserted to 2 - 3 mm short of working length and slowly withdrawn to fill the entire canal with sealer avoiding entrapment of air . the prefit master cone ( 0.06 taper resin coated , iso no .
30 ) was then inserted into the canal to the working length , followed by the passive placement of multiple , accessory 0.02 taper , no .
# 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger .
the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument .
endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) .
all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with
the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures .
after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice .
extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls .
the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured .
the block with embedded root were mounted on to the loading frame of material testing system .
fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed .
the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test ,
the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . a complete graphical representation of the entire testing procedure on a particular specimen ( axial force vs. displacement graph ) .
neither instrumentation nor obturation was done in the samples in this group ; the root canal openings were sealed with cavit .
these teeth received a root filling by cold lateral condensation technique with gutta - percha cones and endoflas fs ( zoe - based sealer ) ( sanlor dental products , usa ) .
the third group received a root filling by cold lateral condensation technique with gutta - percha and ah plus ( dentsply , detrey , germeny ) an epoxy resin - based root canal sealer .
the fourth group received a root filling by lateral condensation using realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) ( figure 2 ) .
after checking the snug fit of the master resilon cone , the realseal primer was dispensed onto a microbrush available in the kit ( figure 3 ) , the excess removed by using paper points .
the apical third of the master cone was coated with the sealer and placed into canal and then a size 20 finger spreader was inserted , rotated and withdrawn .
an accessory resilon cone coated with a thin layer of sealer was placed into the space created by the spreader and process repeated until the canal was completely obturated .
the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess resilon to be removed with a hot instrument .
realseal resilon points and realseal dual cure resin sealer ( pentron clinical technologies , wallingford , ct ) .
the realseal primer dispensed on to a microbrush , the primer applied on the root canal walls with the help of the microbrush , excess primer was removed by using paper points .
the fifth group was root canals filled with cold lateral condensation with endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) ( figure 4 ) .
after checking the snug fit of the master endorez cone , the mixed sealer was injected into the root canal via 30-gauge navitip ( ultradent ) attached to skini syringe ( ultradent ) ( figure 5 ) .
the former was inserted to 2 - 3 mm short of working length and slowly withdrawn to fill the entire canal with sealer avoiding entrapment of air . the prefit master cone ( 0.06 taper resin coated , iso no .
30 ) was then inserted into the canal to the working length , followed by the passive placement of multiple , accessory 0.02 taper , no .
# 20 resin coated gutta - percha cones , to reduce the sealer volume and to avoid scraping off of the resin coating with the use of spreader / plugger .
the coronal portion of the sealer was subsequently light cured for 40 s , to stabilize the material , enabling excess gutta - percha to be removed with a hot instrument .
endorez resin coated gutta - percha points and endorez resin - based sealer ( ultradent , south jordan , usa ) .
all roots were kept at 37c with 100% humidity for 2 weeks to allow the sealers to set completely after which the samples were prepared to be subjected to mechanical strength testing . an acrylic block with a simulated periodontal ligament created with
the help of vinyl polysiloxane light body rubber base impression material was fabricated to allow for adequate stabilization of the specimens during testing procedures .
after the resin had set , the blocks with embedded roots were mounted on to the loading frame of material testing system ( minneapolis , minnesota , usa ) ( figure 6 ) . a stainless steel pin simulating a size 40 spreader was manufactured and fixed into the cross head and placed directly above the root canal orifice .
extreme care was taken to ensure that all the roots were embedded in an exact line parallel to the long axis of the pin in order to prevent any unusual oblique stresses on the root canal walls .
the testing machine was calibrated with these components to vertically drive the pin into the canal at a cross head speed of 5 mm / min during which it penetrated the root canal and force was applied to the root until it fractured . the block with embedded root
fracture was defined as the point at which a sharp and an instantaneous drop>25% of the applied force was observed for each root and so the machine was adjusted to terminate the test when a 25% reduction of force was observed .
the force applied to the root canal was recorded in the form of a graphical representation in addition to the continuous digital display of different test parameters ( graph 1 ) . throughout the test ,
the loads at which different root specimens fractured were recorded in newtons and the data were subjected to statistical analysis . a complete graphical representation of the entire testing procedure on a particular specimen ( axial force vs. displacement graph ) .
firstly one - way anova test followed by post - hoc scheffe multiple comparison tests on both the variables of length and faciolingual diameters of different samples were performed and it was concluded that all the samples of the experimental groups were completely well - balanced concerning the defined constraints .
the results of fracture resistance obtained were measured in newtons for each specimen has been tabulated in ( table 1 ) .
the descriptive statistics of loads of various specimens of different groups are mentioned in ( table 2 ) .
descriptive statistics of the loads to fracture different samples . a graphical representation of the mean load values to fracture roots obturated with different sealers is shown below ( graph 2 ) . a graphical representation of the mean load values to fracture roots obturated with different sealers .
considering this statistical situation a more detailed analysis one - way anova test was called for ( table 3 ) .
further this was substantiated with multiple comparisons through post - hoc tukey hsd multiple comparison test ( table 4 ) .
one - way anova revealed highly significant difference between groups ( p < 0.0001 ) .
statistically significant difference is noted in fracture resistance values of control group and certain test groups i.e. , the control group displayed significantly higher values than both the gutta - percha groups ( group ii and iii ) ( p < 0.05 ) . while groups iv and v displayed values lower than the control group but were statistically insignificant ( p > 0.05 ) . among the test groups , resilon group ( group iv ) and endorez group ( group v ) displayed significantly higher values than the gutta - percha groups ( group ii - endoflas and group iii - ah plus ) ( p < 0.05 ) .
furthermore , there was no statistically significant difference between the values of group iv and group v ( p > 0.05 ) .
similarly , there was no statistically significant difference between the values of group ii and group iii ( p > 0.05 ) .
the primary goal of endodontics is not only to restore the tooth structure but also to increase the inherent strength of the remaining tooth structure .
although the use of gutta - percha with an insoluble root canal sealer can be seen as a gold standard of root canal fillings , the ability of these materials to reinforce an endodontically treated root is discussed with controversy .
certain resin - based materials have been proposed since long to reinforce the endodontically treated teeth like diaket,10 hydron,11 ketac endo aplicap12 ah 26 , ah plus13,14 endoresin.15,16 the development of bonded obturating materials is in congruence with the efforts to provide a more effective seal apically as well as coronally .
the adhesion between dental structures and resin - based sealers is the result of a physicochemical interaction across the interface , allowing the union between filling material and root canal wall.17,18 this process is important in static and dynamic situations . in static circumstances ,
the adhesion eliminates spaces that allow the infiltration of fluids into the sealer / dentine interface.19 in dynamic situations , the adhesion is necessary to avoid the sealer dislodgment during operative procedures .
therefore , the endodontic filling materials may enhance the ability of root filled teeth to resist fracture.20,21 because the resin core , sealant , and the dentinal wall all are attached , it appears logical that they have the potential to strengthen the walls against fracture .
however , very few studies have been conducted on this aspect to support the hypothesis regarding the root reinforcement ability of the newer resin - based systems .
all the root canals were enlarged by a single operator to minimize operator variation.13 canal shape after preparation with hand files can be quite irregular.22 from a fracture mechanics point of view , the presence of structural defects , cracks or canal irregularities is likely to play a major role in determining fracture strength.23 rotary niti canal preparation using profile rotary instruments did not reduce fracture susceptibility of the root and also the roots were significantly weakened by the preparation with greater taper instruments.24 during the root canal preparation freshly prepared 1% sodium hypochlorite was used as an irrigant as it is the most commonly used irrigant , and a low concentration was used to minimize the adverse effect on dentin mechanical properties.1 final rinse was done with edta followed by naocl to enhance the bonding of the materials tested to the dentinal surface of the root .
this was done in accordance with the protocol recommended by weiger et al . to use edta followed by naocl , which seemed to optimize adhesion of sealers to the root canal
walls.25 root canal obturation has been implicated as the major cause of vertical root fracture . in lateral condensation ,
the strain is generated by the wedging effect of the spreader because it laterally compacts the gutta - percha and adapts it to canal wall while vertical condensation creates strains as the mass of gutta - percha is compacted apically with pluggers under consistent vertical load.5,26 however , lateral condensation technique was used in this study because it is a more widely recommended and a proven classic technique,7 which facilitates comparison with previous studies.1,27 furthermore , the principal reason is that endorez system is only provided in the form of cones and not yet available in any other form for vertical or thermal condensation.3 strength testing is the methodology that has been used to study the influence of filling materials on the fracture resistance of teeth submitted to root canal treatment9,12,28 as performed in this study . in this study ,
the force was applied along the long axis of the root with a stainless steel pin , which produced root fracture when contact was made between the pin and the walls of the canal opening .
the method adapted in this study to fracture the root specimens was chosen because it provided force distribution from inside the root canal and fractures occurring as a result of forces transmitted via the obturating material.5 this resembled root fracture of endodontic origin or from a post.27 stresses generated from inside the root canal were transmitted through the root to the surface where the interdentin bonding failed.29 the test was terminated after a 25% drop in the maximum force recorded similar to that used in the previous studies.1,8,13 the method presented in this study for measuring the fracture strength of obturating material has proven to be effective and reproducible . it is simple and easy to duplicate and reliable in accordance with the results of previously published findings .
the following conclusions were drawn from this study :
intact roots have greater fracture resistance than that of the instrumented roots.intact roots have significantly greater fracture resistance than the gutta - percha filled roots.the roots obturated with both the newer resin - based adhesive systems i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots.there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots.there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer .
intact roots have significantly greater fracture resistance than the gutta - percha filled roots . the roots obturated with both the newer resin - based adhesive systems
i.e. realseal and endorez have significantly greater fracture resistance than the gutta - percha filled roots .
there is no statistically significant difference in fracture resistance of the roots obturated with gutta - percha / endoflas fs and gutta - percha / ah plus sealer . there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer and the intact roots .
there is no statistically significant difference in fracture resistance of roots obturated with resin coated gutta - percha / endorez sealer and the intact roots .
there is no statistically significant difference in fracture resistance of roots obturated with resilon / realseal sealer , and resin coated gutta - percha / endorez sealer .
although the present results concerning the adhesive root canal filling materials realseal and endorez to reinforce the endodontically treated roots are very promising some care should be taken in the transfer of these findings to the long term clinical situation . further in vivo and biocompatibility tests involving newer resin - based systems will be necessary to determine whether the results in vitro will be validated .
clinical long - term studies are necessary to collect evidence - based data to support the confident use of these materials . | background : to compare fracture resistance of endodontically treated teeth obturated with different resin - based adhesive sealers with a conventional obturation technique.materials and methods : a total of 60 single canaled teeth were divided into five groups .
the first group was taken as a negative control .
the rest of the groups were shaped using profile rotary files ( dentsply maillefer , ballaigues , switzerland ) .
the second group was obturated with gutta - percha and a zoe - based sealer endoflas fs ( sanlor dental products , usa ) .
the third group was obturated with gutta - percha and an epoxy - based sealer ah plus ( dentsply , detrey , germany ) .
the fourth group was obturated with resilon ( pentron clinical technologies , wallingford , ct ) and realseal sealer ( pentron clinical technologies ) .
the fifth group was obturated with endorez points and endorez sealer ( both from ultradent , south jordan , ut ) .
roots were then embedded into acrylic blocks and were then fixed into a material testing system and loaded with a stainless steel pin with a crosshead speed of 5 mm / min until fracture .
the load at which the specimen fractured was recorded in newtons.results:it was found that forces at fracture were statistically significant for the newer resin systems , resilon , and endorez.conclusion:it was concluded that roots obturated with newer resin systems ( resilon and endorez ) enhanced the root strength almost up to the level of the intact roots . |
to provide a literature review of implant related complications from bilateral newcoloriris implantation ( kahn medical devices , panama city , panama ) .
a pubmed search of peer - reviewed ophthalmology journals for complications related to newcoloriris implants was performed .
including a recent case published by the authors , a total of nine patients ( 18 eyes ) with management of complications such as endothelial cell loss , elevated intraocular pressure ( iop ) , corneal edema , anterior chamber inflammation , decreased visual acuity , and cataract formation were reviewed .
sight - threatening complications including corneal decompensation , iop elevation , uveitis , and hyphema have been described after newcoloriris implantation .
for the purposes of cosmetic eye color change , newcoloriris ( kahn medical devices , panama city , panama ) silicone prosthesis implants were developed in 2004.1 although not approved by the food and drug administration ( fda ) , implantation has been performed with subsequent complications noted in the literature , including endothelial cell loss , elevated intraocular pressure ( iop ) , corneal edema , anterior chamber inflammation , decreased visual acuity , and cataract formation.28 while not specific to the newcoloriris , placement of phakic intraocular lenses have been associated with posterior segment complications , such as giant retinal tears.9 in recent years , newcoloriris implants have been removed following the development of acute and chronic side effects.24,68,10 this paper summarizes the published cases of complications arising from newcoloriris implantation for cosmesis in phakic eyes .
table 1 was compiled from a pubmed search of peer - reviewed ophthalmology journals . to our knowledge ,
only eight cases ( 16 eyes ) with complications following newcoloriris cosmetic implantation surgery had been published prior to the submission of our recent case.29 of the nine known cases ( 18 eyes ) including our own , six of the nine ( 66.7% ) involve female patients .
the only available long - term safety data comes from the manufacturer , which studied twelve female patients ( 24 eyes ) with a mean endothelial cell density loss of 2.6% after eight months of follow - up.1 in this series , only one patient ( two eyes ) required explantation due to uveitis and ocular hypertension .
our review summarizes nine cases ( 18 eyes ) , including one new bilateral case encountered by us that together suggest a need for more information regarding the long - term safety of newcoloriris implantation .
symptomatic patients can be stratified into early ( < 1 month ) or late ( > 6 months ) symptom onset , with seven of nine cases ( 77.8% ) presenting in the first postoperative month .
several of these patients reported immediate complications including hyphema , elevated intraocular pressure , and blurred vision.2,6 others were asymptomatic in the immediate postoperative period , but developed complaints in the following weeks.4,5,8 the most common complaint amongst early and late presenters was decreased visual acuity / blurred vision , which was present in all nine patients ( 18 eyes , 100% ) . at last follow - up ,
nine of the 16 eyes ( 56.25% ) had decreased best corrected visual acuity ( bcva ) , which is the same number of eyes with sub - optimal bcva at presentation ( table 1 ) . in the early presentation cohort , only one of 14 eyes ( 7.1% ) displayed hyphema , which resolved after anterior chamber paracentesis and two 25 mcg tissue plasminogen activator ( tpa ) injections.2 a proportionately higher two of four eyes ( 50% ) in the late presentation group developed hyphema , which resolved with conservative pharmacological management.3 corneal edema and elevated intraocular pressure were more common findings , occurring in 13 of 16 eyes ( 81.25% ) and 12 of 16 eyes ( 75% ) , respectively ( table 1 ) .
exam findings were not available for one patient.7 all published cases indicate anterior chamber inflammation visible by slit - lamp exam .
these procedures included : three trabeculectomies for iop management , two descemet stripping automated endothelial keratoplasties ( dsaek ) , and three peripheral iridotomies , two of which were performed by the implant surgeon.2,3,6,7 hull et al , in their case report of a 27-year - old female who presented with a large peripheral iridotomy , contended that their patient s account of acute pain upon implantation suggests that the iridotomy was performed intraoperatively in response to iris - prolapse.6 aside from the aforementioned procedures , one other patient was undergoing evaluation for descemet s stripping endothelial keratoplasty ( dsek ) secondary to ongoing corneal edema at last follow - up.2
in order to better understand the pathogenesis of the numerous complications seen in newcoloriris - implanted eyes , it is helpful to consider reported irregularities in the implants themselves .
several case reports in the literature identify manufacturing defects that likely contribute to implant dysfunction .
in one such study , scanning electron microscope ( sem ) images revealed surface irregularities and sharp edges that may contribute to the abrasion of the corneal endothelium and iris.4 concerns about the implant s peripheral footplates have also been documented . according to the manufacturer , these footplates are responsible for minimizing the implant s interaction with the trabecular meshwork.11 however , anderson et al report that four of six footplates were missing from one of the explanted prostheses in their case report .
they suspect that part of the implant had been hand - cut , resulting in significant edge irregularity.2 several authors have also demonstrated a tendency of the newcoloriris implant to directly contact the angle structures.3,4,8 pentacam images of our patient similarly illustrate the implant s close proximity to the iris and angle ( figure 1 ) .
these prostheses not only impinge on the angle structures , but shift position with time .
our patient displayed a temporally - shifted implant ( figure 2 ) , while another case in the literature describes a nasally - decentered presentation .
other authors have published images demonstrating improperly - sized implants that exhibit vaulting within the anterior chamber.2 the pathogenesis of the iop increases commonly seen in these patients may be multifactorial . in light of microscopic images showing pigment - laden macrophages and direct pigment deposition on the surface of the implant , some authors have attributed the rise to iris pigment liberation.2,6,8 others have cited the implant s direct contact with angle structures.24,6,8 explantation is an effective means of controlling elevated iop , but some patients may require long - term postoperative pharmacotherapy.2 in some cases , explanted eyes suffered insults that may lead to future complications .
our patient , like the one presented by thiagalingam et al , developed bilateral premature sclerotic changes in his native lenses.8 a 30-year - old female described by castanera et al experienced visual field deficits in her follow - up period.4 other eyes were found to have significant corneal decompensation.2,3,5,8 due to the high likelihood of postexplantation sequelae in these patients , they should receive long - term follow up with iop , bcva , endothelial cell counts , specular microscopy , and gonioscopy .
while anterior chamber depth and baseline endothelial cell density should be evaluated , close attention must be given to the dimensions of the cornea at the limbus .
given the asymmetry of the horizontal and vertical meridians of the cornea , the anterior chamber would not uniformly support a perfectly circular implant .
evaluation of the configuration of the iris root by imaging modalities could reveal variants such as plateau configuration , which would increase the risk of iris chafing .
specific measurement of each individual patient s anatomy may permit custom sizing of the implant in the future , with decreased associated risk of complications .
nevertheless , if preoperative evaluation suggests a patient may be a good candidate for an elective newcoloriris implant , this procedure currently has no proven safety record and may result in urgent explantation and long - term sight - threatening sequelae .
although it is difficult to estimate the extent of complications as it is unknown how many newcoloriris implants have been implanted , review of the literature reveals vision - threatening complications such as elevated iop , intraocular inflammation , and corneal decompensation . with its current design
, implantation should be seriously deliberated and risks such as possible surgery for cataracts , elevated iop , and explantation should be extensively discussed with potential patients . | purpose : to provide a literature review of implant related complications from bilateral newcoloriris implantation ( kahn medical devices , panama city , panama).methods : a pubmed search of peer - reviewed ophthalmology journals for complications related to newcoloriris implants was performed.results:including a recent case published by the authors , a total of nine patients ( 18 eyes ) with management of complications such as endothelial cell loss , elevated intraocular pressure ( iop ) , corneal edema , anterior chamber inflammation , decreased visual acuity , and cataract formation were reviewed.conclusions:sight-threatening complications including corneal decompensation , iop elevation , uveitis , and hyphema have been described after newcoloriris implantation . |
the internal transcribed spacer 2 ( its2 ) of the nuclear rrna cistron is a widely used phylogenetic marker .
as its sequence evolves comparably fast , it is mainly used for low - level analyses .
the hallmarks , namely four helices with the third as the longest , have been found in detailed exemplary studies ( 1 ) as well as in large - scale analyses ( 2 ) .
this lead to the suggestion to enlarge the application field to higher taxonomic levels ( 3 ) .
in addition to these phylogenetic analyses , a specific structural feature between two its2 , a compensatory base change ( cbc ) , can be used to distinguish two species from each other ( 4 ) .
this underlines the importance of considering not only the sequence but also the structure when performing any analysis based on the its2 .
but the proposed correct structure is only rarely automatically found by standard minimum free energy folding ( mfe ) ( 2 ) . to overcome this hindrance for the wider application of the its2
, we developed a homology - based structure modelling approach , which allowed predicting the structure for 20 000 sequences which were not found by rnafold ( 5 ) .
as these can be used as a basis for any phylogenetic analysis , we have developed the its2 database as a resource for sequence and structure information of the its2 ( 6 ) .
here we report modifications and improvements of the database which allowed us to find structural information for 86 784 its2 sequences , which is 55% of all entries concerning its2 in genbank .
in the first version of the database , every sequence whose correct structure could not be found by rnafold was searched against the original set of 5 000 sequences with correct rnafold based structures ( 2 ) to identify possible templates for homology modelling ( models ) . as a first step in the development of the new version of the database
, we checked whether there were additional novel sequences in genbank whose structure could be determined directly by rnafold .
indeed , we found a 2-fold increase in the amount of correctly predicted structures ( table 1 , method 1 ) .
we used this dataset as a starting point for a complete rebuild of the database .
more importantly , this result led us to a change in the logic and therefore to a re - design of the update procedure .
each time the structure of an incoming sequence can be predicted directly by rnafold or in the first round of homology modelling , it is added to the set of models . thus , no core sequence / structure set ( as before the set of 5 000 ) is existent any more but a dynamically growing set of possible structure models . in summary , this approach together with a second iteration of homology modelling allowed us to predict 38 350 structures ( table 1 , methods 2 and 3 ) .
table 1.methods used for its2 structure prediction and number of folded sequences.methoddescriptioncount1direct rnafold10 6672homology modelling , first iteration27 0443homology modelling , second iteration11 3064direct rnafold , sequence discovery by blast5 1965homology modelling , first iteration , sequence discovery by blast1 7306homology modelling , second iteration , sequence discovery by blast17 7767partial structures from homology modelling , both iterations13 065total86 784 methods used for its2 structure prediction and number of folded sequences .
if the boundaries are incorrect , missing or additional sequence fragments might be considered as a specific feature of an organism leading to a wrong phylogenetic classification . with the correct structure at hand ,
the boundaries of the its2 can be exactly determined , again underlining the importance of considering structure for phylogenetic analyses .
accordingly , already in the first version of the database , a clustalw - based approach ( 7 ) was used to extend the sequence if the genbank annotation missed the first or the last helix . as this approach
was limited to cases where ( i ) there exists a feature annotation by genbank and ( ii ) the homology modelling was of high quality for the other helices , we developed a novel , blast - based approach ( 8) for the re - annotation of genbank entries .
for the cases where no structure could be predicted for the its2 as annotated by genbank , the whole genbank entry is retrieved and searched against all sequences with known structures using blast . if a significant hit is found ( e - value 10e ) , the homologous region of the query is cut .
rnafold is used to test whether this fragment can be folded in the correct structure . if not
, we were able to re - annotate the position of the its2 in 6901 genbank entries .
in most cases , this structure - based annotation lead to a slight shift of the 5 ' or / and the 3 ' end of the its2 , but some entries were heavily shifted ( figure 1 ) .
for example the its2 of trifolium affine ( gi:85724133 ) is incorrectly annotated with a length of just 7 bp , its preceding 5.8s ribosomal rna with 9 bp .
these cases underline the advantage of the structure - based annotation compared to one based on sequence information alone .
figure 1.re-annotated sequences , each dot representing a successfully predicted secondary structure x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation .
six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown .
re - annotated sequences , each dot representing a successfully predicted secondary structure x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation .
six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown .
in contrast to the method used in the previous version of the database , the blast - based approach is completely independent of any pre - annotated its2 .
its2 without a feature annotation lead to the new annotation of 17 801 its2 sequences . many of the sequences without predicted structure were fragments , i.e. they missed at least one helix of the structural hallmark and therefore did not fulfil the quality control of the standard homology modelling . still , these sequences could increase the coverage of a systematic analysis .
in contrast to the mfe approach , our homology - modelling algorithm is able to predict the structure of fragments . to assure a sufficient quality , only entries where
at least two consecutive helices could be modelled with sufficient quality ( 75% ) were accepted .
this method resulted in additional 13 065 its2 sequences with structural information ( table 1 , method 7 ) .
the existence of a large number of pairwise alignments allowed us to calculate its2-specific evolutionary models .
based on variants of the methods described in mller and vingron ( 9 ) and mller et al .
( 10 ) , we were able to derive an its2-specific substitution model , which is an important ingredient for phylogenetic analyses .
this model reflects nicely the special features of rna and in particular , its2 sequence evolution .
based on this molecule - specific substitution model , an its2-specific scoring matrix is derived that strongly deviated from the unity matrix as used as default , for example in blast . to test the influence of this matrix compared to the standard identity matrix , we performed all calculations with the standard and the its2-specific matrix , respectively . in the genbank version used in the test run ,
structural information was found for 57 680 sequences whereas the usage of the its2-specific matrix resulted in 76 721 structures .
this underlines the importance of the correct evolutionary model in the homology modelling of its2 and presumably other rna sequences .
accordingly , the its2-specific score matrix is now used in all calculations for the its2 database and can be downloaded from the web site as supplementary data .
here , the user provides an rna sequence with a known structure and other , homologous sequences . for these
, a homology model is calculated based on the known structure . when using this feature , it has to be taken into account that there is , in contrast to the modelling of its2 , no quality measure for the model .
thus , it is the obligation of the user to check the validity of the results .
in the first version of the database , every sequence whose correct structure could not be found by rnafold was searched against the original set of 5 000 sequences with correct rnafold based structures ( 2 ) to identify possible templates for homology modelling ( models ) . as a first step in the development of the new version of the database
, we checked whether there were additional novel sequences in genbank whose structure could be determined directly by rnafold .
indeed , we found a 2-fold increase in the amount of correctly predicted structures ( table 1 , method 1 ) .
we used this dataset as a starting point for a complete rebuild of the database .
more importantly , this result led us to a change in the logic and therefore to a re - design of the update procedure .
each time the structure of an incoming sequence can be predicted directly by rnafold or in the first round of homology modelling , it is added to the set of models . thus , no core sequence / structure set ( as before the set of 5 000 ) is existent any more but a dynamically growing set of possible structure models . in summary , this approach together with a second iteration of homology modelling allowed us to predict 38 350 structures ( table 1 , methods 2 and 3 ) .
table 1.methods used for its2 structure prediction and number of folded sequences.methoddescriptioncount1direct rnafold10 6672homology modelling , first iteration27 0443homology modelling , second iteration11 3064direct rnafold , sequence discovery by blast5 1965homology modelling , first iteration , sequence discovery by blast1 7306homology modelling , second iteration , sequence discovery by blast17 7767partial structures from homology modelling , both iterations13 065total86 784 methods used for its2 structure prediction and number of folded sequences .
if the boundaries are incorrect , missing or additional sequence fragments might be considered as a specific feature of an organism leading to a wrong phylogenetic classification . with the correct structure at hand
, the boundaries of the its2 can be exactly determined , again underlining the importance of considering structure for phylogenetic analyses .
accordingly , already in the first version of the database , a clustalw - based approach ( 7 ) was used to extend the sequence if the genbank annotation missed the first or the last helix . as this approach
was limited to cases where ( i ) there exists a feature annotation by genbank and ( ii ) the homology modelling was of high quality for the other helices , we developed a novel , blast - based approach ( 8) for the re - annotation of genbank entries .
for the cases where no structure could be predicted for the its2 as annotated by genbank , the whole genbank entry is retrieved and searched against all sequences with known structures using blast . if a significant hit is found ( e - value 10e ) , the homologous region of the query is cut .
rnafold is used to test whether this fragment can be folded in the correct structure . if not
, we were able to re - annotate the position of the its2 in 6901 genbank entries .
in most cases , this structure - based annotation lead to a slight shift of the 5 ' or / and the 3 ' end of the its2 , but some entries were heavily shifted ( figure 1 ) .
for example the its2 of trifolium affine ( gi:85724133 ) is incorrectly annotated with a length of just 7 bp , its preceding 5.8s ribosomal rna with 9 bp .
these cases underline the advantage of the structure - based annotation compared to one based on sequence information alone .
figure 1.re-annotated sequences , each dot representing a successfully predicted secondary structure x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation .
six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown .
re - annotated sequences , each dot representing a successfully predicted secondary structure x - axis represents shift in the 5 ' end of the its2 , y - axis change of the length compared to the genbank annotation .
six outliers ( gi : 5814072 , 57999795 , 2896060 , 13507073 , 4006937 , 85724147 ) are not shown .
in contrast to the method used in the previous version of the database , the blast - based approach is completely independent of any pre - annotated its2 .
its2 without a feature annotation lead to the new annotation of 17 801 its2 sequences .
many of the sequences without predicted structure were fragments , i.e. they missed at least one helix of the structural hallmark and therefore did not fulfil the quality control of the standard homology modelling .
in contrast to the mfe approach , our homology - modelling algorithm is able to predict the structure of fragments . to assure a sufficient quality , only entries where
at least two consecutive helices could be modelled with sufficient quality ( 75% ) were accepted .
this method resulted in additional 13 065 its2 sequences with structural information ( table 1 , method 7 ) .
the existence of a large number of pairwise alignments allowed us to calculate its2-specific evolutionary models . based on variants of the methods described in mller and vingron ( 9 ) and mller et al .
( 10 ) , we were able to derive an its2-specific substitution model , which is an important ingredient for phylogenetic analyses .
this model reflects nicely the special features of rna and in particular , its2 sequence evolution .
based on this molecule - specific substitution model , an its2-specific scoring matrix is derived that strongly deviated from the unity matrix as used as default , for example in blast . to test the influence of this matrix compared to the standard identity matrix , we performed all calculations with the standard and the its2-specific matrix , respectively . in the genbank version used in the test run ,
structural information was found for 57 680 sequences whereas the usage of the its2-specific matrix resulted in 76 721 structures .
this underlines the importance of the correct evolutionary model in the homology modelling of its2 and presumably other rna sequences .
accordingly , the its2-specific score matrix is now used in all calculations for the its2 database and can be downloaded from the web site as supplementary data .
here , the user provides an rna sequence with a known structure and other , homologous sequences . for these
, a homology model is calculated based on the known structure . when using this feature , it has to be taken into account that there is , in contrast to the modelling of its2 , no quality measure for the model .
thus , it is the obligation of the user to check the validity of the results .
with the modifications of the its2 database outlined above , the structural features of 86 784 sequences were predicted , which was 55% of all genbank entries concerning the its2 . as this number gives just an overall average , we tested the coverage of predicted structures within all genera .
a clear separation was found between genera where the structure for nearly all sequences could be predicted and others , where no structure was found despite considerable sequence coverage ( figure 2 ) .
we suggest that in these genera the structure of the its2 deviates from the standard .
this notion is supported by their length distribution being nearly equal to the length distribution of successfully folded sequences ( data not shown ) .
furthermore , within those genera without any structural data , there is a strong bias towards metazoans ( 11 ) .
this is consistent with the observation that vertebrates have a more complex structure than the one described by coleman ( 1 ) .
the latter one fits mostly for plants and fungi , taxa whose genera are strongly represented in the overall number of genera with structural data .
figure 2.structure coverage each point indicates one genus . on the y - axis , the square root of the number of sequences in the genus is indicated . on the x - axis ,
the percentage of correct structures for all sequences of the genus is plotted . additionally on top of the scatter plot ,
a similar , less pronounced effect can be seen at 33.3% and 66.6% for genera with three sequences .
the square root of the number of sequences in the genus is indicated . on the x - axis
, the percentage of correct structures for all sequences of the genus is plotted . additionally on top of the scatter plot ,
a similar , less pronounced effect can be seen at 33.3% and 66.6% for genera with three sequences .
how could a user , who is interested in the phylogeny of a specific taxonomic group , use the its2 database ? if he starts with an already known sequence , he can directly extract the corresponding structure from the database ( search by gi / accession / taxon ) .
if he has sequenced his own organisms , he should first homology model the structure of this sequences ( predict its2 structure ) .
second , he can extract its2 sequences and their structures for further organisms in the taxonomic group of interest ( browse taxonomy )
. this will result in a set of its2 sequences with corresponding structures . in the third step ,
these have to be aligned . here , an alignment program , which considers both sequence and structure , like 4sale ( 12 ) , will be suitable .
finally , this sequence structure - based alignment will be the input for standard phylogenetic analyses , e.g. in paup ( 13 ) or phylip ( 14 ) .
furthermore , one is now able to check for cbcs to distinguish possible different species in the dataset ( 4 ) or to calculate cbc trees ( 15 ) . | an increasing number of phylogenetic analyses are based on the internal transcribed spacer 2 ( its2 ) .
they mainly use the fast evolving sequence for low - level analyses .
when considering the highly conserved structure , the same marker could also be used for higher level phylogenies . furthermore
, structural features of the its2 allow distinguishing different species from each other . despite its importance ,
the correct structure is only rarely found by standard rna folding algorithms . to overcome this hindrance for a wider application of the its2 ,
we have developed a homology modelling approach to predict the structure of rna and present the results of modelling the its2 in the its2 database . here , we describe the database and the underlying algorithms which allowed us to predict the structure for 86 784 sequences , which is more than 55% of all genbank entries concerning the its2 .
these are not equally distributed over all genera .
there is a substantial amount of genera where the structure of nearly all sequences is predicted whereas for others no structure at all was found despite high sequence coverage .
these genera might have evolved an its2 structure diverging from the standard one .
the current version of the its2 database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de . |
fetal malnutrition ( fm ) is a significant contributor to perinatal morbidity and mortality.123 it is a state of poor nutrition in - utero resulting from inadequate supply and/or utilization of nutrients1 leading to the fetus failing to acquire adequate amount of fat , subcutaneous tissues , and muscle mass during intrauterine growth.1 fm describes the underweight / wasting aspect of the clinical syndrome seen in malnourished newborns .
this clinical state may be present at almost any birth weight.2 globally , the incidence of fm is between 2% and 10% of total births with highest incidence in developing countries.2 a study conducted in ile - ife , nigeria , reported an incidence of 18.8% for fm in term newborns.3 the assessment of the nutritional status of the fetus has been of considerable interest to clinicians because of its potential serious sequelae on multiple organ systems manifesting as perinatal problems and/or long - term central nervous system sequelae.34 recent studies have also demonstrated the evidence that fm may have a far - reaching effect on adult life such as susceptibility to cardiovascular disease and non - insulin dependent diabetes mellitus.567 various anthropometric indices have been used to identify babies that suffered suboptimal fetal growth ( weight , length , mid - arm circumference ( mac ) and head circumference ( hc),89 proportionality indices ( ponderal index [ pi],8910 mac / hc ratio , body mass index [ bmi ] ) , and clinical assessment of nutritional ( can ) status and its score.91112 can score which contains nine clinical signs , namely , hair , cheeks , neck , arms , chest , abdomen , back , buttocks , and legs , as developed by metcoff11 to differentiate malnourished from appropriately nourished babies4 has also been used widely by researchers to determine fm in the newborn period .
there is a dearth of research in the combined use of can score , bmi , and the other anthropometric indices as a means of assessing fm in newborns in the west african subregion .
the aims of the present study are to assess the nutritional status of term newborns at birth using can score , bmi , pi , mac / hc , and birth weight and to compare the relative efficiency of can score and the anthropometric indices in the identification of fm in term newborns . the findings from this study will provide renewed insight into the easier and better methods of determining fm in term newborns .
the study was carried out on consecutive , singleton , live born babies of 37 completed weeks through 41 weeks of gestation at the inborn unit of luth between may 1 , 2010 , and november 30 , 2010 .
all the anthropometric measurements , except birth weight , were carried out by the investigator with trained assistance where needed , within 48 h of baby 's birth .
all the neonates were weighed completely nude at birth by the delivery room staff using the infant weighing scale ( weighmaster model , usa ) which records the weight to the nearest 10 g. using the olowe intrauterine growth chart,13 birth weights for gestational age below the 3 percentile and above the 97 percentile on the chart were taken as small for gestational age ( sga ) and large for gestational age ( lga ) , respectively .
the hc was measured using a flexible nonstretchable tape measure with due attention to the appropriate prominences.14 the measurement was to the nearest 0.1 cm .
the mac was measured using the same tape at the midpoint between the acromion and the olecranon process with the forearm flexed at 90 at the elbow.14 in the abducted arm with the elbow flexed ( in almost all the term babies ) , a skin crease appears which corresponds approximately with the midpoint of the arm.141516 the readings obtained were recorded to the nearest 0.1 cm .
pi was computed from the formula : pi = weight ( g)/length ( cm ) 100 and pi < 2.2 was considered as malnutrition .
the mac / hc ratio was calculated for each infant and the value plotted on a standard curve designed for nigerian newborn.161718 bmi was calculated for each baby and the value was plotted on the bmi curve designed by brock et al .
for newborn babies.18 can status was done within 48 h of life on the basis of the superficial readily detectable signs of malnutrition in the newborn as described by metcoff [ table 1].4 this consisted of inspection and estimation of loss of subcutaneous tissues and muscles in the designated areas .
hair , cheeks , neck and chin , arms , back , buttocks , legs , chest , and abdomen were examined and then scored .
a maximum score of 4 was awarded to each parameter with no evidence of malnutrition , and the lowest score of 1 was awarded to parameter with the worst evidence of malnutrition .
the total rating of the 9 can score signs was the can score for the subject .
fm was defined as can score < 25.41920 the nine signs for clinical assessment of nutritional status in the newborn statistical analysis was performed using the statistical package for social sciences ( spss)for windows , version 17.0 ( spss inc , chicago released 2008 , ibm corp , new york ) .
student 's t - test was used to compare the mean anthropometry between males and females .
one hundred and thirty - seven were male while 145 were female , with a male : female ratio of 1:1.07 .
eight babies ( 2.8% ) were of low birth weight and sga while 15 ( 5.3% ) were lga .
the anthropometric characteristics of the study population showed that the mean birth weight was 3260 460 g and mean length of 49.37 2.26 cm while the mac and hc were 34.63 1.19 cm and 10.83 1.01 , respectively .
about 53.2% of the babies were born to multiparous mothers and 39% of the mothers had various illnesses during pregnancy ( high blood pressure [ 30% ] , malaria [ 15.9% ] , and retroviral disease [ 37.8% ] ) . the prevalence of fm as detected by can score was 14.5% while bmi detected 13.1% of the study participants as malnourished .
pi and mac / hc detected fm in 10.3% and 2.8% of the study population , respectively .
the incidence of fm is slightly more in male babies , but it was not statistically significant except with mac / hc ratio which detected no female newborn with fm .
relationship between sex and fetal malnutrition two hundred and fifty - nine babies ( 91.8% ) were appropriate for gestational age ( aga ) , 15 ( 5.3% ) were lga , and 8 ( 2.8% ) were classified as sga using olowe 's intrauterine growth chart .
can score identified 15.4% of the aga babies as fm and only one of the sga babies as fm .
tables 3 and 4 show the comparison of can score with anthropometry in detection of fm .
pi , mac / hc , and birth weight showed very low sensitivity of 19.5% , 12.3% , and 2.4% , respectively , in detecting fm ; however , all showed very high specificity ( 90.9% , 98.6% , and 97.1% , respectively ) in detecting fm .
comparison between clinical assessment of nutritional score and anthropometric indices in detection of fetal malnutrition in term newborns the sensitivity , specificity and predictive value of anthropometric indices in determining fetal malnutrition
about 53.2% of the babies were born to multiparous mothers and 39% of the mothers had various illnesses during pregnancy ( high blood pressure [ 30% ] , malaria [ 15.9% ] , and retroviral disease [ 37.8% ] ) .
the prevalence of fm as detected by can score was 14.5% while bmi detected 13.1% of the study participants as malnourished .
pi and mac / hc detected fm in 10.3% and 2.8% of the study population , respectively .
the incidence of fm is slightly more in male babies , but it was not statistically significant except with mac / hc ratio which detected no female newborn with fm .
relationship between sex and fetal malnutrition two hundred and fifty - nine babies ( 91.8% ) were appropriate for gestational age ( aga ) , 15 ( 5.3% ) were lga , and 8 ( 2.8% ) were classified as sga using olowe 's intrauterine growth chart .
can score identified 15.4% of the aga babies as fm and only one of the sga babies as fm .
tables 3 and 4 show the comparison of can score with anthropometry in detection of fm .
pi , mac / hc , and birth weight showed very low sensitivity of 19.5% , 12.3% , and 2.4% , respectively , in detecting fm ; however , all showed very high specificity ( 90.9% , 98.6% , and 97.1% , respectively ) in detecting fm .
comparison between clinical assessment of nutritional score and anthropometric indices in detection of fetal malnutrition in term newborns the sensitivity , specificity and predictive value of anthropometric indices in determining fetal malnutrition
fm is prevalent in the developing countries.321 studies have documented that the nutritional status of the newborn is important for identifying perinatal risks42223 as any deviation from the normal is associated with an increased risk of morbidity and mortality.24 in the present study , we evaluated 282 babies for fm .
the mean birth weight of the babies was 3260 460 g which was higher than 3067 g recorded in ile - ife5 and 3080 g in jos,24 both in nigeria , and 2600 g reported in india.25 it was however comparable with the 3290 g documented by metcofff4 in the usa .
the prevalence of sga in term babies in the present study is low ( 2.8% ) compared to the prevalence reported by adebami et al.3 in ile - ife ( 12.1% ) and mehta et al.25 in india ( 25% ) while metcoff4 documented sga prevalence of 11% in term newborns in oklahoma , usa . the low prevalence of sga in the present study may be due to the different intrauterine growth curves used .
the olowe intrauterine growth chart13 used in this study classified babies < 2 standard deviations below the mean or below the 3 percentile as sga , whereas the brenner intrauterine growth chart which uses the 10 percentile as the cutoff for sga was used in the study by metcoff4 and adebami et al.3 a similar study in india by sankhyan et al.9 had also reported a lower prevalence of sga when local indian intrauterine growth chart was used ( 9.1% ) compared to when international growth charts which had higher cutoff weights at each gestational age was used ( 45.4% ) resulting in higher sensitivity but low specificity .
the prevalence of fm in the present study using can score ( 14.5% ) is slightly lower than 18.8% documented in ile - ife by adebami et al.3 and is 23-fold lower than that documented in india by sankhyan et al.9 and mehta et al.25 the reason for the lower prevalence in our study may be related to the demographics and socioeconomic status of the population studied .
a well - nourished and healthy mother is more likely to deliver a healthy and well - nourished baby .
the prevalence of fm using can score and anthropometry in various gestational age groups of infants had been reported by different researchers .
it has been demonstrated that birth weight alone may not reflect the fetal state of nutrition of a newborn adequately .
previous researchers had demonstrated that not all sga babies had fm and that some aga babies may have features of fm . in the study by metcoff,4 the prevalence of fm
sankhyan et al.9 documented features of fm in 57.1% of sga and 3.8% of aga newborns .
adebami et al.3 detected fm in 11.5% of term aga babies using can score . in the present study , can score identified fm in 15.4% of term aga babies indicating that these babies suffered from fm in spite of having normal birth weight . among sga babies , only one baby ( 2.4% ) was malnourished using can score ; thus , the term fm and sga are not synonymous .
this finding is also similar to that of other researchers such as metcoff that not all sga babies were fm .
the anthropometric parameters are important to reflect intrauterine growth and to define a baseline to follow - up the nutritional progress of the infant .
many authors have proposed that the assessment of body proportions may be more useful than single measurements for age alone for assessing newborn nutrition.892627 bmi provides a high estimate of body fat mass as it has a positive correlation with skinfold thickness and other methods of estimating the percentage of body fat,15 and it takes advantage of the physiologic principle regarding sparing length at the expense of weight during mild to moderate malnutrition.28 in our study , bmi detected fm in 13.1% of the babies and on comparing with can score , it was noted that the sensitivity of bmi in detecting fm was the highest among anthropometric parameters , suggesting bmi may be a sensitive indicator of fm .
soundarya et al.29 also found bmi to be a sensitive indicator of fm in their study and proposed a combination of bmi and pi in the screening of babies for fm .
this is lower than the prevalence recorded by mehta et al.25 ( 29.19% ) and kayshap et al.27 ( 27.8% ) in india .
it is , however , comparable with the lower prevalence of 8.1% documented by adebami et al.3 both mehta et al.25 and kashyap et al.27 combined the late gestational preterm babies of 35 weeks to the analysis of their results and thus may have contributed to their higher prevalence .
some babies with normal pi were found to be malnourished using can score in the present study .
can score identified 34 ( 13.4% ) of the babies with normal pi as malnourished .
the prevalence of fm in the present study using mac / hc ratio was very low ( 2.8% ) .
this is lower than the 49.76% recorded in term babies in india by mehta et al.25 although both studies used different cutoff values in determining fm.252730 it is interesting to note that not all babies classified as malnourished by anthropometry were found malnourished by can score and vice versa .
the present study , like others in the literature , had also shown that can score identified more babies with fm compared to anthropometry making it a reliable tool in the detection of fm .
we concluded that the newborn baby with fm is a high - risk newborn and his postnatal survival greatly depends on careful observation and documentation of the evidence of his adverse intrauterine life and a proactive management of his anticipated complications . a simple and easy way of identifying fm at birth is ideal and will make for judicious use of scarce resources in developing countries .
the different anthropometric indices measure different aspects of the well - being of a newborn while can score measures the visible wasting observed in malnourished newborn .
all the parameters , in one - way or the other , reflect the adverse intrauterine nutrition these newborns suffered .
therefore , the use of multiple methods of determining fm will increase the likelihood of identification of most babies with fm .
the combination of can score and bmi to the routine assessment of newborns at birth may further improve newborn care and buttress the need to look out for these high - risk babies for anticipatory care and follow - up .
| background : fetal malnutrition ( fm ) which describes the underweight / wasting seen in newborns is a significant contributor to perinatal morbidity and mortality and requires proper documentation .
the objective of this study was to assess the nutritional status of term newborns at birth using clinical assessment of nutritional ( can ) status score and four other anthropometric indices and to compare the efficiency of can score and the anthropometric indices in identifying fm in term newborns.materials and methods : the study was conducted on live singleton babies delivered 3742 completed weeks of gestation at the inborn unit of lagos university teaching hospital without major congenital abnormalities or severe perinatal illness . birth weights and lengths
were recorded at birth .
ponderal index ( pi ) , body mass index ( bmi ) , and mid - arm / head circumference ( mac / hc ) ratio were calculated and the values were compared with standard curves .
the can score consisted of inspection and estimation of loss of subcutaneous tissues and muscles .
fm was defined as can score < 25 .
data were analyzed using the statistical package for social sciences statistics software version 17.0.results:two hundred and eighty - two newborns were analyzed .
fm was identified in 14.5% , 10.3% , 13.1% , and 2.8% of newborns using can score , pi , bmi , and mac / hc ratio , respectively
. out of the fm babies identified by can score , pi , mac / hc , and bmi identified 19.5% , 12.3% , and 53.7% of them as fm also .
bmi was the most sensitive anthropometric index for detecting fm.conclusion:can score is a simple clinical tool for identifying fm and when used in conjunction with bmi will enhance fm detection . |
to determine the accuracy of the holladay 1 , hoffer q , sanders - retzlaff - kraff ( srk)/t , and srk ii iol power calculation formulas in patients with high myopia in a subset of indian population .
this study was conducted at sadguru netra chikitsalaya , chitrakoot , madhya pradesh , india .
all consecutive patients attending our out patient department ( opd ) for phacoemusification with axial length ( al ) more than 24.5 mm ( range 24.75 - 32.35 mm ) between may 2009-october 2009 ( in all patients hydrophilic acrylic foldable within the bag iol was implanted ) .
pre - existing astigmatism > 3.0 diopters ( d)corneal scarkeratoconuscomplications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) .
pre - existing astigmatism > 3.0 diopters ( d ) complications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) . in our study , keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed .
the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii , srk - t , holladay 1 , and hoffer q. with each formula , the mean error ( me ) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error . the spherical equivalent
was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery .
repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p < 0.05 was considered as significant .
this study was conducted at sadguru netra chikitsalaya , chitrakoot , madhya pradesh , india .
all consecutive patients attending our out patient department ( opd ) for phacoemusification with axial length ( al ) more than 24.5 mm ( range 24.75 - 32.35 mm ) between may 2009-october 2009 ( in all patients hydrophilic acrylic foldable within the bag iol was implanted ) .
pre - existing astigmatism > 3.0 diopters ( d)corneal scarkeratoconuscomplications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) .
pre - existing astigmatism > 3.0 diopters ( d ) complications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) . in our study , keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed .
the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii , srk - t , holladay 1 , and hoffer q. with each formula , the mean error ( me ) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error . the spherical equivalent
was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery .
repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p < 0.05 was considered as significant .
all consecutive patients attending our out patient department ( opd ) for phacoemusification with axial length ( al ) more than 24.5 mm ( range 24.75 - 32.35 mm ) between may 2009-october 2009 ( in all patients hydrophilic acrylic foldable within the bag iol was implanted ) .
pre - existing astigmatism > 3.0 diopters ( d)corneal scarkeratoconuscomplications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) .
pre - existing astigmatism > 3.0 diopters ( d ) complications significantly affecting the refractive status ( vitreous loss with iol implanted in sulcus or anterior chamber , high wound induced astigmatism ) . in our study , keratometry measurements were retrieved from records and a scan applanation ultrasonography was done on echorule2 and analyzed .
the implanted iol power was used to calculate the predicted postoperative refractive error by four commercially available iol formulas : srk ii , srk - t , holladay 1 , and hoffer q. with each formula , the mean error ( me ) was calculated from the difference between the formula predicted refractive error and actual postoperative refractive error . the spherical equivalent
was measured by a single trained optometrist using an autorefractor and subjective retinoscopy 1 - 2 months after cataract surgery .
repeated measures anova tests were done after making bonferroni corrections to have pair - wise comparisons between the formulas and p < 0.05 was considered as significant .
forty three eyes of 43 patients with axial length more than 24.5 mm in our set - up were retrospectively studied .
the keratometry in most of the patients ( 81% ) was within the normal range of 42.0 - 46.0 d. overall performance of all the formulas in al more than 24.5 mm showed a tendency to cause a hyperopic refractive error post - operatively [ table 1 ] .
overall performance of formulas in eye with al>24.5 mm holladay 1 caused the smallest me , + 0.24 d. fifty three percent of patients had mean absolute error less than 1.00 d. the hoffer q and srk - t caused a little larger hyperopic shift with mes of + 0.58d and + 0.92 d. srk ii caused the largest hyperopic error + 1.23 d. [ table 1 ] .
pair - wise comparisons between different formulas shows that p value between different formulas is significant ( p < 0.05 ) implicating that holladay 1 with least me is significantly different from other formulas and have better performance in patients with al more than 24.5 mm when compared to srk - t , srk ii , hoffer q [ table 2 ] .
pair - wise comparisons measure : me ( mean error ) the performance of each formula in al subcategory in eyes with al between 24.5 - 26.5 mm also found holladay 1 as the best formula [ table 3 ] .
performance of formulas in eye with axial length 24.5 - 26.5 mm pair - wise comparisons between different formulas in a subset of patients with a al 24.5 - 26.5 mm shows that holladay 1 ( me = + 0.4320 d ) and hoffer q ( me = + 0.4695 d ) are comparable , p value not being significant [ table 4 ] .
our preliminary results showed that in our patients with axial myopia with als longer than 24.50 mm , the holladay 1 provided the best predictive result and the hoffer q was comparable to holladay 1 for al 24.5 - 26.5 mm for iol power calculation .
this agrees with the results in several studies in which better performances were found in cases with long eyes with the holladay 1 formula than the srk ii formula .
olsen and co - authors reported that the holladay formula is more accurate than the srk ii . in a study conducted by stopyra , in which a study of patients with al 24.51 - 26.72 mm and found holladay 1 formula was the most accurate with 88.5% of the patients achieving full visual acuity ( 1.0 on snellen ) after cataract surgery .
stopyra also found srk / t formula satisfactory with 63.9% of the patients achieving full visual acuity on snellen but only 36.1% of the patients achieved full visual acuity using hoffer q. kijima et al .
, report the best results using the holladay and srk / t in the midrange al ( 24.5 - 26.9 mm ) group and the srk / t or l - srk in the long al ( longer than 27.0 mm ) group .
tsang cs and co - authors also reported best results in patients with al 25.0 mm using hoffer q and holladay 1 and srk - t were comparable with srk ii being least accurate and in patients with al > 28.0 mm they found holladay 1 and srk / t more accurate than hoffer q. narvaez and co - authors also reported better performance of these formulas when compared with srk - ii . in studies by bang and co - authors on al > 27.0 mm and wang and co - authors on al > 25.0 mm using iol master reported that haigis formula is the best .
ghanem and el - sayed reported that in patients with high axial myopia , the performance of srk - t , hoffer - q , holladay-2 , and haigis formulas are comparable in low plus powered iol implantation and haigis formula is the best formula when minus power iol is implanted .
however , we did not find performance of srk - t formula better in indian myopic population .
our recommendation is to avoid using the srk ii and srk / t formula for iol power calculation in eyes with axial myopia in indian population with al longer than 24.50 mm . in our study , all four formulas had a tendency to cause a postoperative hyperopic refraction .
our study had some limitations : ( a ) it was a single center retrospective study and ( b ) surgeons , and time of the postoperative refraction were not standardized .
to conclude , the results of our study provide useful information to aid the choice of iol power in patients with high axial myopia in indian population during our daily practice with holladay 1 being the most accurate and srk ii and srk / t to be avoided . | efficacy of intraocular lens power calculation formulas in a subset of indian myopic population . retrospectively reviewed 43 patients who underwent phacoemulsification with high axial length ( al ) ( > 24.5 mm , range 24.75 - 32.35 mm ) .
the power of the implanted intraocular lens ( iol ) was used to calculate the predicted post - operative refractive error by four formulas : sanders - retzlaff - kraff ( srk ii ) , srk / t , holladay 1 , and hoffer q. the predictive accuracy of the formulas was analyzed by comparing the difference between the actual and predicted postoperative refractive errors .
repeated measures analysis of variance ( anova ) tests were done to have pair - wise comparisons between the formulas and p < 0.05 was considered significant .
a subcategory of axial length 24.5 - 26.5 mm was also tested .
holladay 1 , hoffer q and srk / t formulas showed a slight tendency toward resultant hyperopia , with mean error of + 0.24 diopters ( d ) , + 0.58 d , and + 0.92 d , respectively . the holladay 1 formula provided the best predictive result overall . |
glycogenic hepatopathy ( gh ) was first identified at 1930 and it is characterized by elevated liver enzyme , hepatomegaly , and glycogen accumulation within hepatocytes and it can be found in patients with type i diabetes mellitus ( dm ) with poorly controlled blood sugar.1,2 clinically , gh has been known to have benign nature ; however it is difficult to distinguish with nonalcoholic fatty liver disease ( nafld ) which could progress into advance liver disease without histological confirmation.2,3 recently , we have diagnosed three cases of gh presented marked serum transaminases elevation more than 30 times of upper normal range as like acute hepatitis with poorly controlled type i dm history . since the first report , about 15 cases suspected as gh have been described in adults over the world . in korea , before this report , only 2 cases of gh have been reported for adults , however , one case showed only mild ast elevation ( 65 u / l ) and normal range of alt and the other case showed mild liver enzyme elevation during diabetic ketoacidosis ( dka ) management.4,5 on the contrary , our three cases of gh in this report showed dramatic elevation of liver enzyme as like acute hepatitis and sustained fluctuation as like relapsing hepatitis , and these findings are very rare and unique even among the all reports of gh over the world .
a 22-year old female was referred to a hepatologist for evaluation of elevated liver enzymes .
when she visited our hospital one month ago , her liver function test had showed abnormal aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) as 169 u / l ( normal value < 35 ) and 172 u / l ( normal value < 35 ) , respectively .
four weeks later , at the time of the referral , her ast and alt were elevated strikingly as 1028 u / l and 365 u / l .
other laboratory findings were total bilirubin ( tb ) 0.5 mg / dl ( normal value < 1.3 ) , alkaline phosphatase ( alp ) 346 u / l ( normal value < 85 ) , -glutamyl transpeptidase ( -gt ) 321 u / l ( normal value < 40 ) , hemoglobin a1c ( hba1c ) 13.8% ( normal value < 6 ) , triglyceride(tg ) 160 mg / dl , high - density lipoprotein ( hdl ) 114 mg / dl creatine kinase ( ck ) 127 ul ( normal value < 220 ) .
auto - antibody including anti - nuclear antibody ( ana ) , smooth muscle antibodies ( asma ) and mitochondrial antibodies ( ama ) were all negative .
the serologic findings for viral infection such as igm anti - hav , hbsag , anti - hcv , hcv - rna , cytomegalovirus and epstein - barr virus were also all negative .
her height , weight , body mass index ( bmi ) and waist circumference were 165.1 cm , 50.8 kg , 18.6 kg / m and 72 cm respectively .
abdominal ultrasonography ( us ) showed just a mild fatty change of liver and mild hepatomegaly ( fig .
she was not an alcohol or drug consumer and was not taking any medication except insulin .
in addition , she did not show any symptom and sign compatible with acute hepatitis .
so , we recommend her for a liver biopsy to elucidate the cause of acute elevation of liver enzyme . in hematoxylin and eosin ( h&e ) stain ,
liver biopsy showed normal liver architecture with no evidence of hepatocellular injury , inflammation , fibrosis , or steatosis .
the periodic acid - schiff ( pas ) staining showed abundant glycogen accumulation within hepatocytes .
eight weeks later , her transaminases were drooped suddenly without any special management . after this event , although we tried to control her sugar level strictly , her sugar control was still poor and hba1c was maintained above 11% with mild fluctuation . until now
, her liver enzymes also have shown severe fluctuation continuously as like relapsing hepatitis ( fig .
a 26-year year old female was referred to a hepatologist for evaluation of elevated liver enzymes .
when she visited our hospital 4 weeks ago , her ast and alt had shown relatively mild elevation as 218 and 213
other laboratory findings were tb 0.5 mg / dl , alp 189 u / l , -gt 294 u / l , hba1c 12.9% , tg 185 mg / dl , hdl 112 mg / dl ck 112 ul .
her height , weight , body mass index ( bmi ) and waist circumference were 154.3 cm , 56.3 kg , 23.6 kg / m and 79 cm , respectively .
in addition , she did not show any symptom and sign compatible with acute hepatitis . in liver biopsy ,
four weeks later , her transaminases dropped spontaneously without any special management . after this event
, her glycemic control has been still poor and hba1c has been maintained above 13% , liver enzyme levels have shown continuous fluctuation ( fig .
a 20-year old female with type i dm during 10 years was referred to a hepatologist for elevated liver enzymes .
when she visited our hospital 8weeks ago , her ast and alt levels were within normal range as 13 and 9 u / l , respectively . at the time of the referral ,
other laboratory finding such as tb ( 0.2 mg / dl ) , alp ( 132 u / l ) , -gt ( 15 u / l ) , tg ( 112 mg / dl ) , hdl ( 36 mg / dl ) , ck 86 ul , thyroid function test , serum ceruloplasmin level were all normal .
her height , weight , body mass index ( bmi ) and waist circumference were 149.8 cm , 47.1 kg and 21 kg / m , respectively .
as former two cases , she did not show any symptom and sign compatible with acute hepatitis and gh was confirmed by liver biopsy . during the follow up
, her glycemic control has been still poor and hba1c has been maintained above 15% .
however , her liver enzyme levels have shown dramatically down and maintenance within normal range ( fig .
a 22-year old female was referred to a hepatologist for evaluation of elevated liver enzymes .
when she visited our hospital one month ago , her liver function test had showed abnormal aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) as 169 u / l ( normal value < 35 ) and 172 u / l ( normal value < 35 ) , respectively .
four weeks later , at the time of the referral , her ast and alt were elevated strikingly as 1028 u / l and 365 u / l .
other laboratory findings were total bilirubin ( tb ) 0.5 mg / dl ( normal value < 1.3 ) , alkaline phosphatase ( alp ) 346 u / l ( normal value < 85 ) , -glutamyl transpeptidase ( -gt ) 321 u / l ( normal value < 40 ) , hemoglobin a1c ( hba1c ) 13.8% ( normal value < 6 ) , triglyceride(tg ) 160 mg / dl , high - density lipoprotein ( hdl ) 114 mg / dl creatine kinase ( ck ) 127 ul ( normal value < 220 ) .
auto - antibody including anti - nuclear antibody ( ana ) , smooth muscle antibodies ( asma ) and mitochondrial antibodies ( ama ) were all negative .
the serologic findings for viral infection such as igm anti - hav , hbsag , anti - hcv , hcv - rna , cytomegalovirus and epstein - barr virus were also all negative .
her height , weight , body mass index ( bmi ) and waist circumference were 165.1 cm , 50.8 kg , 18.6 kg / m and 72 cm respectively .
abdominal ultrasonography ( us ) showed just a mild fatty change of liver and mild hepatomegaly ( fig .
she was not an alcohol or drug consumer and was not taking any medication except insulin .
in addition , she did not show any symptom and sign compatible with acute hepatitis .
so , we recommend her for a liver biopsy to elucidate the cause of acute elevation of liver enzyme . in hematoxylin and eosin ( h&e ) stain ,
liver biopsy showed normal liver architecture with no evidence of hepatocellular injury , inflammation , fibrosis , or steatosis .
the periodic acid - schiff ( pas ) staining showed abundant glycogen accumulation within hepatocytes .
eight weeks later , her transaminases were drooped suddenly without any special management . after this event , although we tried to control her sugar level strictly , her sugar control was still poor and hba1c was maintained above 11% with mild fluctuation . until now
, her liver enzymes also have shown severe fluctuation continuously as like relapsing hepatitis ( fig .
a 26-year year old female was referred to a hepatologist for evaluation of elevated liver enzymes .
when she visited our hospital 4 weeks ago , her ast and alt had shown relatively mild elevation as 218 and 213 u / l , respectively . at the time of the referral ,
other laboratory findings were tb 0.5 mg / dl , alp 189 u / l , -gt 294 u / l , hba1c 12.9% , tg 185 mg / dl , hdl 112 mg / dl ck 112 ul .
her height , weight , body mass index ( bmi ) and waist circumference were 154.3 cm , 56.3 kg , 23.6 kg / m and 79 cm , respectively .
in addition , she did not show any symptom and sign compatible with acute hepatitis . in liver biopsy ,
after this event , her glycemic control has been still poor and hba1c has been maintained above 13% , liver enzyme levels have shown continuous fluctuation ( fig .
a 20-year old female with type i dm during 10 years was referred to a hepatologist for elevated liver enzymes . when she visited our hospital 8weeks ago , her ast and alt levels were within normal range as 13 and 9 u / l , respectively . at the time of the referral , her ast and alt
were elevated strikingly to 1,310 u / l and 346 u / l . other laboratory finding such as tb ( 0.2 mg / dl ) , alp ( 132 u / l ) , -gt ( 15 u / l ) , tg ( 112 mg / dl ) , hdl ( 36 mg / dl ) , ck 86 ul , thyroid function test , serum ceruloplasmin level were all normal .
her height , weight , body mass index ( bmi ) and waist circumference were 149.8 cm , 47.1 kg and 21 kg / m , respectively .
as former two cases , she did not show any symptom and sign compatible with acute hepatitis and gh was confirmed by liver biopsy . during the follow up
, her glycemic control has been still poor and hba1c has been maintained above 15% .
however , her liver enzyme levels have shown dramatically down and maintenance within normal range ( fig .
we reported three cases of gh which were associated with type i diabetes mellitus , causing a clinical picture of acute and relapsing hepatitis .
gh can cause severe , but reversible elevations of serum transaminase levels in patients with poorly controlled type i diabetes due to hepatic glycogen accumulation .
long - standing hyperglycemia , over insulin use , glucose given to control hypoglycemia and increased concentrations of the counter regulatory hormones have been known as risk factors for hepatic glycogen accumulation in patients with type i dm by their actions on the glycogen phosphorylase and synthase enzymes.6 - 8 the typical clinical findings of gh are hepatomegaly and elevated liver enzymes , especially transaminases.2,3,9 - 14 however , there is no histological evidence that suggests the enzyme elevations are due to liver necrosis , so their elevation is considered as a result of hepatocyte 's membrane injury to cause enzyme " leakage " instead of cell death .
histological features in gh are characterized by large , swollen , glycogen - laden hepatocytes and glycogenated nuclei without significant fatty change , inflammation , lobular spotty necrosis or fibrosis.1 the abnormality of transaminase in gh has been known to be easily treatable by improvement of glycemic control in a short time within 2 to 14 weeks.4,5,12,15 however , as seen in our case , the changes of liver enzyme levels showed somewhat different pattern compared with previous reported cases .
the sudden elevation and dropping of transaminase did not showed any definitive correlation with the glycemic control ( the change of hba1c ) and continuous fluctuation of transaminase as like relapsing hepatitis in case 1 and 2 is also unusual pattern compared to previous reports .
in addition , there was no insulin dose - up even in acute flare - up phase of transaminase .
so , it suggests that the changes of transaminases can be related with not only poor glycemic control or insulin therapy but also another factor which has not been clarified .
in addition , in all cases , the ast dominant elevation compared to alt is also impressive finding . therefore , to elucidate the risk factors and more complete pathophysiology , more advanced systemic review and research have to be performed .
clinically , to distinguish gh from nafld is important , because as mentioned above , the prognosis of both diseases are different .
gh has been known to have benign nature whereas nafld could progress into advance liver disease such as cirrhosis or hepatocellular carcinoma.2,3 including our cases , overall gh in diabetic patients with hepatomegaly and abnormal liver enzymes within 5 - 10 times of upper normal range have often been assumed to have nafld rather than gh in the initial diagnostic process .
gh is a rare condition and many clinicians are unaware of it if no biopsy is obtained.16 as mentioned above , especially in case of mild transaminases elevation in diabetic patients , liver biopsy is essential in differential diagnosis of gh with nafld . in addition , us finding is not helpful in distinguishing nafld from gh because both fatty liver and glycogen overload could show similar us finding as mild hyperechoic hepatic parenchyma.3,6,10,11 consequently , the condition is often misdiagnosed and it result in wasting medical resources .
it may suggest that many cases of gh have been missed due to a lack of interest by clinician .
clinician 's awareness of gh should prevent diagnostic delay and misdiagnosis and will provide better insight and management for the gh . | glycogenic hepatopathy ( gh ) is an uncommon cause of serum transaminase elevation in type i diabetes mellitus ( dm ) .
the clinical signs and symptoms of gh are nonspecific , and include abdominal discomfort , mild hepatomegaly , and transaminase elevation . in this report
we describe three cases of patients presenting serum transaminase elevation and hepatomegaly with a history of poorly controlled type i dm .
all of the cases showed sudden elevation of transaminase to more than 30 times the upper normal range ( like in acute hepatitis ) followed by sustained fluctuation ( like in relapsing hepatitis ) .
however , the patients did not show any symptom or sign of acute hepatitis .
we therefore performed a liver biopsy to confirm the cause of liver enzyme elevation , which revealed gh .
clinicians should be aware of gh so as to prevent diagnostic delay and misdiagnosis , and have sufficient insight into gh ; this will be aided by the present report of three cases along with a literature review . |
mammalian cells , bacterial strains , and culture
conditions hela human cervix carcinoma cells ( atcc ccl-2 ) were
cultured in dmem plus glutamax ( amersham biosciences ) supplemented with 1%
sodium pyruvate ( amersham biosciences ) and 10% fetal calf serum .
l.
monocytogenes type strain egd ( bug 600 ) was grown overnight at 37 c
in brain heart infusion media ( difco laboratories ) , diluted 15 in fresh
brain heart infusion , and cultured until a600 nm =
0.8 .
gentamicin survival assays gentamicin survival assays were
performed as previously described
( 16 ) . in brief , cells were
incubated in dmem with listeria at a multiplicity of infection of 50
for 1 h at 37 c and 10% co2 , washed with dmem without
antibiotic , and subsequently incubated with fresh gentamicin - containing
complete media ( 10 g / ml ) for an additional 1 h. cells were washed and then
lysed with distilled h2o .
the number of viable bacteria released
from the cells was assessed by plating on brain heart infusion agar plates .
each experiment was done in triplicate , and triplicates were performed at
least three times independently .
survival assays involving drug treatments were performed as described for
other listeria in vitro systems
( 17 ,
18 ) , where cells were treated
with various concentrations of drugs 30 min prior to infection . upon
infection
, host cells were incubated in drugs ( at molar concentrations varying
from 200 to 0.01 m ) throughout the gentamicin survival assays
as detailed above .
final values are expressed as the number of intracellular
bacteria per drug treatment relative to dmso - treated cells .
forchlorfeneuron ,
cytochalasin d , latrunculin b , nocodazole , and paclitaxel were all suspended
in dmso and handled as suggested by the manufacturer ( sigma ) .
antibodies and reagents primary antibodies used in this
study included rabbit polyclonals anti - sept9 ( r69 )
( 14 ) , anti - sept11 ( gift from
makoto kinoshita ) , and anti - sept2 ( gift from william trimble ) . mouse
monoclonal anti - inlb has been described elsewhere
( 19 ) .
mouse monoclonal
anti - gapdh ( glyceraldehyde-3-phosphate dehydrogenase ) , used as a control
throughout western blot experimentation , was purchased from abcam ( 6c5 ) .
secondary antibodies used were cy5-conjugated ( jackson immunoresearch
laboratories ) , alexa 488-conjugated , and alexa 546-conjugated goat anti - rabbit
and goat anti - mouse antibodies ( molecular probes ) .
f - actin was labeled with
alexa 488-fluorescein isothiocyanate and alexa 546-phalloidin ( molecular
probes ) . for immunoblotting
, total cellular extracts were blotted with the
above - mentioned antibodies , followed by peroxidase - conjugated goat anti - mouse
and anti - rabbit antibodies ( biosys laboratories ) .
preparation of inlb - coated beads coating of latex beads with
purified inlb and incubation of host cells with inlb - coated latex beads were
performed as previously described
( 14 ,
20 ) , where 1-m diameter
latex beads were coated following the manufacturer 's instructions ( molecular
probes ) .
prior to addition to sirna - treated hela cells , inlb - coated beads were
briefly sonicated , diluted in dmem , and added to hela cells at a multiplicity
of 100 beads per cell . for kinetic analysis by immunofluorescence and scanning electron
microscopy , host cells were stored at 4 c for 5 min before incubation
with beads , then beads diluted in dmem were added and cells were centrifuged
at 1000 g for 1 min at 4 c .
cells were then incubated at 37 c and 10%
co2 for 5 min , after which they were washed twice with dmem and
processed for immunofluorescence or scanning electron microscopy analysis .
immunofluorescence and confocal
microscopy immunofluorescence analysis was performed as previously
described ( 16 ) .
briefly ,
10 - 10 hela cells were plated onto glass coverslips in
6-well plates ( techno plastic products ) and used for experiments 48 h later .
cells on coverslips were fixed 15 min in 4% paraformaldehyde , then washed with
1 phosphate - buffered saline , and processed for immunofluorescence .
after 10 min of incubation in 50 mm ammonium chloride , cells were
permeabilized 4 min with 0.1% triton x-100 , and then incubated in 1
phosphate - buffered saline .
hybridization of primary and secondary antibodies
was performed in 1 phosphate - buffered saline .
vectashield hard set
mounting medium with or without dapi was applied ( vector laboratories ) .
images were acquired on a fluorescence inverted microscope axiovert 200 m
( carl zeiss microimaging , inc . ) equipped with a cooled digital charge - coupled
device camera ( cool snaphq , photometrics ) driven by metamorph
imaging system software ( universal imaging corp ) .
immunofluorescence and
confocal images were visualized and processed using the imagej public domain
image processing software
( rsb.info.nih.gov/ij/ ) . to localize and track changes in septin filament distribution and to
segment cells after sirna treatment
, a journal was created using metamorph
( version 7.1 , universal imaging ) .
confocal z - stack images of sirna - treated
cells were catalogued where images from sets of at least two biological
replicates per sirna treatment were analyzed .
endogenous f - actin and sept9
were visualized for immunolabeling with fluorescent phalloidin and anti - sept9
antibodies . to define their location ,
after
deconvolution ( huygens deconvolution software ; scientific volume imaging ) , the
confocal z - stack images of cells were segmented , and fibers were localized in
each cell . for each segmented cell , quantitative parameters ( coordinates and
area )
were exported to an excel file . for each segmented fiber , quantitative
parameters ( coordinates , area , and shape factor )
were also exported to an
excel file , where fibers were analyzed per cell , and cells could subsequently
be attributed to class 1 , 2 , or 3 .
when comparing the class frequencies for
sirna treatments , p values were accordingly obtained by chi square
test .
hela cells ( 0.8 10 )
were plated in 6-well plates ( techno plastic products ) and transfected the
following day using oligofectamine ( invitrogen ) following the manufacturer 's
instructions .
scramble sequence , custom designed from septin sequences
( ( sense ) auaagcgacguccgcguggtt and ( antisense ) ccacgcggacgucgcuuautt ) , was
applied as our control throughout experimentation
( 15 ) .
custom scramble
sequence , as well as pre - designed sirnas for sept9 ( id#18228 ) and sept11
( id#125139 ) were all from ambion , and handled according to manufacturer 's
instructions .
cells were tested 72 h after sirna transfection . to measure the
efficiency of sirna knockdown , the relative amounts of sept9 and sept11
proteins
were quantified using genetools ( syngene ) , normalized to give 100%
for the controls
, sirna sequence for sept2 ( id#14709 )
( 15 ) , sept6 ( id#136764 ) , and a
second sirna sequence for sept11 ( id#125138 ) , was applied .
scanning electron microscopy for scanning electron
microscopy analysis , non - infected sirna - treated hela cells , and sirna - treated
hela cells incubated with inlb - coated beads , were prepared as described above ,
subsequently washed in phosphate - buffered saline , prefixed in 2.5%
glutaraldehyde in 0.1 m cacodylate buffer for 30 min , and then
rinsed in 0.2 m cacodylate buffer .
after post - fixation in 1% osmium
tetraoxide ( in 0.2 m cacodylate buffer ) , samples were dehydrated in
a series of ethanol concentrations .
specimens were critical point - dried using
carbon dioxide , and then coated with gold and examined / photographed with a
jeol jsm-6700f scanning electron microscope . fluorescence resonance energy transfer as previously
described by our laboratory
( 21 ) , pi3k activation can be
measured by the production of its lipid products phosphoinositide
3,4-bisphosphate and phosphoinositide 3,4,5-trisphosphate , which recruit the
serine / threonine kinase akt from the cytosol to the membrane , with the akt
pleckstrin homology ( ph ) domain specifically interacting with phosphoinositide
3,4-bisphosphate and phosphoinositide 3,4,5-trisphosphate
( 22 ,
23 ) .
the quantification of
fret between two coexpressed fluorescent chimeras of the akt ph domain ,
yfp - aktph and cfp - aktph , can be used as readout of pi3k activity . without
stimulation ,
the fluorescent chimeras are too dispersed in the cytosol to
undergo fret . upon pi3k activation ,
the fluorescent aktph chimeras concentrate
at the plasma membrane , consequently leading to an increase in the fret
signal .
sirna - treated hela cells were cotransfected using jetpei ( polyplus
transfection ) according to the manufacturer 's instructions with plasmids
encoding yfp - aktph and cfp - aktph .
details on fret image acquisition ,
processing , and statistical analysis were introduced in mostowy et
al .
, cells
were automatically classified as not responsive to inlb stimulation if a
maximum slope during fret , i.e. a peak of signal intensity , could not
be detected above background values during inlb stimulation .
final kinetics
was obtained for 19 and 11 experiments , representing at least 3 biological
replicates for each sirna treatment , for control and sept11 sirna - treated
cells , respectively . to automatically detect whether a cell could be
classified as having responded or not to inlb stimulation , we designed our own
analysis software carried out using an excel macro .
our analysis software is
based on these following criteria . from the original kinetic signals
( i.e. the mean ( ea + ed)/2
over the entire cell ) , a first median linear filter ( formula :
yi = ( i - n/2,i+n/2
yi)/n , n = 3 ) was applied to smooth artifact
arising from the acquisition of original signal .
the slope of each filtered
curve was subsequently calculated for each time point and was plotted as the
processed
4b ) . starting
from t0 ( here defined as the time frame immediately
following inlb stimulation , frame 16 ) , tmax was detected
when the slope again reached a minimum , where minimum slope is based on a null
second derivative function .
tmax - t0
was used to define the duration of fret response ( i.e.time of
fret , where units are frames per 15 s ) . between these two time points ,
the amplitude of the original signal ( i.e. the mean
( ea + ed)/2 over the entire cell ) was
calculated as a characteristic to qualify the response
( i.e.amplitude of fret ) . as a final characteristic
, the
maximum slope was calculated between t0 and
tmax ( i.e.maximum slope during
fret ; this value also served to determine if the fret response of that
cell was positive ) . in the case
where tmax could not be
detected based on these criteria , such cells were then classified as not
responding to inlb stimulation .
sept9 and sept11 have non - overlapping roles in filament
organization we previously found that sept9 interacts with septins
from other groups to form a complex , and in agreement with other reports
( 10 ,
24 ) identified sept11 as a
binding partner of sept9 ( 15 ) .
in non - dividing , non - infected hela cells ,
both sept9 and sept11 colocalize
with actin filaments , typically in the nuclear region and along the cell
periphery , and rarely colocalize with microtubules
( 15 ,
24 ,
25 ) .
to establish the respective role of sept9 and sept11 in actin and septin
filament formation , we treated hela cells , a cell line routinely used for
sirna depletion experiments , with sirna against sept9 or sept11 .
sirna
depletion of each targeted septin protein was assessed following western blot
( fig .
knockdown
efficiency was quantified as 95 2% for sept9 and 80 13% for
sept11 .
we then labeled sirna - treated cells with fluorescein
isothiocyanate - phalloidin and anti - sept9 antibody
( fig .
1b ) , and
systematically analyzed actin and septin filament remodeling upon septin
depletion .
confocal z - stack images of sirna - treated cells were then
categorized by a septin filament tracking algorithm into three different
pre - assigned classes of sept9 filament distribution
( fig .
1c ) : cells
presenting filaments around the cell periphery and under the nucleus were
designated as class 1 , cells with filaments only around the cell periphery ,
but not under the nucleus were designated as class 2 , and cells no longer
presenting any filaments were designated as class 3 .
the frequency of each
filament class among septin - depleted cells was then statistically compared
with values from control cells ( fig .
actin filaments had also disappeared . in contrast , sept9
and actin filaments were not reduced in cells depleted for sept11 ( p
> 0.75 ) , which retained their capacity to present both septin and actin
filaments under the nucleus ( p > 0.20 ) .
these results were also
observed using a second sirna sequence for sept11 ( p > 0.15 and
p > 0.30 , respectively ) ( data not shown ) .
thus our analysis , in contrast to previously published results for sept2 ,
sept6 , and sept7 ( 26 ,
27 ) , showed that different
septin inactivations have different effects on the organization of septin and
actin filaments at least in hela cells . to further appreciate the impact of
depleting different septin subunits ,
we have assessed the impact of sept2 ,
sept9 , and sept11 depletion on septin expression and filament formation
( supplemental fig .
these data suggest that
sept11 , unlike sept2 or sept9 , is dispensable for septin and actin filament
formation .
listeria invasion of non - phagocytic cells relies
upon the polymerization and depolymerization of actin
( 13 ,
28 ) in concert also with the
function of sept2 ( 15 ) .
to
assess the functional consequence of sept11 depletion on listeria
invasion , we employed the classic gentamicin survival assay
( 16 ) . in this assay
,
internalized bacteria are plated and counted 24 h after host cell lysis .
colony counts revealed that sept11 sirna treatment increased listeria
invasion into hela cells ( p < 0.0005 )
( fig .
2 ) , as opposed to results
obtained for sept2 ( 15 ) .
significantly increased listeria invasion was also observed using a
second sirna sequence for sept11 ( p < 0.01 ) ( data not shown ) .
considering that sept2 depletion decreased listeria entry
( 15 ) , but sept11 depletion
increased listeria entry
, these data strongly suggest that different
septin inactivations can differentially modulate listeria
invasion .
. a , western
blot ( wb ) of hela cells transfected with sirna against control
( ctrl ) , sept9 , or sept11 .
the blots were probed with antibodies specific to
gapdh , sept9 , sept11 , and actin .
b , septin depletions have different effects on cell shape and actin
and septin filament distribution .
endogenous f - actin and sept9 were visualized
by immunostaining with anti - f - actin ( green ) and anti - sept9 antibodies
( red ) for control ( ctrl)- , sept9- , and sept11-depleted
cells .
c , different classes of sept9 filament
distribution . in sirna - treated hela cells , septin and actin filaments were
distributed into different patterns .
cells presenting filaments around the
cell periphery and under the nucleus were designated as class 1 , cells with
filaments only around the cell periphery , but not under the nucleus were
designated as class 2 , and cells no longer presenting any filaments were
designated as class 3 .
confocal z - stack images of sirna - treated
cells were categorized by a filament tracking algorithm into the three
pre - assigned classes of filament distribution ( fig .
the
percentage of septin - depleted cells observed to still have filaments ,
i.e. % of cells in class 1 and class 2 versus % of cells in
class 3 , was statistically compared with expectations as derived from control
cells by chi square test ( p value(c1+c2 versus
c3 ) ) . to assess whether sept11-depleted cells retain sept9 filaments
under the nucleus
, the distribution of sept9 filaments in sept11-depleted
cells , i.e. % of cells in class 1 versus % of cells in class
2 , was statistically compared with expectations as derived from control cells
by chi square test ( p value(c1 versus c2 ) ) .
a , western
blot ( wb ) of hela cells transfected with sirna against control
( ctrl ) , sept9 , or sept11 .
the blots were probed with antibodies specific to
gapdh , sept9 , sept11 , and actin .
b , septin depletions have different effects on cell shape and actin
and septin filament distribution .
endogenous f - actin and sept9 were visualized
by immunostaining with anti - f - actin ( green ) and anti - sept9 antibodies
( red ) for control ( ctrl)- , sept9- , and sept11-depleted
cells .
c , different classes of sept9 filament
distribution . in sirna - treated hela cells , septin and actin filaments were
distributed into different patterns .
cells presenting filaments around the
cell periphery and under the nucleus were designated as class 1 , cells with
filaments only around the cell periphery , but not under the nucleus were
designated as class 2 , and cells no longer presenting any filaments were
designated as class 3 .
confocal z - stack images of sirna - treated
cells were categorized by a filament tracking algorithm into the three
pre - assigned classes of filament distribution ( fig .
the
percentage of septin - depleted cells observed to still have filaments ,
i.e. % of cells in class 1 and class 2 versus % of cells in
class 3 , was statistically compared with expectations as derived from control
cells by chi square test ( p value(c1+c2 versus
c3 ) ) . to assess whether sept11-depleted cells retain sept9 filaments
under the nucleus
, the distribution of sept9 filaments in sept11-depleted
cells , i.e. % of cells in class 1 versus % of cells in class
2 , was statistically compared with expectations as derived from control cells
by chi square test ( p value(c1 versus c2 ) ) .
gentamicin
survival assays for l. monocytogenes egd were performed in hela cells
treated with control ( ctrl ) sirna , or sirna targeted against sept11 .
graphs represent the relative number of intracellular bacteria found
inside sirna - treated cells after the survival assay , where cfu counts obtained
from sept11-depleted cells were normalized to ctrl sirna - treated cells . on the
graph ctrl sirna
is figuratively presented as 1 , and data represent the mean
from n 9 experiments .
gentamicin
survival assays for l. monocytogenes egd were performed in hela cells
treated with control ( ctrl ) sirna , or sirna targeted against sept11 .
graphs represent the relative number of intracellular bacteria found
inside sirna - treated cells after the survival assay , where cfu counts obtained
from sept11-depleted cells were normalized to ctrl sirna - treated cells . on the
graph ctrl sirna
is figuratively presented as 1 , and data represent the mean
from n 9 experiments .
latex beads coated with purified recombinant inlb protein
have been extensively used to study specifically the inlb - mediated entry of
listeria into hela cells
( 15 ,
19 ,
30 ,
31 ) . to understand why l.
monocytogenes invasion is increased upon sept11 depletion , we
investigated particle uptake using 1-m inlb - coated latex beads .
sirna - treated cells were incubated with inlb beads and were examined by a
double immunofluorescence microscopy technique to distinguish
intra - versus extracellular beads and provide a measure of
inlb - induced phagocytosis ( fig .
3a ) . at 5-min postincubation , sept11-depleted cells
presented an increased capacity to internalize beads compared with control
cells ( p < 0.05 ) . on average , control cells internalized 6 beads
per cell , whereas sept11-depleted cells internalized 24 beads per cell
( p < 0.001 ) .
scanning electron microscopy was also used to examine the uptake of inlb
beads ( fig .
the membrane surface of sept11-depleted cells presented smoother
membrane with a heterogeneous distribution of microvilli - like structures
compared with control cells ( supplemental fig .
, the majority of beads were already internalized for control
and sept11-depleted cells , but in agreement with the above immunofluorescence
experiments , the number of internalized inlb beads per sept11-depleted cell
was significantly higher than in control cells .
as previously reported for sept2- , sept6- , and sept7-depleted cells
( 26 ,
27 ) , we found that hela cells
become large and flat upon sept11 depletion
( fig .
, differences in the number of internalized inlb beads per sirna - treated
cell could reflect changes in cell size . to evaluate this , we measured the
surface area of non - infected sirna - treated hela cells after fixation
( fig . 3c and
supplemental fig .
the surface area for
sept11-depleted cells was almost twice as large as control cells ( p
< 0.05 ) . by comparison ,
sept2-depleted cells
( 15 ) occupied approximately
the same surface area as control cells ( p > 0.73 ) ( data not
shown ) .
thus , inlb invasion differences in sept11-depleted cells can in part
be explained by differences in surface area . taken together , these results
definitively establish that sept11-depleted hela cells are competent to
internalize listeria and 1-m inlb beads . signaling events in response to met stimulation by inlb are not
affected by the depletion of sept11listeria enter hela
cells via interactions between the cell surface receptor met and the bacterial
ligand inlb ( 32 ) .
given the
positive impact of sept11 depletion on particle entry , we hypothesized that
signaling events induced upon inlb - met interaction could be increased upon
sept11 depletion . to test this
, we used a fret - based approach previously
developed in our laboratory that focuses on phosphoinositide 3-kinase
activation , a signaling pathway induced upon inlb - met interaction
( 15 ,
21 ) .
the fret assay is based
on the fact that 3-phosphoinositides produced by pi3k upon inlb - induced
met activation recruit the serine / threonine kinase akt from the cytosol to the
plasma membrane . previous work with this assay revealed that sept2 has a
critical role in these signaling events
( 15 ) .
sept11-depleted cells and control cells were cotransfected with plasmids
encoding yfp - aktph and cfp - aktph .
the fret stoichiometry measurement between
yfp - aktph and cfp - aktph was then performed to determine fret efficiency
( fig .
4 , a and
b , and supplemental movies s1a and
s1b ) . as shown in fig . 4 ,
c and d , cells depleted of sept11 responded to
inlb stimulation as well as control cells .
thus sept11 , in contrast to sept2
( 15 ) , does not have an
essential role in early signaling events in response to met stimulation by
inlb .
septins are gtp - binding proteins that form filaments and are increasingly
recognized as cytoskeleton components
( 5 ,
7 ) .
although several studies
have reported on septin function , little information is available for sept11 .
our results show that sept11 is dispensable for
inlb - mediated invasion , and surprisingly , its absence even leads to an
increase in invasion .
figure 3.sept11 inactivation increases the entry of inlb - coated beads .
a , quantification of inlb - induced phagocytosis for control
( ctrl ) and sept11-depleted hela cells . for internalization assays ,
beads were analyzed by immunofluorescence ( see experimental
procedures ) for being extracellular or intracellular in at least 50
host cells counted for each of n 2 separate experiments per
sirna treatment .
the left graph depicts the total % of internalized
1-m inlb - coated beads at 5-min postincubation of sirna - treated cells ,
calculated from the ( total number of internalized beads)/(total number of
cell - associated beads ) 100 .
the right graph
depicts the average number of internalized 1-m inlb - coated beads per cell
at 5-min postincubation of sirna - treated cells .
graphed data represent this
average value s.e . , where results were analyzed for statistical
significance using the two sample z - test .
b , uptake of inlb beads by
sirna - treated cells . control ( ctrl ) and sept11-depleted hela cells
were incubated with 1-m inlb beads for 5 min , and cells were processed for
scanning electron microscopy .
c , comparing the average surface areas of
sirna - treated cells . to quantify the surface area of sirna - treated hela cells ,
bar
graphs depict the average cell occupied area ( m )
s.e . for control ( ctrl)(n = 77)- and sept11 ( n =
30)-depleted cells .
the cell - occupied area for each septin - depleted cell type
was then statistically compared with control cells by student 's t
test .
sept11 inactivation increases the entry of inlb - coated beads .
a , quantification of inlb - induced phagocytosis for control
( ctrl ) and sept11-depleted hela cells . for internalization assays ,
beads were analyzed by immunofluorescence ( see experimental
procedures ) for being extracellular or intracellular in at least 50
host cells counted for each of n 2 separate experiments per
sirna treatment .
the left graph depicts the total % of internalized
1-m inlb - coated beads at 5-min postincubation of sirna - treated cells ,
calculated from the ( total number of internalized beads)/(total number of
cell - associated beads ) 100 .
the right graph
depicts the average number of internalized 1-m inlb - coated beads per cell
at 5-min postincubation of sirna - treated cells .
graphed data represent this
average value s.e . , where results were analyzed for statistical
significance using the two sample z - test .
control ( ctrl ) and sept11-depleted hela cells
were incubated with 1-m inlb beads for 5 min , and cells were processed for
scanning electron microscopy .
c , comparing the average surface areas of
sirna - treated cells . to quantify the surface area of sirna - treated hela cells ,
bar
graphs depict the average cell occupied area ( m )
s.e . for control ( ctrl)(n = 77)- and sept11 ( n =
30)-depleted cells .
the cell - occupied area for each septin - depleted cell type
was then statistically compared with control cells by student 's t
test . a dispensable role for sept11 in septin filament
formation
sept11 can bind sept9 in vitro
( 10 ,
15 ,
24 ) . in hela cells ,
in contrast , sept11 depletion did not alter the presence and
colocalization of sept9 and actin filaments
( fig . 1
that
sept9 , but not sept11 , is required for septin filament formation strongly
suggests non - overlapping functions for these different septins . of note ,
neither sept9 nor sept11 depletion had any noticeable effect on microtubules
( supplemental fig .
septins differentially control the inlb - met - mediated entry of listeria
into host cells to investigate how sept11 can regulate the
internalization process , we performed our functional analysis on hela cells ,
because they allow the specific analysis of the inlb - met entry pathway used by
listeria ( 28 ) .
our
present study together with previous work
( 15 ) reveals that septins
differentially modulate the inlb - met - mediated entry of l.
monocytogenes , with an increase in sept11-depleted cells and a decrease
in sept2-depleted cells .
moreover , fret analysis confirmed that sept11 , unlike
sept2 , is not required for the appropriate signaling cascade in response to
met stimulation by inlb .
these data reveal a different effect of sept11 and
sept2 depletion on the interaction of inlb with its host receptor met , and
consequently on bacterial entry .
septin filaments colocalize in hela
cells ( data not shown ) , however we can not exclude different localization
between sept2 and sept11 at this stage . using sirna , we observe that sept11
filament formation relies on sept2 expression , but sept2 filaments continue to
form in the absence of sept11 ( supplemental fig .
structural data have shown that septins are ordered in the
isolated complex as sept7-sept6-sept2-sept2-sept6-sept7
( 9 ) .
as the central subunit in
this septin complex , sept2 can not be replaced in the conditions tested
( 15 ) .
conversely , it has been
proposed that members of the sept6 group are interchangeable , at least in the
context of the sept2-sept6-sept7 complex
( 3 ,
12 ) .
thus , some of the
phenotypes we observe here in cells depleted for sept11 could be because the
sept6 group members are partially redundant .
nevertheless , cells depleted for
sept11 do not present all the control cell phenotypes . in particular they have
an increased cell size and are more permissive to inlb - mediated invasion ,
suggesting that other sept6 group members can not fully substitute for sept11 .
the extent to which sept6 group members share properties and/or functionally
compete with each other is difficult to currently appreciate .
figure 4.sept11-depleted cells respond to inlb stimulation . a ,
representative fret efficiency images for control ( ctrl ) and
sept11-depleted hela cells expressing yfp - aktph and cfp - aktph stimulated with
inlb .
fret efficiency at two time points are presented for each cell type :
t0 ( frame 16 ) and tmax ( frame
corresponding to the maximum induction of fret efficiency ) after stimulation .
pseudocolor scale represents the range of fret efficiency values from
original signal ranging from blue ( low ) to red ( high ) .
movies for the entire time course of
inlb stimulation for these cells can be observed in the supplemental movies
s1a and s1b .
processed fret response was plotted against time
( i.e. frame per 15 s ) , for the representative sirna - treated cells
depicted in fig .
values from the control ( ctrl ) cell are
plotted in black , and the sept11-depleted cell is in yellow .
c , percentage of cells that respond to inlb treatment . processed fret
response was determined for 11 or more experiments for each sirna treatment .
cells were classified as not responding to inlb stimulation if the slope of
processed fret response could not be distinguished above baseline values .
the
percentage of sept11-depleted cells classified as not responding to inlb
stimulation was statistically compared with expectations as derived from
control cells by chi square test .
characteristics of the fret response
( time , amplitude , and maximum slope ) were evaluated for control
( ctrl)- and sept11-depleted cells that respond to inlb treatment , and
a ,
representative fret efficiency images for control ( ctrl ) and
sept11-depleted hela cells expressing yfp - aktph and cfp - aktph stimulated with
inlb .
fret efficiency at two time points are presented for each cell type :
t0 ( frame 16 ) and tmax ( frame
corresponding to the maximum induction of fret efficiency ) after stimulation .
pseudocolor scale represents the range of fret efficiency values from
original signal ranging from blue ( low ) to red ( high ) .
movies for the entire time course of
inlb stimulation for these cells can be observed in the supplemental movies
s1a and s1b .
processed fret response was plotted against time
( i.e. frame per 15 s ) , for the representative sirna - treated cells
depicted in fig .
values from the control ( ctrl ) cell are
plotted in black , and the sept11-depleted cell is in yellow .
c , percentage of cells that respond to inlb treatment . processed fret
response was determined for 11 or more experiments for each sirna treatment .
cells were classified as not responding to inlb stimulation if the slope of
processed fret response could not be distinguished above baseline values .
the
percentage of sept11-depleted cells classified as not responding to inlb
stimulation was statistically compared with expectations as derived from
control cells by chi square test .
. characteristics of the fret response
( time , amplitude , and maximum slope ) were evaluated for control
( ctrl)- and sept11-depleted cells that respond to inlb treatment , and
why are sept11-depleted cells more permissive to listeria
invasion ? to explain differences in l. monocytogenes
infection arising from the sept11 sirna treatment , we analyzed phenotypes
previously reported for septin - depleted cells .
of these , arrest in cytokinesis
is a classic phenotype of septin depletion
( 1 ) , itself typically assessed
in mammalian cells by the increase of binucleated cells
( 25 ,
33 - 35 ) .
in agreement with these data ,
s4a ) . to determine if
binucleation was influencing listeria infection , we fixed and
fluorescently labeled cells after gentamicin treatment to determine the
frequency of infected cells that were binucleated . however , binucleated cells
were as susceptible to infection as cells with a single nucleus ( supplemental
fig .
the sept11 phenotype described here is different from the effects of global
cytoskeleton disruption on bacterial entry .
as well established
( 17 ,
18 ) , inhibition of actin even
by low concentrations of cytochalasin d or latrunculin b was found to have
potent negative effects on bacterial invasion of hela cells ( supplemental fig .
depolymerization of microtubules by nocodazole , or
stabilization of microtubules with paclitaxel , also resulted in a significant
reduction in the number of invading listeria
( 18 ) ( supplemental fig .
we finally tested the impact of a novel drug , forchlorfeneuron ,
to specifically investigate septin function
( 29 ) .
forchlorfeneuron
treatment also had a negative impact on bacterial invasion ( supplemental fig .
as sept11 depletion increases entry , our study
highlights a novel type of control for a cytoskeleton component .
sept11-depleted cells are larger than control cells ,
which in part may explain the higher permissivity .
nevertheless , the fact that
sept11-depleted cells are more permissive suggests a role for sept11 in
limiting entry into cells .
quite strikingly , preliminary work has revealed
that sept6-depleted hela cells also are increased for listeria
invasion ( p = 0.001 ) ( supplemental fig .
s5e ) . these results
strongly support that all sept6 group members play a similar role in
restricting entry .
whether this is due to septin association with the plasma
membrane or septin function in regulating cytoskeleton dynamics is currently
under investigation .
on the basis of different recruitment we have previously distinguished
septin from actin function during bacterial entry and identified sept2 as
essential for bacterial entry
( 15 ) . here
we demonstrate that
sept11 depletion does not decrease , but surprisingly increases , the
inlb - mediated entry of listeria . taken together , these data highlight
the specific roles for two different septins , sept2 and sept11 , during the
inlb - mediated internalization process .
these findings for the first time
distinguish unique septin roles in regulating the efficiency of
receptor - mediated bacterial invasion .
| septins are filament - forming gtpases implicated in several cellular
functions , including cytokinesis .
we previously showed that sept2 , sept9 , and
sept11 colocalize with several bacteria entering into mammalian non - phagocytic
cells , and sept2 was identified as essential for this process . here , we
investigated the function of sept11 , an interacting partner of sept9 whose
function is still poorly understood . in uninfected hela cells ,
sept11
depletion by sirna increased cell size but surprisingly did not affect actin
filament formation or the colocalization of sept9 with actin filaments .
sept11
depletion increased listeria invasion , and incubating sept11-depleted
cells with beads coated with the listeria surface protein inlb also
led to increased entry as compared with control cells .
strikingly , as shown by
fluorescence resonance energy transfer , the inlb - mediated stimulation of met
signaling remained intact in sept11-depleted cells . taken together , our
results show that sept11 is not required for the bacterial entry process and
rather restricts its efficacy . because sept2 is essential for the
inlb - mediated entry of listeria , but sept11 is not , our findings
distinguish the roles of different mammalian septins . |
arrow injuries are an extinct form of injury in most parts of the developed world but are still seen , albeit infrequently in developing countries .
reports of penetrating injuries of the craniofacial region secondary to projectiles are few and far between .
we report the case of a patient who was attacked by a projectile fired from a crossbow .
immediate surgery under general anesthesia was required to remove the arrow , with utmost care to avoid any neurovascular compromise to the facial nerve , as well as minimize post operative complications such as otitis media and subsequent meningitis .
a 40-year - old female , of the bhil community , from malwa region , presented to the emergency department of our hospital , following an attempted murder , six hours ago , using a crossbow arrow .
the attack was due to an old rivalry between two families . at her arrival to the hospital , she was fully conscious and the neurological and systemic physical evaluations were normal , with a glasgow coma scale of 15/15 .
the arrow was impacted firmly in her left occipito - temporal region , narrowly missing her cervical spine [ figures 1 , 2 ] .
the missile had entered posterior to the left ear , and was lodged midway between the cervical spine and left angle of mandible , narrowly missing the neurovascular bundles situated at this level . her respiration was settled and all her vital parameters were within normal limits , except for mild tachycardia .
arrow impacted firmly in the patient 's left occipito - temporal region patient in sitting position : arrow impacted in her left occipito temporal region x ray of the patient : showing the depth of arrow penetration with a bent arrow tip , indicative of the force of impact she was immediately taken into the operation theater , and under general anesthesia , using nasal intubation ; the precariously placed arrow was carefully removed , via adequate dissection from its surrounding structures , with utmost care to avoid any neurovascular damage , in the presence of a neurosurgeon .
after removal of the arrow , the local area was irrigated with antiseptic solution , and tissue approximation was completed in two layers [ figure 4 ] .
arrow removal : the residual bed visible beneath the postoperative period was uneventful , with the patient being started on antibiotics , analgesics and serratiopeptidase preparation .
she had no neuro deficit and was discharged on the fifth postoperative day , without event , and advised regular follow ups .
arrow injuries are an extinct form of injury in most parts of the developed world but constitute 0.1% of emergency admissions in the developing world . the various reasons for the injuries vary , such as armed robberies in 41% , communal clashes 20.5% , dispute between farmers and cattle herdsmen 13% , and cattle theft 8% .
various factors are responsible for the severity of the injury such as distance of the assailant from the target , the force and trajectory of the arrow , as well as the physical characteristics of the arrow . despite a relatively low velocity ( compared with gunshots )
, the sharpness and propulsion force of the crossbow may be sufficient to enable penetration of the skull at short range .
the major risk is a direct injury of cerebral parenchyma , and a vascular injury is sometimes revealed at the removal of the foreign body .
in addition , otitis media and subsequent meningitis following penetrating facial injuries are not uncommon .
post - traumatic meningitis ( ptm ) can lead to devastating results and mortality rates up to 65% have been reported.[35 ] while the time between injury and infection may be brief , there are numerous cases where ptm has been diagnosed years after injury .
causative agents for ptm include a wide range of both gram - positive and gram - negative organisms .
streptococcus pneumoniae is the most common agent found in most series and is isolated in 52 - 100% of cases .
the diagnosis of ptm is usually made by csf cultures with positive results seen in 73 - 100% of patients .
cultures of csf may fail to yield an isolate , however , and negative cultures have been reported in up to 27% of cases .
moreover , the facial nerve also lies in close proximity to the site of injury , as in this case , and it is of significance to avoid any injury to it and thus avoid any subsequent neuro deficit .
antibiotics to be given post operatively should be chosen in accordance with the clinical situation and ability to penetrate the blood - brain barrier .
jones found that ptm onset within 3 days in patients with non - penetrating , non - depressed injuries was uniformly pneumococcal and recommend empiric therapy against this organism . while the infecting organism is likely to be from the nasopharynx or external auditory canal , nosocomial bacteria can not be excluded until culture results are known . in patients with penetrating injuries , prolonged hospitalization , delayed ptm onset , or antibiotic prophylaxis ,
definitive therapy should include broad spectrum drugs since the risk of infection with gram - negative or resistant microorganisms is higher .
thus , in the management of arrow injuries lies the significance of avoiding any undue time delay , as well as any neurological or vascular compromise , so as to obtain the best possible outcome .
the morbidity - free outcome of surgical removal , in case of penetrating arrow injuries , despite the delay in presentation and moreover in the emergency surgical practice , are the salient points to be remembered whilst managing such cases , for what the mind knows is what the eyes see and what the eyes see is what can be practiced | arrow injuries are an extinct form of injury in most parts of the developed world , but are still seen , albeit infrequently in developing countries .
reports of penetrating injuries of the craniofacial region secondary to projectiles are few and far between .
the morbidity - free outcome of surgical removal , in case of penetrating arrow injuries , despite the delay in presentation and , moreover , in the emergency surgical practice , are the salient points to be remembered whilst managing such cases , for what the mind knows is what the eyes see and what the eyes see is what can be practiced. we report the case of a patient who was attacked by a projectile fired from a crossbow .
immediate surgery under general anesthesia was required to remove the arrow , with utmost care to avoid any neurovascular compromise to the facial nerve , as well as minimize postoperative complications such as otitis media and subsequent meningitis . |
they are opportunistic and associated with some health - related infections ( 1 , 2 ) .
such bacterial infections , particularly in patients hospitalized at intensive care units , are very dangerous ( 3 ) .
in addition , colonization rates are rising among hospitalized patients , especially those hospitalized for a long time or those who are on extended antibiotic therapy or receiving anti - cancer treatment ( 2 , 4 ) .
this organism can infect the respiratory tract , blood , soft tissues , urinary tract and the central nervous system .
acinetobacter calcoaceticus baumannii complex has emerged as a highly troublesome pathogen in hospitals , and is resistant to multiple antimicrobial agents ( 5 ) .
the various species of acinetobacter are mostly intrinsic with acquired resistance to common antimicrobial agents , thus , the treatment of respective infections is very difficult ( 6 ) . in the recent years
, there have been reports on the incidence of acinetobacter infections with multi - drug resistance ( mdr ) around the world ( 7 ) . excessive use of antimicrobial drugs in the treatment of such infections has caused problems in treatments leading to growing concerns in many communities .
health communities are concerned about the resistance of acb complex to all conventional antibiotics , even to broad - spectrum antibiotics such as carbapenems and sulbactam ( 8) . at present
, these organisms only have little resistance to colistin and polymyxin b. keeping the spread of acinetobacter under control is very difficult because these organisms disseminate easily in hospital environments ( 8) .
epidemiological studies of nosocomial infections are essential to prevent these infections . however , phenotypic methods such as antibiotic resistance tests , because of phenotypic characterization variation and low differentiation power , are not sufficient alone in epidemiological studies ( 9 ) . to obtain more reliable results ,
pulsed field gel electrophoresis ( pfge ) is the most widely used method for valuable typing of epidemiological studies in different regions and many researchers have used this method to evaluate the similarities between the strains of acb complex ( 8 , 10 - 13 ) .
comparison of pulse - field patterns is a useful method for obtaining strain relationships in a certain species .
pulsed field gel electrophoresis was used in this study as a reliable method for differentiating between the strains of acb and distinguishing respective clonal relationships , as well as the relationship between genetic and phenotypic patterns .
in addition , the present study was designed for the isolation of strains from clinical samples , identification using appropriate methods and evaluation of their antibiotic resistance patterns . in doing so
during a period of nine months in 2011 , 117 isolates were collected from the samples of patients with suspected acinetobacter infections , who were referred to nemazee hospital of shiraz , southern iran .
all presumptive acb complexes , which were gram - negative coccobacilli , oxidase negative and non - lactose fermentative , were identified at the species level by using biochemical test and conventional analytical profile index ( api ) ( microgen gna - idtm ) ( 14 ) . as the cases enrolled in this study were only patients with nosocomial infections , the inclusion criteria were antibiotic use during
the last 15 days and infections noted 48 hours after admission or within four weeks after a previous discharge ( 15 ) . informed written consents were obtained from all patients .
the design and protocol of the study were also approved by the ethics committee of professor alborzi clinical microbiology research center .
susceptibility of all isolates to different antibiotics was determined by the disk diffusion method , as recommended by the national committee for clinical laboratory standards ( clsi ) , ( 16 ) with commercial antimicrobial disks ( mast .
the antibiotics consisted of : gentamicin ( gm ) , ticarcillin ( tc ) , ceftazidime ( caz ) , co - trimoxazole ( ts ) , imipenem ( imi ) , piperacillin tazobactam ( ptz ) , amikacin ( ak ) , aztreonam ( atm ) , sulbactam ( sam ) , meropenem ( mem ) , tobramycin ( tn ) , cefotaxime ( ctx ) , ceftriaxone ( cro ) , colistin ( co ) and polymyxin b ( pb ) .
escherichia coli atcc 25922 was used for quality control purposes in antibacterial susceptibility determination . in this study
subsequently , the colonies were mixed with 1 cc of buffer suspension ( 10 mm tris hcl [ ph 8 ] , 100 mmol ethylenediaminetetraacetic acid ( edta ) ) .
next , 1 cc of 2% low melting point agarose ( lma ) was added to the tube containing the microbial solution .
the resulting mixture was transferred to a gel mold until the gel was formed completely .
then the gel was transferred to the flask to which we added 25 cc of lysing buffer ( 50 mm tris hcl [ ph 8 ] , 50 mmol edta , 1% lauroylsarcosine ) , and 125 of 20 mg / cc proteinase k ( fermentase , lithuania ) ) so that the gel floated on the solution .
next , each dna plug was initially washed with sterile distilled water , and then , with sterile tris edta washing buffer ( 10 mm tris hcl [ ph8 ] , 1 mmol / liter edta ) on an incubated shaker at 50c .
subsequently , the dna plug was transferred to a sterile eppendorf tube and preserved in the heater for 18 hours at 37c ( takara biotechnology , dalian , co , ltd ) .
dna preparations were transferred to the wells of a nucleic acids agarose ( molecular grade , amersham bioscience , sweden ) .
next , the buffer tank containing edta , boric acid and tris - base at ph 8 was prepared for isolating bacterial genome and transferred to the pfge instrument ( gene navigator system , amersham bioscience , sweden ) .
electrophoretic conditions used , were as follows : initial switch time of five seconds ; second switch time of 20 seconds ; final switch time of 40 seconds ; temperature of 6c , run time of 33 hours ; angle of 120 and gradient of 6 v / cm . in each set a 1000 bp - lambda ladder ( biolabs , new england )
photocapt software ( vilber loumart , marnesla valle , france ) was used to determine the molecular weights of the sample profiles .
the bands were evaluated according to the dna marker and were scored across all samples .
subsequently , the data were used to calculate pair - wise similarity coefficient following jaccard s method . to generate a dendrogram using the average linkage procedure ,
the analysis of the similarity coefficients matrices was performed using unweighted pair - group method analysis ( upgma ) . to calculate correlations among the variables ,
the correlations were subjected to eigenvector analysis to evince the first three uttermost elucidative principal components . to study the patterns of variations observed among the isolates , the three principal components were plotted .
the numerical taxonomy system ( ntsys ) pc software version 2.02i ( exeter software , new york ) was used to conduct all the numerical analyses .
during a period of nine months in 2011 , 117 isolates were collected from the samples of patients with suspected acinetobacter infections , who were referred to nemazee hospital of shiraz , southern iran .
all presumptive acb complexes , which were gram - negative coccobacilli , oxidase negative and non - lactose fermentative , were identified at the species level by using biochemical test and conventional analytical profile index ( api ) ( microgen gna - idtm ) ( 14 ) . as the cases enrolled in this study were only patients with nosocomial infections , the inclusion criteria were antibiotic use during
the last 15 days and infections noted 48 hours after admission or within four weeks after a previous discharge ( 15 ) . informed written consents were obtained from all patients .
the design and protocol of the study were also approved by the ethics committee of professor alborzi clinical microbiology research center .
susceptibility of all isolates to different antibiotics was determined by the disk diffusion method , as recommended by the national committee for clinical laboratory standards ( clsi ) , ( 16 ) with commercial antimicrobial disks ( mast .
the antibiotics consisted of : gentamicin ( gm ) , ticarcillin ( tc ) , ceftazidime ( caz ) , co - trimoxazole ( ts ) , imipenem ( imi ) , piperacillin tazobactam ( ptz ) , amikacin ( ak ) , aztreonam ( atm ) , sulbactam ( sam ) , meropenem ( mem ) , tobramycin ( tn ) , cefotaxime ( ctx ) , ceftriaxone ( cro ) , colistin ( co ) and polymyxin b ( pb ) .
in this study , we used the seifert procedure with some modifications ( 17 ) .
subsequently , the colonies were mixed with 1 cc of buffer suspension ( 10 mm tris hcl [ ph 8 ] , 100 mmol ethylenediaminetetraacetic acid ( edta ) ) .
next , 1 cc of 2% low melting point agarose ( lma ) was added to the tube containing the microbial solution .
the resulting mixture was transferred to a gel mold until the gel was formed completely .
then the gel was transferred to the flask to which we added 25 cc of lysing buffer ( 50 mm tris hcl [ ph 8 ] , 50 mmol edta , 1% lauroylsarcosine ) , and 125 of 20 mg / cc proteinase k ( fermentase , lithuania ) ) so that the gel floated on the solution .
next , each dna plug was initially washed with sterile distilled water , and then , with sterile tris edta washing buffer ( 10 mm tris hcl [ ph8 ] , 1 mmol / liter edta ) on an incubated shaker at 50c .
subsequently , the dna plug was transferred to a sterile eppendorf tube and preserved in the heater for 18 hours at 37c ( takara biotechnology , dalian , co , ltd ) .
dna preparations were transferred to the wells of a nucleic acids agarose ( molecular grade , amersham bioscience , sweden ) .
next , the buffer tank containing edta , boric acid and tris - base at ph 8 was prepared for isolating bacterial genome and transferred to the pfge instrument ( gene navigator system , amersham bioscience , sweden ) .
electrophoretic conditions used , were as follows : initial switch time of five seconds ; second switch time of 20 seconds ; final switch time of 40 seconds ; temperature of 6c , run time of 33 hours ; angle of 120 and gradient of 6 v / cm . in each set a 1000 bp - lambda ladder ( biolabs , new england )
photocapt software ( vilber loumart , marnesla valle , france ) was used to determine the molecular weights of the sample profiles .
the bands were evaluated according to the dna marker and were scored across all samples .
subsequently , the data were used to calculate pair - wise similarity coefficient following jaccard s method . to generate a dendrogram using the average linkage procedure ,
the analysis of the similarity coefficients matrices was performed using unweighted pair - group method analysis ( upgma ) . to calculate correlations among the variables ,
the correlations were subjected to eigenvector analysis to evince the first three uttermost elucidative principal components . to study the patterns of variations observed among the isolates , the three principal components were plotted . the numerical taxonomy system ( ntsys )
pc software version 2.02i ( exeter software , new york ) was used to conduct all the numerical analyses .
in total , 117 samples from patients aged between 16 days and 95 years , with suspected acb complex infection were obtained ; of which 93 samples were acb complex while others were gram - negative organisms ( such pseudomonas and e. coli ) . among acb complex isolates ,
31 ( 33.4% ) samples were from sputum and throat secretions , 23 ( 24.8% ) from wound , 19 ( 20.4% ) from blood cultures , nine ( 9.6% ) from urine , seven from trachea ( 7.6% ) , and four from body fluids ( 4.2% ) .
samples were collected from different wards of nemazee hospital with corresponding distributions presented in figure 1 . to avoid multiple entries from a single patient ,
the similarities among the patterns of the 93 isolates were calculated by the dice coefficient and then regrouped using the unweighted pair group method with arithmetic mean ( upgma ) .
antibiotic sensitivity patterns of the isolates were determined by the standard disc diffusion method and the results are shown in table 1 . high resistance was observed among the tested antibiotics , except co and pb . over 90% of the strains exhibited resistance to antibiotics ctx ,
abbreviations : ak , amikacin ; atm , aztreonam ; caz , ceftazidime ; co , colistin ; cro , ceftriaxone ; ctx , cefotaxime ; gm , gentamicin ; imi , imipenem ; mem , meropenem ; ptz , piperacillin tazobactam ; pb , polymyxin b ; sam , sulbactam ; tc , ticarcillin ; tn , tobramycin ; ts , co - trimoxazole .
ninety - eight percent of the isolates were resistant to three or more antibiotics and were designated as mdr ( 18 ) .
thirty - five patterns of resistance were recognized for the acb complex strains ( table 2 ) and the most common pattern was resistance to 13 antibiotics ; atm , ak , cro , ptz , tc , sxt , imi , gm , caz , mem , tob , ctx and sam .
both patterns were the dominant patterns in the emergency coronary unit ( icu ) internal patients .
abbreviations : ak , amikacin ; atm , aztreonam ; caz , ceftazidime ; co , colistin ; cro , ceftriaxone ; ctx , cefotaxime ; gm , gentamicin ; imi , imipenem ; mem , meropenem ; ptz , piperacillin tazobactam ; pb , polymyxin b ; sam , sulbactam ; tc , ticarcillin ; tn , tobramycin ; ts , co - trimoxazole . using this technique and considering the designed dendrogram ( figure 1 ) , in total ,
47 genetic patterns were obtained and assigned letters from z to g. with cut off level 80% , clone p ( p1 , p2 , p3 , p4 , p5 ) holds the largest number of organisms .
eleven samples with strains of clone p were isolated from patients in the internal icu , eight from icu surgical , and one sample from general icu , pediatric , plastic surgery and neurology each .
band sizes obtained by pfge ranged from 6 to 590 kbp and band number ranged from 8 to 18 .
the most frequent patterns were those with 12 and 13 bands detected in 24.7% and 20.4% of the strains , respectively . the patterns with 8 and 18 bands accounted for 1.1% of the total isolated strains .
in total , 117 samples from patients aged between 16 days and 95 years , with suspected acb complex infection were obtained ; of which 93 samples were acb complex while others were gram - negative organisms ( such pseudomonas and e. coli ) . among acb complex isolates ,
31 ( 33.4% ) samples were from sputum and throat secretions , 23 ( 24.8% ) from wound , 19 ( 20.4% ) from blood cultures , nine ( 9.6% ) from urine , seven from trachea ( 7.6% ) , and four from body fluids ( 4.2% ) .
samples were collected from different wards of nemazee hospital with corresponding distributions presented in figure 1 . to avoid multiple entries from a single patient ,
the similarities among the patterns of the 93 isolates were calculated by the dice coefficient and then regrouped using the unweighted pair group method with arithmetic mean ( upgma ) .
antibiotic sensitivity patterns of the isolates were determined by the standard disc diffusion method and the results are shown in table 1 . high resistance was observed among the tested antibiotics , except co and pb . over 90% of the strains exhibited resistance to antibiotics ctx ,
abbreviations : ak , amikacin ; atm , aztreonam ; caz , ceftazidime ; co , colistin ; cro , ceftriaxone ; ctx , cefotaxime ; gm , gentamicin ; imi , imipenem ; mem , meropenem ; ptz , piperacillin tazobactam ; pb , polymyxin b ; sam , sulbactam ; tc , ticarcillin ; tn , tobramycin ; ts , co - trimoxazole .
ninety - eight percent of the isolates were resistant to three or more antibiotics and were designated as mdr ( 18 ) .
thirty - five patterns of resistance were recognized for the acb complex strains ( table 2 ) and the most common pattern was resistance to 13 antibiotics ; atm , ak , cro , ptz , tc , sxt , imi , gm , caz , mem , tob , ctx and sam .
both patterns were the dominant patterns in the emergency coronary unit ( icu ) internal patients .
abbreviations : ak , amikacin ; atm , aztreonam ; caz , ceftazidime ; co , colistin ; cro , ceftriaxone ; ctx , cefotaxime ; gm , gentamicin ; imi , imipenem ; mem , meropenem ; ptz , piperacillin tazobactam ; pb , polymyxin b ; sam , sulbactam ; tc , ticarcillin ; tn , tobramycin ; ts , co - trimoxazole .
using this technique and considering the designed dendrogram ( figure 1 ) , in total , 47 genetic patterns were obtained and assigned letters from z to g. with cut off level 80% , clone p ( p1 , p2 , p3 , p4 , p5 ) holds the largest number of organisms .
eleven samples with strains of clone p were isolated from patients in the internal icu , eight from icu surgical , and one sample from general icu , pediatric , plastic surgery and neurology each .
band sizes obtained by pfge ranged from 6 to 590 kbp and band number ranged from 8 to 18 .
the most frequent patterns were those with 12 and 13 bands detected in 24.7% and 20.4% of the strains , respectively .
the patterns with 8 and 18 bands accounted for 1.1% of the total isolated strains .
acinetobacter has been one of the main causes of nosocomial infections in the recent years .
these opportunistic bacteria are the second agent isolated from hospitalized patients after pseudomonas strains . in a report published in 2004 by the center for disease control and prevention ( cdc ) ,
a. baumannii was the cause of 80% of infections ( 19 ) . in the present study , based on biochemical tests and using standardized methods , 100% of the isolates
were found to be acb complex , which is almost consistent with the results of other studies ( 17 , 20 ) .
acinetobacter baumannii is the most common isolate obtained from the skin , blood , sputum , pleural fluid and urine ( 21 ) .
as many studies have shown , the respiratory tract is the body site where the organism is most frequently isolated in the icu ( 22 - 25 ) . in this study
, the majority of studied patients were hospitalized in the icu and most samples were collected from throat and sputum cultures and the rest of the samples were isolated from wound , blood , urine and tracheal fluid .
nosocomial infections caused by the strains of these bacteria are increasingly becoming resistant to a range of antibiotics .
for example , previous iranian studies on resistance to mem and imi reported lower resistance rates than that of our study ( 26 , 27 ) . in another study conducted in turkey ( 26 ) , our neighboring country , all the samples were found to be sensitive to these antibiotics . as seen in table 1 , among the acb complex except against co and pb , high rates of antibiotic resistance were observed , indicating that these two antibiotics could be the most effective antibacterial agents against the respective infections .
similarly , there have been several reports of resistance to these antibiotics ( 28 , 29 ) , with some being alarming ( 30 ) . in some other studies , resistance to co was lower than that in our study ( 31 , 32 ) . according to the present study , co was the least administered antibiotic , i.e. , out of the 93 patients , only seven patients received this antibiotic , and consequently the least resistance to this antimicrobial agent was observed .
although in the present study , resistance to ptz was high , the highest resistance was observed to third generation cephalosporins , including ts , atm , and tc ( 90% ) . in some studies ,
high percentage of resistance to ts , ptz , ctx and caz was observed among the examined isolates ( 33 , 34 ) .
resistance to tn and sam in the present study was comparable to the rates reported by previous studies from iran ( 26 , 27 ) . in this study ,
a total of 35 phenotypic patterns were observed among the 93 strains , of which the predominant pattern was repeated in 21 strains , which was resistance to 13 antibiotics .
appearance of varying resistance patterns in different nations might be due to factors including financing policies , doctors and patients visiting and prescribing habits ( 37 ) .
there are many causes responsible for emerging mdr acb complex infections in hospitals including length of hospitalization and staying at the icu , which was an average of 24 days in the present study .
given the easy spread of acinetobacter and its ability to survive in the environment for 13 days ( 19 ) , there was enough time for the transmission of infection .
other factors such as number of hospital beds , and invasive and non - invasive procedures performed on the patients at the icu , could be the cause of emerging mdr abc strains .
since the majority of patients in this study were from the icu with invasive procedures , the presence of mdr abc was not unexpected .
the majority of samples were isolated from the respiratory tract , therefore , it could be suggested that the methods associated with mechanical ventilation can facilitate the transmission of mdr abc amongst patients in a ward . in this study ,
similar to previous reports ( 30 , 31 , 38 ) , a high prevalence of mdr isolates was found among the samples ( 98.9% ) .
one of the reasons for the selection of abc in hospital epidemics is the outbreak of mdr caused by this organism , as clearly demonstrated by previous studies ( 5 , 39 ) .
the results of pfge and similarity of genetic patterns of mdr strains confirmed this point .
a specific phenotypic pattern was not observed in the epidemic clone p. as observed , most strains in this clone exhibited resistance to all aminoglycosides and carbapenems .
simultaneous presence of resistance to the two types of antibiotics can give rise to increasing epidemic infection in the studied hospital .
since these two antibiotics were the most widely used agents to treat suspected patients in the studied hospital , it can be suggested that based on the selective pressure related to antibiotic exposure , clonally related strains of acinetobacter with different susceptibility patterns can be simultaneously traced in a hospital . in many countries ,
various studies have shown genetic similarity among most strains of a. baumannii in a single hospital or different hospitals ( 13 , 31 , 40 ) .
indeed , isolates with the same clonality have caused epidemics . in the present study , using the pfge method as the gold standard typing method for acinetobacter ( 31 ) , we obtained 47 patterns of which type p with 23 strains by cut off level of 80% , included most strains with the same genetic pattern . using this method ,
numbers of dna molecular bands were between 8 and 18 and bands ranged in size from 6 to 590 kbp .
changes in the genetic diversity of strains in different geographical areas can cause different patterns of bands in different laboratories .
a close genetic relationship among abc strains showed the distribution of organisms in the studied hospital environment . regarding clone p ,
most isolates were separated from icus and three phenotypic profiles of no.2 , 5 and 7 were observed among the strains . in other words ,
it can be stated that the mechanisms of plasmids resistance and changes in gene regulation or point mutations are indistinguishable by pfge . in some clones ,
observations were different , i.e. strains with different genetic patterns had similar phenotypic patterns , which can be attributed to plasmid or transposons that can carry multiple resistance genes and then make the strains resistant to multiple antibacterial agents simultaneously , and due to the different genetic mechanisms , different strains have similar phenotypic patterns . with respect to the above mentioned findings , identifying
the bacterial resistance patterns of abc in various hospitals over a specified time period seems to be necessary .
as revealed by the present study , all the investigated acinetobacter isolates were highly resistant to the examined antibiotics because of unwise and untimely use of antibiotics to treat infections . in view of the easy spread of abc in nosocomial infections , accurate identification , antibiotic patterns evaluation and updating infection control procedures
the results of pfge and the presence of closely related bacterial strains suggest that preventive measures such as education of clinicians , patients and visitors , hand hygiene , isolation of patients , periodic culture of patients and staff , changes in antibacterial usage , and regular disinfection of medical devices particularly for invasive procedures are warranted . | backgroundacinetobacter calcoaceticus baumannii ( acb ) complex are gram - negative opportunistic bacteria with low virulence properties .
their resistance to antibiotics has become a matter of concern in hospital infections.objectivesthe present study aimed to determine the prevalence and antimicrobial susceptibility of acb isolates collected from the nemazee hospital of shiraz .
in addition , pulsed field gel electrophoresis ( pfge ) was used to determine the genetic patterns of these strains.patients and methodsin this cross - sectional study , 93 strains of acb complex were isolated from patients of nemazee hospital , shiraz , iran .
the antibiotic susceptibility patterns of the isolates to the following 15 antibiotics were determined : gentamicin , ticarcillin , ceftazidime , co - trimoxazole , imipenem , piperacillin tazobactam , amikacin , aztreonam , sulbactam , meropenem , tobramycin , cefotaxime , ceftriaxone , colistin , polymyxin b. pulsed field gel electrophoresis was used to determine the clonal relationship of these strains.resultsmost of the isolates were found to be resistant to cefotaxime , co - trimoxazole , ceftriaxone , aztreonam , ceftazidime and ticarcillin ( 90% ) , and the least resistance was observed to colistin and polymyxin b. among the 93 tested samples , 35 antimicrobial susceptibility patterns and 47 pfge patterns were obtained.conclusionshigh resistance to antibiotics was observed among the strains of acb complex and the least resistance was towards colistin and polymyxin b , indicating that these antibiotics could be effective for treatment , in case there is no other choice .
using pfge , the similarity between some strains of acinetobacter was determined , which indicated epidemics in different parts of the hospital ; such epidemics can in turn lead to increased incidence of acinetobacter infections . |
ten years later , levels of serum tumor marker ca15 - 3 progressively increased until 2n although other serum tumor markers ( a - fetoprotein , carcinoembryonic antigen , carbohydrate antigen 19 - 9 ) were normal .
axial contrast - enhanced multislice ct scan showed a solitary and hypovascular 4-cm lesion located in the right lobe of the liver ( segment vii ) .
liver mri found this lesion in segment vii to be associated with multiple and small lesions with high signal intensity scattered throughout the liver especially in the subcapsular area in t2-weighted images ( fig .
after contrast injection , the multiple masses showed irregular progressive enhancement from arterial phase through delayed phase .
gastroscopy and coloscopy did not find new tumors and pet scan showed hyperfixation only on the hepatic lesion in segment vii .
the diagnosis of a single liver metastasis of breast cancer was suspected although its characteristics were unusual ( hypovascular lesion ) .
the diagnosis of von meyenburg complexes ( vmc ) was also suspected preoperatively , but the presence of multiple secondary hepatic lesions could not be dismissed because of breast cancer history .
percutaneous biopsy of the largest hepatic lesion found fibrosis but no malignant infiltration and could neither confirm the diagnosis of metastasis of breast cancer nor the diagnosis of vmc . since this tumor had unusual characteristics , we performed laparoscopic assessment to obtain histological proof of malignancy . during laparoscopy , multiple gray - white nodular lesions ( about 0.5 - 1 cm in diameter )
a liver biopsy was performed because we feared multiple liver metastases ( contraindication of curative resection ) .
histological examination confirmed the diagnosis of multiple benign vmc ( biliary hamartomas ) encompassed by major fibrous stroma .
after conversion to laparotomy , right hepatectomy was performed and the pathological findings of the specimen confirmed the liver metastasis of the breast cancer in segment vii ( without argument for cholangiocarcinoma ) associated with multifocal , irregularly dilated tinged bile ducts .
vmc is a cluster of benign liver malformations including biliary cystic lesions , with congenital fibrocollagenous stroma .
this entity corresponds to ductal plate malformation , which explains why they can be found in association with other congenital intrahepatic abnormalities , such as congenital hepatic fibrosis , caroli 's syndrome and polycystic liver disease .
vmc are usually asymptomatic but clinical findings including jaundice , cholangitis , epigastralgias and fever have been reported .
when these lesions are multiple , deep and larg ( many millimeters in diameter ) , they can simulate secondary metastatic lesions particularly in case of cancer history and so contraindicate curative resection .
ultrasound can show hypoechoic ( larger cysts ) and/or hyperechoic structures ( smaller cysts ) with comet - tail echoes . in some case , these hyperechoic structures correspond to small stones inside the dilated bile ducts .
nonenhanced ct shows hypodense , small hepatic nodules , scattered throughout both liver lobes , measuring less than 15 mm in diameter .
the best imaging to diagnose vmc is mri with typically hypointense and well - defined lesions in t1-weighted images and hyperintense lesions in t2-weighted images [ 3 , 6 , 7 ] .
semelka et al . reported a thin rim enhancement on early postgadolinium images that persisted until late postgadolinium images .
vmc was suspected preoperatively in our patient , but confirmation of the diagnosis required further investigations particularly because of cancer history .
macroscopically , vmc are grayish white nodules measuring 1 to 15 mm in size that are scattered throughout the liver , particularly below the capsule of the liver , in the periportal region of hepatic lobules .
a definitive diagnosis of vmc requires histopathologic examination of the specimen because its findings are nonspecific and easily confused with malignant liver diseases .
microscopically , vmc are characterized by cystic dilatations of the bile duct or clusters of mature bile duct of various sizes , periductal glands and encompassed by fibrous stroma .
preoperative discovery of multiple gray - white nodular lesions scattered on the surface of the liver should not always contraindicate curative liver resection . | von meyenburg complexes ( vmc ) are a cluster of benign liver malformations including biliary cystic lesions , with congenital fibrocollagenous stroma .
this rare entity can mimick multiple secondary hepatic lesions .
we report a case of a 56-year - old woman who had multiples liver lesions 12 years after operation for breast cancer .
biopsy of the hepatic lesion confirmed the diagnosis of vmc .
preoperative discovery of multiple gray - white nodular lesions scattered on the surface of the liver should not always contraindicate curative liver resection .
the diagnosis of vmc should be known and confirmed with liver biopsy . |
chronic myelogenous leukemia ( cml ) is a malignant clonal disorder of hemopoietic stem cells characterized by abnormal proliferation and accumulation of immature granulocytes .
leukostasis is one of the complications of cml and is characterized by partial or total occlusion of microcirculation by aggregation of leukemic cells and thrombi leading to respiratory , ophthalmic or neurologic symptoms .
avascular necrosis of bone occurs in the areas with poor collateral circulation when the only branch supplying blood is unable to fulfill the requirements of the bone .
commonly affected areas are head of the femur , head of the humerus , femoral condyles and tibial plateau .
very few cases dealing with cml and avascular necrosis due to leukostasis have been reported . here ,
we report a rare case of 21 year - old male patient with avascular necrosis of right femoral head owing to leukostasis as the initial presentation of cml .
a 21-year - old male patient was presented with pain in the right hip joint for 2 months .
he was a soldier and went to the dispensary of the unit that he belonged to .
he was referred to our hospital for further evaluation of abnormal complete blood cell count which was performed at the dispensary .
the patient suffered from weight loss , mild dyspnea , fatigue , intermittent fever , and night sweat .
, it was observed that the patient was in poor general condition with pallor skin and anemic conjunctiva .
laboratory investigations revealed leukocytosis with leukocyte count of 96,800/mm , increased platelets with a count of 684,000/mm , and hemoglobin level of 10.4 g / dl .
bone marrow examination revealed that the entire marrow space was packed with granulocytes and megakaryocytes .
the bone marrow aspiration showed 0.4% myeloblasts , 6% promyelocytes , 13.2% myelocytes , 15.2% metamyelocytes , 14.7% band neutrophils , 40% segmented neutrophils , 2.7% eosinophils , 0% basophils , 0.8% monocytes , 0.4% pronormoblasts , 0% lymphoblasts , 0% immature lymphocytes , 0.4% lymphocytes , 0.4% plasma cells , and 0% histiocytes suggesting chronic phase of cml .
chromosomal analysis of bone marrow revealed philadelphia chromosome , t ( 9;22 ) and molecular cytogenetics also showed bcr - abl positive .
treatment with hydroxyurea was induced , combined with immediate evaluation of right hip joint pain .
radiolucency superimposing the right femoral head was seen on the pelvic x - ray ( figure 1 ) .
magnetic resonance imaging ( mri ) of both the hips revealed inhomogenously decreased signal intensity in the both proximal femur and acetabulum on t1 weighted images .
the outer areas surrounding these areas had increased signal densities on t2 weighted images ( figure 2 ) . on application of contrast medium , area with former decreased signal density on the cranial part of the right femoral head and some smaller areas of the left femoral head , showed a lack of signal enhancement .
bilateral joint effusion , worse in the right than the left , was also seen .
the leukocyte count decreased from 96,800/ul to 26,300/mm with the treatment of hydroxyurea ( 3 g / day ) for 2 weeks and subsequently hydroxyurea was replaced with imatinib mesyalte . with this
the size of the spleen decreased to 3 cm below the costal margin within 1 month .
following normalization of the peripheral blood counts , the patient was transferred to the department of orthopedics .
he underwent a bipolar hemiarthoplasty of the right hip joint , because the necrosis of the right hip joint was more severe than the left and he had no pain in the left hip joint .
the postoperative course was complicated due to bleeding from the operative site possibly due to thrombocytopenia which may have been induced by imatinib mesylate .
temporarily , the medication of imatinib mesylate was stopped , and the patient was transfused with platelets and packed red cells .
the biopsy revealed focal subchondral necrotic areas in the right hip joint ( figure 3 ) .
the patient was on a continuous follow up and showed no complications with complete cytogenetic response and has been on medication of imatinib mesylate ( 400 mg / day ) .
cml is characterized by a chronic phase which lasts from months to years , and is followed by accelerated myeloproliferative phase and subsequently blast crisis . in the chronic phase ,
manifestations include anemia , splenomegaly , bleeding , and constitutional symptoms such as fatigue , lethargy , weight loss , or low - grade fever . as in the present case study our case of a 21-year - old male , symptoms secondary to leukostasis may also be the presenting features1 ) .
since there are no reliable clinical criteria , in practice , leukostasis is empirically diagnosed when patients present with leukemia , hyperleukocytosis and respiratoy or neurologic distress2 )
. however , some patients have clinically suspected or pathologically proven leukostasis with leukemic blast counts significantly lower than 100,000/mm3 ) .
litchmann proposed a pathophysiological model of leukostasis , based on the rheological consequences of hyperleukocytosis .
he determined that the viscosity of leukocyte suspensions in vitro increased dramatically when the fractional volume of leukocytes ( also known as leukocrit ) exceeded 12~15 ml / dl4 ) .
the concentration of leukocytes necessary to produce such leukocrits is a function of the mean cell volume ( mcv ) of the cells . since leukemic myeloblasts have larger mcv than leukemic lymphoblasts
, leukostasis is more frequently observed in patients with acute or chronic myelogenous leukemia than in patients with acute or chronic lymphoblastic leukemia5 ) .
however , leukemic blast concentrations necessary to reach leukocrit of 12~15 ml / dl are rarely seen .
therefore , additional factors possibly related to the adhesive or invasive properties of leukemic blasts , may be more important .
there are indications that leukemic blast - endothelial cell interactions may be triggered by locally released chemoattractant factors6 ) .
several adhesion molecules ( cd54 , cd62e , cd62p , cd106 ) are up - regulated in endothelial cell of aml patients with leukocytosis6 ) .
blasts appear to have the ability to secrete cytokines such as tnf- and interleukin-1 and therefore bring about the activation of endothelium .
for instance , activation of vascular endothelium by tumor necrosis factor - alpha ( tnf- ) has been reported to increase the adhesion of myeloid blasts8 ) .
once leukocytosis is developed , signs and symptoms are usually related to the involvement of the respiratory or central nervous system .
additional but less common clinical findings include neck vein distension , gallop rhythm and electrocardiographic sign of right ventricular overload , myocardial ischemia , priapism , acute limb ischemia , bowel infarctions , renal vein thrombosis , sudden bilateral deafness and avascular necrosis of bone2 ) . until now , in cml ,
avascular necrosis of femoral head due to leukostasis has not been adequately documented , probably due to the rarity of its occurrence .
after reviewing the world medical literature , we encountered only few cases of cml associated with avascular necrosis of femoral head9 - 13 ) .
the current standard of care for hyperleukocytosis and leukostasis should be the immediate initiation of intravenous fluids , allopurinol , hydroxyurea and the correction of coagulopathy and thrombocytopenia2 ) . due to paucity of data on the efficacy of leukapheresis in reducing early mortality and/or improving overall survival ,
unfortunately there are no evidences to prove that any of these measures have produced a reduction in the number of early deaths .
a variety of traumatic and nontraumatic factors contribute to the etiology of avacular necrosis . in adult patients , corticosteroid
use and excessive alcohol intake are reported to account for more than 90 percent of cases15 ) .
additionally , other causes of avascular necrosis are systemic lupus erythematus , antiphospholipid antibody , trauma , sickle cell hemoglobinopathes , gaucher 's disease , caisson disease ( dysbarism ) , transplantation , interferon- , inherited thrombophilia , or hiv infection16 - 19 ) .
avascular necrosis usually occurs in the anterolateral femoral head , although it may also affect the humeral head , femoral condyles , proximal tibia , vertebrae , and small bones of the hand and foot20 ) .
many patients have bilateral involvement at the time of diagnosis , including disease of the hips , knees , and shoulders .
the goal of therapy for avascular necrosis is to preserve the naive joint for as long as possible . | chronic myelogenous leukemia ( cml ) is a malignant clonal disorder of hemopoietic stem cells characterized by abnormal proliferation and accumulation of immature granulocyte .
leukostasis is one of the complications of cml and is characterized by partial or total occlusion of microcirculation by aggregation of leukemic cells and thrombi leading to respiratory , ophthalmic or neurologic symptoms .
we experienced a rare case of avascular necrosis of the femoral head as the initial presentation of chronic myelogenous leukemia .
a 24-year - old male patient was admitted to our hospital with pain in the right hip joint .
the patient was diagnosed to be suffering from chronic myelogenous leukemia by packed marrow with granulocytic and megakaryocytic hyperplasia and the presence of philadelphia chromosome .
the right hip joint pain was attributed to avascular necrosis of the femoral head . and
the avascular necrosis could be considered as the complication of chronic myelogenous leukemia due to microcirculatory obstruction of the femoral head .
the avascular necrosis of the right femoral head was treated with bipolar hemiarthoplasty . |
acute lung injury ( ali ) , induced by pneumonia , sepsis , multiple trauma , aspiration of gastric contents and severe burns , is a critical illness syndrome marked by excessive production of proinflammatory mediators , massive infiltration of leukocyte , and rapid alveolar injury , resulting in respiratory failure and high short - term mortality [ 1 , 2 ] .
sepsis is a common and life - threatening medical condition , with more than 50% of patients admitted to an intensive care unit ( icu ) developing ali . despite recent advances in antibiotic therapy and intensive care ,
septic lung injury in mice is associated with excessive production of proinflammatory mediators including tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , cyclooxygenase-2 ( cox-2 ) , and inducible nitric oxide synthase ( inos ) .
interfering with proinflammatory cytokines to relieve lung injury has been widely applied , while less attention was paid to anti - inflammatory molecules .
previous studies have demonstrated that upregulating anti - inflammatory molecules can significantly attenuate sepsis - induced inflammatory response [ 3 , 4 ] .
heme oxygenase ( ho-1 ) , also called heat shock protein 32 ( hsp32 ) , is an inducible enzyme catalyzed rate - limiting step in the oxidative degradation of heme to free iron , biliverdin , and carbon monoxide .
recent findings showed that ho-1 and its subsequent metabolites exerted anti - inflammatory and antiapoptotic activities .
overexpression of ho-1 exhibited a blunted activation of nf-b and less production of cytokines induced by lps [ 6 , 7 ] .
therefore , specific induction of ho-1 expression may be a new target in the treatment of septic lung injury .
polydatin ( 3 , 4 , 5-trihydroxystibene-3--mono - d - glucoside , pd , figure 1(a ) ) , the glycoside of resveratrol , is a crystal component extracted from the root stem of the perennial herbage polygonum cuspidatum sieb.et zucc , which is traditionally used for patients with chronic bronchitis , hepatitis , and shock .
previous studies indicated that pd had various activities such as inhibiting the platelet aggregation , lowering the level of blood lipid , reducing lipid peroxidation , dilating blood vessels , and protecting from myocardial ischemia / reperfusion damage [ 911 ] .
a number of studies have reported that pd and its derivatives have anti - inflammatory activities [ 8 , 9 ] .
however , the efficacy , as well as the mechanisms of action of pd on acute lung injury , remains unknown . on the basis of these reported results , we used a typical septic - induced ali model , cecal ligation and puncture procedure , to examine the effects of pd on septic acute lung injury and to further study its possible molecular mechanisms
mice weighing 18~22 g were obtained from the experimental animal center of chongqing medical university .
they were maintained in a controlled environment at a temperature of 2025c and 50 5% relative humidity under a 12 h dark / light cycle , and were acclimatized for at least l week before use .
all experimental procedures involving animals were approved by the animal care and use committee of chongqing medical university .
pd ( c20h22o8 , mw : 390.40 , purity 95% ) was purchased from nanjing debiochem co. ltd .
znpp ix was obtained from sigma ( st . louis , mo , usa ) .
bicinchoninic acid ( bca ) protein assay kit was purchased from pierce ( rockford , il , usa ) .
tnf- and il-6 enzyme - linked immunosorbent assay ( elisa ) kits were obtained from the bender medsystems ( vienna , austria ) .
rabbit anti - cox-2 , inos , ho-1 , and ho-2 antibodies were obtained from abcam ( cambridge , ma , uk ) .
-actin antibodies were purchased from cell signaling technology ( boston , ma , usa ) .
transam nf-b transcription factor assay kit was purchased from active motif ( carlsbad , usa ) .
the clp protocol was performed as previously described [ 7 , 12 ] . under sodium pentobarbital ( 30 mg / kg , i.p . ) anesthesia ,
the cecum was ligated by silk 4 - 0 and punctured twice with an 18-gauge needle , and then the cecum was gently squeezed to extrude a small amount of feces .
the cecum was repositioned , after which the abdomen was closed in two layers with 4 - 0 silk thread .
control operated mice underwent the same surgical procedures , but the cecum was neither ligated nor punctured . after surgery , animals were put back to their home cages with free access to water .
mice were randomly divided into six groups : control group , clp group , clp - pd ( 15 mg / kg ) group , clp - pd ( 45 mg / kg ) group , clp - pd ( 100 mg / kg ) group , and clp - pd - znpp ix group . the control and
the clp - pd group was injected i.p . with the same volume of pd ( 15 , 45 , and 100 mg / kg , resp . ) 1 h before clp .
injection of the same volume of the mixed drugs of pd ( 100 mg / kg ) and znpp ix ( 40 mg / kg ) 1 h before clp .
znpp ix was dissolved in 0.2 m sodium hydroxide and added hcl to adjust ph to 7.4 .
the survivors were sacrificed at 24 h and blood and lung specimen were obtained for following analysis .
the different doses of pd or znpp ix alone did not induce lung injury ( data not shown ) .
the blood was enucleated at 24 h after clp for serum tnf- and il-6 assay .
the blood samples were centrifuged at 3000 rpm , 10 min . then the serum was separated and transferred to other tubes and frozen at 20c .
serum tnf- and il-6 were determined using commercially available elisa kits according to manufacturer 's recommendations .
the left lung was excised , washed in pbs , gently dried using blotting paper , and weighed .
the change in the ratio of wet weight to dry weight was used as an indicator of lung edema formation .
balf was obtained by washing the airways three times with 1.5 ml of saline through a tracheal cannula .
the supernatant was harvested for total proteins analysis using the bca protein assay kit and the pellet was smeared onto slides for cell classification and counting in balf with a modified giemsa stain .
the lung specimens were excised and fixed for one week in 10% formaldehyde at room temperature , and then embedded in paraffin . serial paraffin sections ( 5 m ) were stained with hematoxylin - eosin routinely for conventional morphological evaluation under a light microscope ( olympus , tokyo , japan ) .
the severity of lung injury was judged by using a blind semiquantitative scoring system according to the following pathological features : ( i ) focal alveolar membrane thickening , ( ii ) capillary congestion , ( iii ) intra - alveolar hemorrhage , ( iv ) interstitial , and ( v ) intra - alveolar neutrophil infiltration .
each feature was scored from 0 to 3 based on its absence ( 0 ) or presence to a mild ( 1 ) , moderate ( 2 ) , or severe ( 3 ) degree , and a cumulative total histology score was determined .
the protein of lung samples were prepared by the protein extract kit ( 20 mm tris , 150 mm nacl , 1 mm edta , 1 mm egta , 1% tritonx-100 , 2.5 mm sodium pyrophosphate , 1 mm na3vo4 , 1 mm -glycerolphosphate , 1 g / ml leupeptin , and aprotinin ) .
40 g protein extracts were fractionated on 12% polyacrylamide - sodium dodecyl sulfate ( sds ) gel and then transferred to nitrocellulose membrane .
the membrane was blocked with 5% ( w / v ) fat - free milk in tris - buffered saline ( tbs ) containing 0.05% tween 20 , followed by incubation with a rabbit primary polyclonal antibody at 4c overnight .
then the membrane was treated with horseradish peroxidase - conjugated goat anti - rabbit secondary antibody .
antibody binding was visualized with a chemiluminescence system and short exposure of the membrane to x - ray films ( kodak , japan ) .
pge2 and no production in lung tissues were measured by commercial kits according to the manufacturer 's instructions .
the amount of total protein in lung tissues was quantified by the bca protein assay kit .
pge2 was measured by an enzyme - linked immunosorbent assay ( elisa ) using an antibody raised against mouse pge2 , absorbance was read at 450 nm . no was analyzed with a nitrate / nitrite colorimetric assay .
firstly , nitrate was converted to nitrite by nitrate reductase , and 100 l of griess reagent was added to 100 l tissue supernatants , which convert nitrite to a deep - purple azo compound .
the absorbance of the latter was measured at 550 nm using a plate reader .
briefly , frozen lung samples were homogenized in lysis buffer ( 250 mm tris.hcl , ph 7.4 ; 150 mm nacl ; 250 mm sucrose ; 0.5 mm pmsf ; 1 g/l leupeptin ; 1 g/l aprotinin ) .
microsomal fraction was obtained by successive centrifugations and washed with 0.15 m kcl followed by centrifugation ( 105,000 g for 30 min ) .
the pellet was solubilized in 0.1 m potassium phosphate by sonication and stored at 80c .
the reaction was carried out in the mixture containing 2 - 3 mg / ml protein microsomal fraction ; 1 mm glucose-6-phosphate ; 0.2 unit / ml glucose-6-phosphate dehydrogenase ; 0.8 mm nadph ; 0.025 mg / ml hemin at 37c for 45 min .
after chloroform extraction , the extracted bilirubin was calculated by the difference in absorbance between 464 and 530 nm .
nuclear protein extracts were obtained from lung tissues using a nuclear extract kit ( active motif , usa ) according to the manufacturer 's instructions .
the p65 dna binding activity was assessed by the transam nf-b kit ( active motif , usa ) according to the manufacturer 's instructions .
the study data were expressed as mean standard deviation ( sd ) and differences between group means were calculated by one - way analysis of variance ( anova ) and student 's t - test .
no death of mice was observed in the control group . when clp was performed on mice , lethality of mice was 50% at 24 h and reached to 90% at 120 h. pretreatment with pd dose - dependently decreased the lethality induced by clp .
however , the clp - pd - znpp ix group had a higher mortality than the clp - pd ( 100 mg / kg ) group , which indicated a significant difference ( p < 0.05 ) ; ( figure 1(b ) ) .
because the lethality of septic mice is associated with acute lung injury , we examine the pathology of lung tissues by he staining .
in contrast to the structure of pulmonary alveoli in the control group , the alveoli in the clp group were destructed , showing severe pathological abnormality including pulmonary interstitial hyperemia and edema , alveolar fluid exudation , and massive infiltration of inflammatory cells .
however , the pathological injury of lung tissues in the clp - pd - znpp ix group were exacerbated when compared with the clp - pd ( 100 mg / kg ) group , indicating ho-1 inhibitor znpp ix might abrogate the pulmonary protective effect of pd in septic mice ( figures 2(a ) and 2(b ) ) . in agreement with this pathological analysis ,
the wet / dry ratio of lung tissue showed a similar result ( figure 2(c ) ) . to further access the extent of the airway and lung injury
, we examined the amount of leukocytes and total proteins in the balfs 24 h after clp .
as expected , the clp group had significantly higher levels of leukocytes and total protein in the balf than the control group .
pretreatment mice with pd lowered infiltrating leukocytes and total proteins in the balf especially at 100 mg / kg .
however , znpp ix administration eliminated the inhibitory effects of pd on infiltration of leukocytes and total proteins in the balfs of septic mice .
it has been shown that systemic proinflammatory cytokines have a central role in sepsis [ 15 , 16 ] ; we investigated next the effects of pd on clp - induced systemic tnf- and il-6 production .
as shown in figure 4 , the serum levels of tnf- and il-6 in the clp group were remarkably increased as compared with these in the control group . however , the elevations of these proinflammatory cytokines were inhibited by pretreatment with pd in a dose - dependent manner . on the contrary , the inhibitory effects of pd on the clp - induced tnf- and il-6 productions were blocked by znpp ix administration . in comparison to the control group , in the clp group , the lung cox-2 and inos protein expression were significantly elevated , pretreatment with pd dose - dependently suppressed these upregulated proteins . however , the clp mice were pretreated with pd in combination of znpp ix , cox-2 and inos expressions in the lung tissues was blunted ( figure 5(a ) ) .
similar results were observed in pge2 and no productions ( figures 5(b ) and 5(c ) ) .
we also observed the change of ho-1 , which has been regarded as an anti - inflammatory molecule .
western blotting and activity analysis showed that the protein expression and activity of ho-1 in the lungs were not detected in the control group , but in the clp - operated mice , ho-1 expression and activity had a slight increase .
pretreated with pd enhanced the expression and activity of ho-1 in lungs of septic mice in a dose - dependent manner .
further , the increase of ho-1 activity by pd was almost completely abolished by znpp ix administration , but ho-1 protein expression was not affected by znpp ix .
however , no changes in ho-2 protein expression were observed in the lungs of all these groups ( figure 6 ) .
the p65 is a main subunit of nf-b , which is activated to translocate to the nucleus .
as shown in figure 7 , in the clp group , nf-b activity were markedly increased compared with the control group . however , pd pretreatment dose - dependently inhibited clp - induced nf-b activity in the lung .
furthermore , the beneficial effect of pd in the nf-b activity of septic mice was abrogated by znpp ix .
in the present study , mice in the clp model group have a high lethality and severe pathological lung injury including massive infiltration of inflammatory cells , hyperemia and edema of pulmonary interstitial .
pretreated mice with pd dose - dependently decreased clp - induced lethality , accompanied with alleviated severe lung pathological injury and leukocytes number and total proteins in balfs . collectively , these results indicated that pd alleviated clp - induced septic lung injury , which is in accordance with a previous study that pd had prophylactic and therapeutic effects on acute lung injury in rats with endotoxic shock .
in fact , sepsis is the intense systemic inflammatory response syndrome , which is associated with the overproduction of proinflammatory cytokines .
these cytokines lead to recruitment of leukocytes , tissue damage , and multiple organ failure [ 18 , 19 ] .
previous studies have indicated that proinflammatory cytokines especially tnf- and il-6 play crucial roles in septic lung injury [ 8 , 20 ] .
tnf- has been also regarded as one of the major mediators of systemic progression and tissue damage in many severe diseases [ 21 , 22 ] .
an earlier study had proven that injecting tnf- into experimental animals caused a syndrome that is indistinguishable from septic shock .
in addition , previous studies indicated that the plasma level of il-6 correlated with mortality in septic patients , and plasma levels of il-6 might be used as a diagnostic marker for the presence of bacteremia .
antibody to block il-6 improved further survival of mice in a bacterium - derived sepsis model [ 15 , 23 ] . to determine whether these cytokines are associated with the protective effect of pd , we examined serum tnf- and il-6 levels in mice .
these data supported that the protective effects of pd on septic lung injury in mice might be achieved by inhibition of these proinflammatory cytokines .
apart from proinflammatory cytokines , some enzymes such as cox-2 and inos were also involved in the pathogenesis of sepsis .
cox-2 is an inducible enzyme in the conversion of arachidonic acid to inflammatory prostaglandins , which is usually absent or minimally present at baseline in most normal tissues but is highly induced in response to inflammatory stimuli . although some studies showed that cox-2 had anti - inflammatory actions ,
accumulating data suggested that the cox-2/pge2 plays a vital role in augmenting inflammatory response and respiratory damage in sepsis [ 26 , 27 ] .
the different role of cox-2 might be associated with different types of inflammatory cells and prostaglandins during inflammatory process .
administration of ns-398 , a specific cox-2 inhibitor , inhibited endotoxin - induced pge2 and improved survival and restored leukocyte counts in septic mice [ 25 , 28 , 29 ] .
nitric oxide ( no ) , generated by the inducible nitric oxide synthase ( inos ) , plays an important role in vascular tone , leukocyte rolling , and microvascular leakage .
inos has been identified in many cell types such as epithelial cells or alveolar macrophages and is activated in septic mice .
knockout of inos expression in mice drastically alleviated pulmonary inflammation in response to intravenous exposure to lps .
furthermore , inhibition of inos activation significantly decreased the mortality and lung injury in septic mice [ 3133 ] . based on these data above , we determined these proteins expression in the lung by immunoblotting .
our results showed that pd was able to robustly inhibit the expression of inos and cox-2 protein mediated by clp in a dose - dependent manner , which was in line with its protection on septic lung injury , indicating that the protective effects of pd on sepsis - induced lung injury might be by inhibiting cox-2/pge2 and inos / no pathways .
nf-b is considered to an important transcriptional factor involved in the production of proinflammatory cytokines [ 24 , 34 ] .
normally , nf-b is sequestered in an inactive form in the cytoplasm , when it is activated by inflammatory stimuli ; nf-b translocates to the nucleus and subsequently initiates the transcription of inflammatory genes .
activation of the nf-b pathway has been implicated in the expressions of proinflammatory mediators , such as tnf- , il-6 , cox-2 , and inos . increased activation of nf-b is found in alveolar macrophages , peripheral blood mononuclear cells , and neutrophils from patients with sepsis [ 35 , 36 ] .
a greater or more persistent nuclear accumulation of nf-b is associated with higher mortality and more persistent organ dysfunction , including pulmonary injury .
therefore , inhibiting nf-b activation , which mediates the expression of proinflammatory mediators , appears to be a logical therapeutic target for controlling hyperinflammatory response including ali .
thus , we confirmed whether pd could modulate the activation of these upstream signal molecules .
we found that in parallel with the above results of proinflammatory mediators , pd also inhibited remarkably clp - induced nf-b activation .
these data suggested that the protective effects of pd on septic lung injury might be linked to inhibition of the activation of nf-b signaling pathway , resulting in suppression of proinflammatory mediators .
alternatively , sepsis - induced inflammation is tightly regulated by some anti - inflammatory molecules that modulate the intensity of inflammation and promote its eventual resolution and return it back to homeostasis .
previous studies have demonstrated that ho-1 is pivotal for downregulating the incremented inflammatory process and maintaining homeostasis for the correct function of vital organs [ 17 , 36 ] . to clarify the role of ho-1 in the protective effects of pd on septic lung injury
pretreatment clp mice with increasing concentration of pd upregulated ho-1 but not ho-2 expression in the lungs in a dose - dependent manner .
further , we administered mice with znpp ix , the specific inhibitor of ho-1 , to block ho-1 activity .
results indicated that znpp ix administration abrogated these benefits of pd not only in the mortality but also in the acute lung injury and inflammatory response .
all these data suggested that the protective mechanisms of pd against septic lung injury might , at least partly , be by upregulation of ho-1 .
previous studies indicated some transcription factors , as nuclear factor e2-related factor-2 ( nrf2 ) , activator protein-1 ( ap-1 ) , and nf-b , are involved in this induction of ho-1 [ 3840 ] . in this study , we found that clp induced a slight increase in ho-1 expression and activity , although this increase had not a statistical significance compared with the control group .
however , it is impossible that upregulation of ho-1 by pd is mediated by nf-b pathway , since pd inhibited clp - induced nf-b activation .
recent studies show that pd has obvious antioxidant property thought increasing the activities of superoxide dismutase ( sod ) and catalase ( cat ) [ 41 , 42 ] .
in fact , nrf2 is an important regulator against oxidative stress , which increases the expression of ho-1 , sod , and cat .
thereby , we speculate that pd may be through the mechanism of nrf2 activation to upregulation of ho-1 . certainly , to verify this hypothesis , further investigation needs to be done . to conclude
, we have demonstrated that pd could effectively exert protective roles against clp - induced septic lung injury , which alleviated lung pathological injury , reduced proinflammatory mediators , and attenuated lung inflammatory responses .
therefore , it is expected that pd might contribute to delaying ali progression and prolonging life in patients with lung damage . | the present study was carried out to investigate the effects and mechanisms of polydatin ( pd ) in septic mice .
the model of cecal ligation and puncture ( clp-)induced sepsis was employed .
pretreatment of mice with pd ( 15 , 45 , and 100 mg / kg ) dose - dependently reduced sepsis - induced mortality and lung injury , as indicated by alleviated lung pathological changes and infiltration of proteins and leukocytes .
in addition , pd inhibited clp - induced serum tumor necrosis factor- ( tnf- ) and interleukin-6 ( il-6 ) production , lung cyclooxygenase-2 ( cox-2 ) and inducible nitric oxide synthase isoform ( inos ) protein expressions and nf-b activation .
notably , pd upregulated the expression and activity of heme oxygenase ( ho-)1 in lung tissue of septic mice .
further , the protective effects of pd on sepsis were abrogated by znpp ix , a specific ho-1 inhibitor .
these findings indicated that pd might be an effective antisepsis drug . |
the migration of an encircling silicone band through a rectus muscle is a rare postoperative complication associated with the scleral buckling procedure .
however , both kreis et al . and tan et al . have reported cases of this rare complication . here , we describe what we believe to be the first reported case of a scleral buckling - associated encircling silicone band that migrated through the rectus muscles , despite the band being securely surgically fixed to the sclera in the scleral tunnel ; i.e. , a review of the video recording of the surgery confirmed that the encircling band had been placed under the medial rectus muscles during the scleral buckling surgery .
a 58-year - old man presented with a rhegmatogenous retinal detachment in his left eye .
pars plana vitrectomy with the placement of an encircling silicone band ( # 240 ; mira , inc . , waltham , ma . , usa )
the solid silicone band was placed under the four rectus muscles and surgically fixed in a scleral tunnel at all four quadrants ( fig 1 ) .
the scleral tunnels were prepared by making two vertical incisions of one - half of the scleral thickness , and then performing a lamellar dissection with a surgical blade .
the anterior extent of each tunnel was 2.02.5 mm posterior to the rectus muscle insertions .
one year later , the patient complained of a mild foreign body sensation in his left eye . upon examination ,
the encircling band was found to be exposed on the nasal side of conjunctiva next to the limbus ( fig 2 ) .
the patient did not report any diplopia , and the movement of the associated eye in the field of the medial rectus muscle was found to be normal .
two weeks later , the encircling silicone band was surgically removed without any complications . during that surgery
, it was noted that the encircling band had cheese - wired through the medial rectus muscle , which had become reattached close to its original insertion with no thinning . at the patient 's most recent follow - up ,
although it has been reported that a solid silicone band can cheese - wire forward through the rectus muscle postoperatively [ 3 , 4 ] , the underlying mechanism of that migration has yet to be elucidated .
it has been suggested that a migration of an encircling band may occur if the band is embedded too deeply , placed anterior to the equator , and not securely anchored to the sclera . in the case shown in this present study , the encircling band was fixed in the scleral tunnel at all four quadrants .
a previous study has shown that as with the use of sutures , the use of a scleral tunnel is an alternative and secure method of anchoring an encircling band . however , in the case shown in this present study , we theorize that the band migration possibly resulted from the scleral flaps being thin .
an alternative explanation as to why the band migrated is that it might have been placed over the rectus muscle .
however , a review of the video recording of the surgery confirmed that the encircling band had actually been placed beneath the muscle .
it has been reported that migrating bands can induce ocular motility disturbances [ 3 , 4 ] .
tan et al . reported a case with ocular motility disturbances in whom reattachment of the cheese - wired lateral rectus muscle was not observed . in the case shown in this present study ,
the cheese - wired medial rectus muscle was found to have become reattached close to its original attachment site .
we theorize that this was the reason why the ocular motility in our patient was preserved .
reportedly , the slow process of cheese - wiring migration through rectus muscle allows sufficient time for the muscle to reattach spontaneously behind the migrating band . in conclusion ,
the findings of this study show that migration of an encircling element can occur within 1-year after placement , and with subtle symptoms such as foreign body sensation . to prevent cheese - wiring migration , a scleral tunnel that is both deep and long enough to securely fix the silicone band
even when an encircling silicone band is surgically secured around the eye , periodic and detailed postoperative follow - up examinations and careful surgical procedures should be performed to ensure no migration of the band .
| the migration of an encircling silicone band through a rectus muscle is a rare postoperative complication associated with scleral buckling surgery for retinal detachment . in this present study
, we describe what we believe to be the first reported case of a patient who experienced postoperative migration of an encircling silicone band through the rectus muscle , despite the band being surgically secured to the sclera in a scleral tunnel . a 58-year - old man presented with a rhegmatogenous retinal detachment in his left eye .
pars plana vitrectomy was performed with the placement of an encircling silicone band , and the patient 's retina was successfully reattached .
one year postoperatively , the encircling band became exposed on the nasal side of the conjunctiva next to the limbus without any symptoms .
two weeks later , the exposed encircling band was surgically removed without any complications .
the findings of this study show that even when an encircling silicone band is surgically secured around the eye , periodic and careful postoperative follow - up examinations should be performed to ensure no migration of the band . |
the use of biomass as a source of energy used in heating systems , power and cogeneration plants or vehicle motors is forecast to grow considerably in the next decades ( nakicenovic and swart , 2000 ) .
bioenergy is promoted for several reasons , e.g. reducing the dependency on exhaustible fossil energy , greenhouse gas ( ghg ) emissions and import dependency as well as creation of jobs and income in rural areas .
however , in recent years the awareness has been growing that energy crop production could have far - reaching adverse consequences if natural and socioeconomic constraints are not respected .
examples include the discussion on possible impacts of bioenergy on food prices ( world bank , 2008 ) , ecological consequences of agricultural intensification ( tilman , 1999 ; tilman et al . , 2002 ) , deforestation and high ghg emissions from land - use change ( fargione et al
. , 2008 ; searchinger et al . , 2008 ) , soil degradation ( lal , 2006 ; van vuuren et al . , 2009 ) , water demand of energy crops ( gerbens - leenes et al . , 2009a ) and pressures on biodiversity ( gibbs et al . , 2008 ) .
some of these potential adverse impacts might even annihilate the environmental benefits of bioenergy ( tilman et al .
forging bioenergy strategies that maximize benefits and minimize adverse effects is a formidable challenge , aggravated by a limited understanding of the magnitude and geographic distribution of future potentials , and their large uncertainties ( haberl et al . , 2010 ) .
biomass currently contributes approximately 46 ej / yr of technical energy in the form of solid , liquid and gaseous fuels from primary ( harvest ) as well as secondary and tertiary ( waste and residue utilization ) sources ( iea , 2006 ) .
recent estimates of additional future bioenergy potentials range from approximately 30 to over 1000 ej / yr for 2050 ( beringer et al . , 2011 ; berndes et al . , 2003 ; campbell et al .
, 2008 ; field et al . , 2008 ; haberl et al . , 2010c ; haberl et al . ,
, 2003 ; hoogwijk , 2004 ; smeets et al . , 2007 ; van vuuren et al . , 2009 )
. the enormous differences between these studies largely result from differing assumptions regarding the large number of factors that influence the availability of biomass for energy and material use , such as the availability and suitability of land , achievable yield levels of energy crops , technological progress in agriculture and livestock breeding , and on the impact of competing land uses such as the demand for food and feed , biodiversity and forest conservation and soil protection . in this context , we here study the following factors that influence global primary energy crop potentials in 2050 .
first , we analyze possible constraints on the area available to grow energy crops resulting from ( 1 ) future changes in food systems , i.e. food demand ( diets ) , food and feed crop yields , feed demand of livestock ( fodder crops and roughage ) depending on livestock feeding efficiency , ( 2 ) political factors , i.e. sufficient stability of nations to allow investments required for establishing bioenergy plantations ( exclusion of failed states ) and ( 3 ) competing area demands for biodiversity conservation ( e.g. nature reserves ) .
our study is based on the conservative assumption that additional deforestation is excluded for both food and energy crop production in order to avoid long carbon payback times from direct and indirect land - use change ( fargione et al . , 2008 ; searchinger et al . , 2008 ;
second , we evaluate the influence of energy crop yields on future bioenergy potentials by assuming different plausible levels of energy crop yields .
bioenergy potentials from forestry as well as from agricultural residues , manures and other wastes , which are substantial ( haberl et al . ,
food first approach , i.e. only areas not required to feed the world population forecast to exist in 2050 according to the specified diet are assumed to be available for bioenergy .
we use a simple and transparent biophysical biomass - balance model ( erb et al . , 2009a ; haberl et al . , 2010
; haberl et al . , 2012a ) that is based on material flow accounting ( mfa ) principles and large , consistent land - use socioeconomic and ecological biomass - flow databases ( including net primary production ; erb et al .
we aim at analyzing the effect of various constraints on future primary bioenergy potentials , but it is neither our objective to forecast bioenergy supply in 2050 , nor to assess its social implications , e.g. economic and social effects of land competition ( e.g. land rents , food and bioenergy prices or expulsion of peasants ) .
thus , we here aim at exploring the biophysical option space for primary bioenergy production in 2050 , using food - first and zero - deforestation assumptions as framework condition of our analysis .
we do not aim at assessing economic bioenergy potentials , e.g. modeling energy demand and supply in dependency of price developments , nor technical bioenergy potentials , i.e. maximum potentials related to technological capabilities .
the biomass - balance model is based on the thermodynamic principles underlying material and energy flow accounting ( mefa ) methods , basically the conservation of mass and energy and the entropy law ( ayres and simonis , 1994 ; fischer - kowalski , 1998 ; fischer - kowalski and httler , 1998 ; haberl , 2001 ; haberl et al . ,
the model calculates the global primary energy crop potential based on assumptions on diets , agricultural technology and cropland area assumed for the year 2050 ( fig .
the model was constructed using a comprehensive land use , npp and biomass - flow database for the year 2000 ( erb et al .
the database integrates global land use and socioeconomic data with npp data across a range of spatial scales , from the grid level ( 5 min resolution , 1010 km at the equator ) to the country level ( 160 countries ) .
it fulfills multiple consistency criteria across scales and domains : biomass flows are traced from the net primary production ( npp ) of each of the five land - use classes ( built - up and infrastructure , cropland , forestry , unused and unproductive , all other land ) to national - level data on primary and final biomass consumption .
the database was used to derive factors and multipliers to match the demand for final products of biomass ( food , fibers ) with gross agricultural production and land use for eleven world regions ( for reference see haberl et al . , 2012a and http://www.uni-klu.ac.at/socec/downloads/wp116_web.pdf ) .
that is , in a first step , the model was calibrated for the year 2000 using the above - described databases . in a second step ,
several critical factors and input parameters were modified in order to reflect assumed changes from 2000 to 2050 ( see below ) .
the biomass - balance model uses a spatially explicit land - use dataset ( erb et al .
, 2007 ; see table 1 ) that distinguishes five land - use classes ( 1 ) infrastructure and urban areas , ( 2 ) cropland , ( 3 ) forestry , ( 4 ) unused areas ( wilderness , including unproductive areas ) , and ( 5 ) all other land ( denoted as grazing land in the original paper ) at a resolution of 5 arc minutes ( approximately 10 per 10 km at the equator ) . the area of class 5 all other land was calculated by subtracting from each gridcell 's total area the area covered by all other four land - use classes .
forests were either subsumed in class 3 ( forestry ) or class 4 ( unused land ) , depending on presence or absence of human artefacts as identified in the underlying
the land - use dataset was extensively cross - checked against independent datasets ( erb et al . , 2007 ) .
land - use class 5 includes only non - forested land used by humans , ranging from high quality meadows and grazing land to shrub - dominated or otherwise marginally productive areas that allow only extensive grazing , including also savannahs , abandoned farmland , as well as degraded and extensively used areas and semi - deserts .
furthermore , this land use class is classified according to its suitability for livestock grazing , discerning four quality classes , from 1 ( best ) to 4 ( poorest ) , based on land - cover information from the glc-2000 map ( mayaux et al . , 2006 ) and npp data ( for reference see erb et al . , 2007 ) .
wilderness , occupies approximately one quarter of the global ice - free land surface ( i.e. excluding greenland and antarctica ) but holds little promise for energy crops because most of this land is extremely unproductive ( e.g. , arctic or alpine tundra or extreme deserts ) .
wilderness class also includes pristine forests which were excluded from the here presented analysis due to very long carbon payback times associated with their use and biodiversity conservation considerations .
areas potentially available in the year 2050 for growing energy crops are estimated as the sum of ( a ) cropland not required for food , feed and fiber production and infrastructure and ( b ) land potentials existing in the all other land category .
infrastructure and urban areas in the year 2050 ( table 1 ) were extrapolated from values for 2000 based on the medium un population forecast and urbanization trends ( un , 2007 ) . with respect to
the all other land category , area can only be assumed to be available for energy crops if roughage demand for livestock is satisfied , according to the food first principle ( see fig .
1 ) . in order to estimate the area that could be made available for energy crops in 2050 on such lands , depending on roughage demand
, we assumed that all other land of the highest grazing suitability ( grazing class 1 ) could be intensified to an extent currently observed in countries with high grazing intensity , i.e. a harvest level of up to 67% ( developing countries ) and 75% ( industrialized countries ) of aboveground npp .
we then assumed that the area set free through this assumed intensification would be available for energy crops in 2050 ( additional constraints
the trend scenario for 2050 was derived based on the un medium population forecast ( un , 2007 ) and fao agricultural projections .
the fao study world agriculture towards 2030/2050 , which was used here , assumes a global growth in cropland area of 9% ( table 1 ) and a growth of cropland yields of 54% ( weighted average of all crops and regions ) from the year 2000 to 2050 ( bruinsma , 2003 ; fao , 2006 ) .
the trend diet ( table 2 ) was constructed by assuming that each region would attain the food supply enjoyed in the country within each region that currently has the
this procedure yielded results that were very similar to the fao forecast ( fao , 2006 ) , despite the different methodology ( erb et al . , 2009a ) .
2 ) were extrapolated to 2050 separately for grazers ( cattle , sheep , goats , etc . ) and monogastric species ( pigs , poultry , etc . ) based on region - specific trends derived from fao statistics for the year 19612000 ( erb et al . , 2009a ; krausmann et al . , 2008
this estimates were complemented by a second variant in which lower feeding efficiencies , i.e. those currently achieved in organic farming , were assumed to prevail in 2050 ( see erb et al .
in addition to the cropland scenario displayed in table 1 we also considered a massive cropland expansion variant in which fao assumptions on cropland expansion in each region were doubled ( and held constant in regions where the fao forecasts a declining cropland area ) . this variant assumes a global growth of cropland area of 19% until 2050 .
this massive cropland expansion variant is still lower than the cropland expansion assumed to occur in some other global scenario studies ( iaastd , 2009 ) .
the largest expansion of cropland areas is assumed to occur in sub - saharan africa and latin america , as these are the regions generally thought to have the largest cropland potentials ( iiasa and fao , 2000 ; ramankutty et al . , 2002 ) .
it was assumed that the additional cropland has a 10% lower productivity than the cropland of the trend expansion because it would be located on less productive lands .
we assumed that the area expansion of cropland and infrastructure would consume the areas with the highest suitability for grazing in the other land class .
this assumption seems justified as regions with much land best suited for grazing according to our land - use dataset are also those in which gaez ( iiasa and fao , 2000 ) and other studies ( ramankutty et al . ,
in contrast , regions with little cropland expansion potential according to iiasa and fao ( 2000 ) and ramankutty et al .
( 2002 ) also have small areas of high - quality grazing land according to the erb et al .
land in grazing class 1 was found to be large enough to account for this change with the exception of the region
north africa and western sahara. in this region , cropland and infrastructure area expansion is assumed to consume land of grazing quality class 2 as well . reduced roughage production in 2050 as a result of the consumption of grazing areas by cropland was explicitly accounted for in the biomass - balance model in the respective regions . grazing land expansion through forest clearance was not considered . despite this restrictive assumption ,
grazing area was not found to limit roughage supply in any of the scenarios . in order to assess the possible effect of changes in diet on the area available for energy crops , three alternative diet variants were defined ( table 2 ) . in the
rich variant we assumed global convergence towards diets currently enjoyed in western europe and the usa , although these levels are not fully reached in 2050 . in the
less meat variant we assumed the same level of calorie and protein supply as in the trend scenario but with a lower level of animal - based products ( milk , meat and eggs ) .
the fair and frugal variant results in a convergence towards a global daily per - capita calorie intake of 2 800 kcal / cap / day ( 11.72 mj / cap / d ) , paired with a relatively low level of animal product supply .
this latter diet is nutritionally sufficient ( also in terms of protein supply , which is assumed to be based to a large extent on pulses and other protein - rich vegetables ) but requires globally equal distribution of food in order to avoid malnutrition in parts of the global population .
conversely , even at the highest supply level malnutrition can not be entirely ruled out if inequality in per - capita supply is assumed to remain at its current level ( erb et al . , 2009a ) .
in order to reflect changes in cropland yields , we defined three alternative variants regarding future cropland yields ( table 3 ) . the maximum yield level ( max )
was defined based on the global orchestration scenario in mea ( millennium ecosystem assessment , 2005 ) which assumes yields that exceed the level forecast by the fao in 2050 by 9% .
a minimum yield level was defined by calculating crop yields in 2050 under wholly organic conditions ( org ) , i.e. by assuming that 100% of the cropland area would be cultivated according to standards of organic agriculture . according to a literature review ( erb et al .
, 2009a ) , yields in organic agriculture are approximately 40% lower than those of industrialized agriculture if calculated over the whole crop rotation cycle , i.e. taking fallow cycles or the cultivation of n - fixating leguminous plants into account ( halberg et al . , 2006 ; connor , 2008 ; von fragstein und niemsdorff and kristiansen , 2006 ) .
this yield reduction was only applied to high - input cropland systems that mainly prevail in industrialized countries , and not to low - input agriculture . in developing countries ,
organic agriculture would allow further yield growth because the nutrient status of croplands is often very poor and can be improved with organic techniques ( halberg et al .
thus , the deviation of the organic yield scenario from the baseline was based on the regional mix between high - input and low - input agriculture ( table 3 ) .
the intermediate ( int ) yield scenario was calculated as the arithmetic mean between fao and org .
it may be interpreted as a scenario in which cropland agro - ecosystems are not pushed to their very limits due to environmental considerations , or as a trajectory in which yield levels forecast by the fao are not achieved for economic ( lack of investment ) or biophysical ( physiological limits , soil degradation , etc . ) reasons .
the biomass - balance model was used to determine the feasibility of combinations of assumptions on diets ( 4 variants ) , food and fodder crop yields ( 4 variants ) , cropland expansion ( 2 variants ) and livestock feeding efficiency ( 2 variants ) .
scenarios in which global cropland area demand exceeded global cropland availability by more than 5% were labeled as non - feasible and not further considered .
we considered a difference of supply and demand smaller than 5% not to be significant given the uncertainties in the biomass balance model .
the matching between supply and demand was performed on the global level ; regional imbalances were assumed to be equalized by trade .
roughage supply was not found to be a limiting factor , as a consequence of the large extent of global grazing areas ( erb et al . , 2007 ) and low average grazing intensities in most regions .
feasible we calculated the area available for energy crops as the sum of ( a ) cropland area not required for food , feed and fiber and ( b ) area available for energy crops on all other land while covering roughage supply for livestock on smaller , but more intensively used areas ( see above ) .
feasible scenarios as follows . in the trend scenario we assumed that aboveground biomass yields of energy crops are related to natural productivity ; that is , net primary production or npp ( field et al . , 2008 ; campbell et al . ,
as we were interested only in primary energy potentials , we did not differentiate between energy crop plants or bioenergy utilization pathways ( first / second generation ) . on croplands , we assumed biomass yields equal to the aboveground npp of potential vegetation ( i.e. the vegetation assumed to exist in the absence of land use ) . on all other land areas we used data on the aboveground npp of the currently prevailing vegetation .
all npp data were taken from prior work ( haberl et al . , 2007 ) .
( 2008 ) , who used the same approach , argued that this assumption may be regarded as optimistic because cropland npp is currently 35% lower than the potential npp of the respective areas .
this assumption implies that energy crops will achieve substantially higher yields in 2050 than those currently achieved by food crops in the global average , compared to the natural potential of the areas on which they are grown .
first generation biofuels are based on food crops , therefore our energy crop yield assumptions may be regarded as optimistic for most first - generation crops with the exception of sugarcane ( wbgu , 2009 ) .
low yield variant based on the conservative assumption that energy crop yields in 2050 would only achieve a level of 65% of the potential aboveground npp of the area on which they are planted .
however , much previous work on energy crop potentials was based on much higher yield expectations .
( 2009 ) assumed second generation energy crop yields of 13 kg dry matter per m and year which is approximately 1.54.5 times larger than the potential aboveground npp of the cropped area .
calculations with the dynamic global vegetation model lpjml suggest that the biological productivity of second generation biofuel crops such as c4 grasses and short - rotation coppice ranges from 0.8 to 1.2 kg / m / yr of dry matter biomass , i.e. slightly higher than potential npp , largely depending on the level of irrigation ( wbgu , 2009 ; beringer et al . , 2011 ) .
in line with a recent study ( johnston et al . , 2009 ) suggesting that yield assumptions for first generation biofuels had been largely overestimated , we did not assume high energy crop yield estimates as implemented in image ( van vuuren et al . , 2009 ) but rather those resulting from lpml ( beringer et al .
, 2011 ; wbgu , 2009 ) by assuming a high yield variant in which energy crops yields were 1.3 times higher than potential aboveground npp .
we assumed that dry - matter biomass has a gross calorific value of 18.5 mj / kg to derive primary energy supply potentials from the biomass flow data .
the conversion from primary biomass to final or useful energy can entail significant losses , in particular for first generation bioenergy , depending on the respective technology ( campbell et al .
, 2008 ; field et al . , 2008 ; wbgu , 2009 ) , which are not deducted here .
we also do not deduct direct or indirect energy inputs ; e.g. energy used for cultivation , fertilizer production or processing in other words , we report gross biomass production potentials of energy crops . in order to consider potential constraints from biodiversity conservation , we followed an approach used in recent studies ( beringer et al . , 2011 ; brooks et al . , 2006 ; wbgu , 2009 ) based on ( a ) biodiversity hotspots , ( b ) wilderness areas and ( c ) protected areas . for ( a )
biodiversity , we used data on biodiversity hotspots ( myers et al . , 2000 ) , endemic bird areas ( stattersfeld et al . , 1998 ) , centers of plant diversity ( wwf and iucn , 1994 ) and the global 200 dataset ( olson and dinerstein , 2002 ) . in order to consider ( b ) wilderness areas , we used biodiversity wilderness areas ( mittermeier et al . , 2003 ) , frontier forests ( bryant et al . , 1997 ) and the last of the wild wilderness area map ( sanderson et al . , 2002 ) .
in addition , we ( c ) superimposed the protected areas with iucn levels i and ii provided by the world database on protected areas ( unep - wcmc , 2010 ) .
we used the number of conservation data sets for a given area as a proxy for calculating a per - cent reduction in the area availability for energy crops .
less restrictive , variant 100% of the area of each pixel was excluded only if more than one wilderness layer was present , 50% of the area if two biodiversity hotspots were present , and 80% if there were three or more .
each of these rules was applied separately , and the most restrictive result was implemented . for the second , more restrictive , variant we decreased the area available for energy crops by 100% if a pixel was classified as
all land areas , regardless of the presence of any biodiversity hotspot or protected area , suffer a 10% decrease in available energy crop area . with the presence of one ( two , three , four ) biodiversity hotspot(s ) , the share of area assumed to be available for energy crops was reduced by 20% ( 30% , 50% , 80% ) .
again , the three calculations were performed separately and the most restrictive result was used . in order to exclude areas
where investment in energy crop plantations is highly unlikely due to lack of stable legal and political framework conditions , we excluded failed states ( newman , 2009 ) based on the list published by the fund for peace for the year 2010 ( www.fundforpeace.org )
. this list ranks 177 countries by analyzing 12 primary social , economic and political indicators , each of which split into a set of sub - indicators , which are summed up to a failed - state index .
the 30 countries with the highest index were treated as areas without any energy crop potential due to lack of stability required for investments into energy crop plantations .
in the trend scenario which assumes cropland expansion and yields as forecast by the fao , livestock efficiencies as well as diets in line with the fao projections and ignores constraints from biodiversity conservation and unstable political framework conditions , we find a global primary energy crop potential of 77 ej / yr .
this result is based on the trend variant of energy crop yields ( the notion of
scenario here stands for a unique combination of variants , i.e. assumptions on the different factors discussed in the
3 shows a map displaying the spatial distribution of the bioenergy production in this scenario which underlines that a considerable proportion of the total potential is located in sub - saharan africa and latin america .
4 displays the bioenergy potential in the 43 ( of a total of 64 ) scenarios which were classified as
feasible , showing a large range from 26 to 141 ej / yr from dedicated energy crops .
4 vary according to changes in diets , cropland expansion , food and feed crop yields and feed conversion efficiencies of livestock as discussed above .
they do not consider constraints from biodiversity conservation or lacking political stability and are based on the trend assumption on energy crop yields .
4a ) : richer diets result in lower energy crop potentials , while more frugal diets , especially those with less animal products , leave more space to plant energy crops .
the interrelation between food crop yields and energy crop potentials is less straightforward , however . while there is a general tendency that energy crop potentials increase with food crop yields ( fig .
4b ) , variability between the highest and lowest values also increases the highest and lowest scenario in terms of energy crop potential are both based on the max yield variant .
5a shows how constraints resulting from political instability ( failed states ) and exclusion of areas for biodiversity conservation affect the energy crop potential in 2050 .
these calculations were all based on the trend scenario with respect to food system and energy crop yields .
the exclusion of failed states reduces the global energy crop potential by 18% ; a large part of this reduction results from exclusion of areas in sub - saharan africa .
the exclusion of areas for biodiversity conservation reduces the global bioenergy potential by 932% , depending on the ambition of conservation efforts .
the combined effect of constraints from conservation and political stability reduces the global energy crop potential by 2545% .
the low assumption a relation between energy crop yields and the npp of potential vegetation on the areas planted with energy crops equal to that currently found on the world 's average croplands reduces the energy crop potential in 2050 by 26% compared to the trend scenario , while high energy crop yields could boost the potential by up to 48% to 114 ej / yr . under such optimistic assumptions , yield growth could approximately compensate the losses resulting from exclusion of areas in failed states and high - biodiversity areas .
in interpreting the results , it is important to bear in mind that we here calculated primary biomass supply potentials .
some conversion pathways , e.g. production of liquid biofuels , can only use parts of the plants and entail considerable conversion losses , e.g. in liquefaction .
moreover , biomass production as well as conversion to fuels requires energy inputs ( pimentel et al . , 1973 ; wbgu , 2009 ) .
hence , the net energy ( cleveland , 2007 ) delivered to society is considerably lower than the primary energy crop potentials calculated here ( e.g. , blottnitz and curran , 2007 ; cleveland et al .
the optimization of biomass utilization pathways in order to maximize the amount of useful energy delivered per unit of primary biomass harvested is beyond the scope of this paper , but is of course highly important in designing sustainable bioenergy strategies ( e.g. , see haberl and geissler , 2000 ; wbgu , 2009 ) .
the primary energy crop potentials calculated in this study on basis of a biophysical analysis , is 77 ej / yr in the trend scenario , with wide ranges resulting from different assumptions on the food and agricultural system , political stability required for investment security , and biodiversity conservation , are similar to those identified with the dynamic global vegetation model lpjml ( beringer et al . , 2011 ; wbgu , 2009 ) , higher than those assumed to exist on abandoned land , i.e. 2741 ej / yr ( campbell et al . , 2008 ; field et al . , 2008 ) , and considerably lower those identified in earlier studies ( hoogwijk et al . , 2003 ; hoogwijk , 2004 ; smeets et al . , 2007 ) .
our result is similar to a recent model - based assessment ( popp et al .
2011a ) which reported a global economic bioenergy potential in 2050 of approximately 70 ej / yr if deforestation was excluded ( 100 ej / yr if not ) based on a combination of a plant growth model ( lpjml ) with a land allocation model ( magpie ) and an energy model ( remind ) .
another study of global bioenergy trajectories under different carbon tax systems using the minicam integrated assessment model ( wise et al . , 2009 ) found that bioenergy supply might grow to over 100 ej / yr if only fossil - fuel based c emissions were limited , which would , however , trigger considerable deforestation . when all c emissions were limited , including those from land - use change , bioenergy supply in 2050 was much lower ( below 40 ej / yr ) .
assumptions on the energy crop yields that can be achieved in 2050 are among the most important reasons for the discrepancies in estimates of global bioenergy potentials .
previous modeling studies may have been overly optimistic in their assumptions on future energy crop yields .
( 2007 ) suggested that in 2050 up to 1272 ej / yr of bioenergy could be produced from energy crops grown on up to 36 million km ( 27% of the global land surface excluding antarctica and greenland ) .
however , this result was based on a yield assumption that was 3.8 times higher than current npp of these areas ( haberl et al . ,
2010 ) as discussed above , yield assumptions in past studies of energy crop potentials may have been too high ( johnston et al . ,
2009 ) , although vegetation models suggest that high - yielding energy crops such as c4 grasses or short - rotation coppice can achieve yields that exceed natural productivity by perhaps 2040% ( beringer et al . , 2011 ; wbgu , 2009 ) .
water supply may become an issue here , as high - yields can only be achieved if sufficient water is available ( fader et al .
, 2010 ; gerbens - leenes et al . , 2009b ; rost et al . , 2008 ) .
on the other hand , while it may be technically possible to achieve high yield levels or improve livestock feeding efficiencies to an extent that would allow to plant more area with energy crops than assumed here , the environmental and economic costs of such strategies could be substantial and need to be considered .
our results suggest that the future global energy crop potential strongly depends on changes in the food system .
assumptions on diet have a strong effect on the area demand for food production ( stehfest et al . , 2009 ) , and thus on the energy crop potential .
rich diet leaves little space for bioenergy plantations , irrespective of food and feed crop yields , while the fair & frugal diet allows for large bioenergy crop potentials .
as one moves from rich to poorer diets , the range between the lowest and highest potential also increases ( fig .
this is a result of the fact that the fair & frugal diet leaves a lot of area available for bioenergy if the most intensive technologies with respect to food and feed crop yields and feeding efficiency are adopted .
however , while such a combination would allow a high level of energy crop production , it may also be perceived as being particularly unlikely . in the case of
the fair & frugal diet , substantial energy crop potentials exist even if organic yields and low feeding efficiency are assumed .
changes in the assumptions on food and feed crop yields also have a substantial effect on the energy crop potential : higher yield levels leave more space for energy crop plantations if everything else remains constant . however , it does not seem likely that future diets are independent on food crop yields and vice versa .
higher yields are likely to be linked with richer diets , for example because food consumption and agricultural technology are both linked to affluence : rich societies can afford to eat better , but they also acquire the means to increase their agricultural yields ( godfray et al .
4b ) , the lowest bioenergy potential estimate found in any of the scenarios was based on high food and feed crop yields .
the reason is that the rich diet can only be provided with high yields .
this diet variant combined with lower yields were all classified as not feasible and consequently excluded .
a rich diet leaves little space for energy crop plantations due to the high roughage demand for livestock , irrespective of yield levels of food crops . in other words ,
pushing yields to their limits also allows richer diets , which then in turn may reduce the energy crop potential if people adopt diets that include more animal products .
it is beyond the scope of this article to assess whether , and under which conditions , it is more favorable in terms of ghg emissions / uptake to use the area that becomes available due to high yields and/or more vegetarian diets for bioenergy provision instead of using it for c sequestration ( stehfest et al . , 2009 ;
our study , however , reveals that this is not the only trade - off related to bioenergy production that requires systematic attention .
our analysis clearly illustrates trade - offs between maximizing energy crop supply and adopting environmentally less burdensome agricultural technologies . at present , agricultural intensification results in higher yields but is often associated with substantial environmental pressures resulting from fertilizers , pesticides , machinery use , etc
. impacts of intensive agriculture include soil degradation , nitrogen leaching , risks from pesticides , among others ( iaastd , 2009 ; popp et al . , 2011b ; smeets et al . , 2009 )
everything else being equal , lower food and feed crop yields leave less space for energy crop plantations , and lower energy crop yields also result in a lower bioenergy potential .
many technologies proposed to reduce environmental pressures from agriculture , e.g. organic farming , tend to reduce yields , which mean that more area is needed to produce the same amount of food . to what extent these trade - offs can be mitigated through
sustainable intensification ( godfray et al . , 2010a ; tilman , 1999 ) remains to be seen .
an additional trade - off exists between biodiversity conservation and maximization of energy crop production .
our results suggest that the exclusion of wilderness areas , protected areas and biodiversity hotspots could lower the global energy crop potential by 932% , depending on the ambition of conservation efforts .
this calculation does not include the possibility that conservation targets could also constrain food crop production , which might indirectly result in additional restrictions for planting more energy crops . a focus on biomass residues and wastes instead of purpose - grown bioenergy plants would allow to tap into a substantial energy potential of up to 100 ej / yr ( haberl et al . , 2010 ) and would deliver energy without increased pressures on ecosystems resulting from additional land demand .
however , use of some residues may pose a risk for the maintenance of soil fertility ( blanco - canqui and lal , 2009 ; lal , 2006 ) , hence secure upper limits to extraction of agricultural residues need to be respected .
where these limits are depends on soil characteristics , climate and many other region - specific factors ; this is an area where more research is needed .
about one quarter of the global energy crop potential calculated for 2050 in the trend scenario is located in countries currently classified as failed states. no projection is available to what extent lacking political stability might affect global energy crop potentials in 40 years , so the share of the global potential that is inaccessible due to political constraints is at present unknown .
however , our calculations support the conclusions of previous assessments ( beringer et al . , 2011 ; wbgu , 2009 ) that such constraints might be substantial .
the design of sustainable bioenergy crop production policies needs to resolve intricate trade - offs between food and energy supply , renewable energy and biodiversity conservation or yield growth and reduction of environmental pressures from intensive agriculture .
our assessment indicates that policy strategies are needed that succeed in simultaneously optimizing production and consumption systems and considering the many potential conflicting uses of biomass , land and water .
increasing crop yields seems to be essential , given the growing world population , the growing energy demand in the light of global environmental changes .
but such strategies will only be successful if not overcompensated by consumption surges and if sustainable methods for intensification can be developed .
our analysis also reveals that successful policies will have to optimize the increasingly globalized , i.e. spatially separated , land use and biomass utilization chains in integrative ways ( erb et al .
in this context , our analysis reveals one particular intricacy : whereas the largest bioenergy demands are expected in regions where energy potentials are low such as europe and parts of asia , the largest energy potentials are found in regions such as sub - saharan africa .
expanding agricultural land use in these regions will require massive investments , which might not be affordable for local governments or land users and are not attractive for international investors if political stability is low .
moreover , sensitive legislation will be required to avoid land - use changes with negative social effects , e.g. see the current land grab discussions ( friis and reenberg , 2010 ; zoomers , 2010 ) . by necessity , growing energy crops requires land .
if substantial amounts of bioenergy should be derived from purpose - grown energy crops , the land area required needs to be significant as well .
the gross energy content of all biomass harvested by humans globally for food , feed , fiber and bioenergy was approximately 230 ej / yr in the year 2000 ( krausmann et al . , 2008 ) .
this biomass was harvested on almost three quarters of the global land surface ( excluding antarctica and greenland ) through cropping , grazing and forestry ( erb et al .
ej / yr of energy crops in 2050 , in addition to the growth of biomass required to adequately feed a world population of perhaps 9.1 billion , is therefore an endeavor that should not be underestimated .
| the future bioenergy crop potential depends on ( 1 ) changes in the food system ( food demand , agricultural technology ) , ( 2 ) political stability and investment security , ( 3 ) biodiversity conservation , ( 4 ) avoidance of long carbon payback times from deforestation , and ( 5 ) energy crop yields . using a biophysical biomass - balance model ,
we analyze how these factors affect global primary bioenergy potentials in 2050 .
the model calculates biomass supply and demand balances for eleven world regions , eleven food categories , seven food crop types and two livestock categories , integrating agricultural forecasts and scenarios with a consistent global land use and npp database .
the trend scenario results in a global primary bioenergy potential of 77 ej / yr , alternative assumptions on food - system changes result in a range of 26141 ej / yr . exclusion of areas for biodiversity conservation and inaccessible land in failed states reduces the bioenergy potential by up to 45% .
optimistic assumptions on future energy crop yields increase the potential by up to 48% , while pessimistic assumptions lower the potential by 26% .
we conclude that the design of sustainable bioenergy crop production policies needs to resolve difficult trade - offs such as food vs. energy supply , renewable energy vs. biodiversity conservation or yield growth vs. reduction of environmental problems of intensive agriculture . |
only 10 cases without predisposing factors such as arthropathy or infectious joint alterations have been described until now.[17 ] it is potentially serious injury that can be associated with concurrent injuries to ribs , lungs , spine , great vessels , or myocardium .
only a few case of patients treated surgically are reported in the literature . in this study
a 49-year - old woman was referred to our department after a frontal car crash .
she had a blood pressure of 120/80mmhg , a pulse rate of 80 beats / min , a respiratory rate of 20 breaths / min , and o2 saturation at 100% .
careful palpation revealed a painful and important deformity which is projected to the manubriosternal joint .
lateral chest radiograph showed a type i manubriosternal joint dislocation : posterior displacement of the sternal body in relation to the manubrium [ figure 1 ] , the mechanism of which was a direct impact to the sternum at the scoreboard ( front passenger without seat belt , no airbag ) .
chest x - ray : posterior manubriosternal dislocation after 24 h of observation , the treatment was performed under general anesthesia in a supine position .
after removal of hematoma , we found the sternal body dislocated posterior to the manubrium [ figure 2 ] .
after debridement of the joint and liberation of the intact , but stripped anterior periost , the sternum was reduced to the manubrium using a reduction clamp .
the body was stabilized to the manubrium using two blount staples [ figures 3 and 4 ] .
operative view showing a posterior manubriosternal dislocation reduction and stabilization with staples postoperative x - ray showing the double staples stabilization postoperative recovery was uneventful , and the patient was discharged on the third postoperative day .
four months after the initial treatment , the outcome was satisfactory , the patient is asymptomatic and the manubriosternal joint is well aligned and stable .
the manubriosternal joint may be synovial , synchondrotic , or synostotic junction and because of the powerful ligamentous attachments in this region , dislocation of the manubriosternal joint is very much a rarity .
dislocation is most common with the synovial type of joint , whereas synchondral and synosteal types typically fracture through the manubrium rather than dislocate at the joint .
manubriosternal dislocation is an uncommon chest injury , typically resulting from a high - energy trauma .
however , dislocation can be caused by a minor trauma among patients with preexisting manubriosternal arthropathy ( most commonly , rheumatoid arthritis and extreme forms of kyphosis ) .
only 10 cases without predisposing factors such as arthropathy or infectious joint alterations have been described until now , we represent the 11 case in the literature .
thirupathi and husted divided manubriosternal dislocations into two types : type 1 , in which the sternum is dislocated posteriorly with respect to the manubrium , and type 2 , in which the manubrium is dislocated posteriorly with respect to the sternum .
generally , type 1 injuries are caused by a direct impact to the body of the sternum .
it is possible to have a type 1 dislocation in the absence of any direct sternal trauma .
type 2 injuries are due to hyperflexion of the upper thoracic spine that transmits a downward and posterior force to the manubrium via the first ribs . on physical examination , manubriosternal dislocation
detecting this dislocation on frontal chest radiography is difficult , but lateral chest radiographs can confirm the diagnosis .
ct scan or mri readily demonstrated the lesion and shows any associated mediastinal injury , which can include potentially life - threatening injuries to the aorta , internal mammary artery , trachea , and esophagus .
there are a few cases of manubriosternal dislocation in the literature , making it difficult to establish the incidence and the type of associated mediastinal injuries .
treatment of a manubriosternal dislocation depends on the stability of the injury and the presence of associated injuries .
unstable injuries and those with an associated mediastinal injury may require open reduction and fixation . if untreated correctly , these disorders can lead to chronic pain , periarticular calcification with ankylosis , and progressive deformity . published literature on surgical repair of manubriosternal dislocation
is sparse and unfamiliar to most surgeons . it has not been possible to establish an optimal , standardized operative procedure so far because of the small number of reported cases
however , different methods were used for the fixation of the manubriosternal joint : kirchner wires , plates , suture with pds ropes or braided polyester thread , all of which have been reported to be successful .
we report an alternative technique which consists in simple stabilization with staples . in our opinion
, this material is advantageous because it is more stable than sternal wires , and does not break as easily while being tied .
finally , ulceration of the skin with this method does not occur , as it may be observed with sternal wires .
we are the first to use a stapling technique ; it allows us to make quick and efficient surgical gesture with excellent stability of the manubriosternal joint .
we strongly believe that this method is safe and reliable , and favors immediate physiotherapy . in our case ,
operative treatment can result in an early relief of complaints and return to a functional level . | traumatic manubriosternal dislocation is a rare lesion . in the literature ,
only a few case reports of patients treated surgically are published . in this case , we report an unstable posterior dislocation of the manubriosternal joint in a 50-year - old women caused by direct trauma .
an open reduction was performed , and the manubriosternal joint was fixed by two staples .
we are the first to use this alternative technique . at the last follow - up , the result was very good without complication . |
a 15-day - old female neonate patient was brought with history of a sudden onset proptosis of the left eye for three days .
ocular examination revealed an axial proptosis of the left eye with limited ocular movements in all directions [ fig . 1 ] .
systemic examination revealed no abnormalities except the presence of a tooth bud abscess in the left maxillary alveolar ridge with overlying facial swelling [ fig . 2 ] .
left eye proptosis with orbital cellulitis showing tooth bud with abscess in left alveolar maxillary area hematological investigations showed the following results : hemoglobin- 14.5 gm% , pcv- 57.4% , total leucocyte count- 17,000/mm , neutrophils- 52% , lymphocytes- 48% , platelet count- 483,000/mm , urea- 28 mg / dl , creatinine- 0.8 mg / dl .
microbiological investigation of both conjunctival and oral swabs grew staph aureus that was sensitive to amikacin , vancomycin , ofloxacin , gentamicin , cefotaxime , co - trimoxazole and resistant to penicillin and cefalexin .
ct scan of the head and orbit showed a dense soft tissue lesion of size 2.5 1.2 cm in the extraconal space of the left orbit and pre - septal region causing anterolateral displacement of the globe .
soft tissue density lesion of size 25 12 mm in extraconal space in the left orbit and pre - septal region causing anterolateral displacement of globe .
features suggestive of the left eye orbital cellulitis with pockets of abscess formation the neonate was admitted and administered intravenous injections of vancomycin of 60 mg / kg / per day in two divided doses with amikacin of 20 mg / kg three times daily and oral metronidazole of 25 mg / kg / three times daily for 14 days .
the tooth bud abscess improved with this conservative management . within 24 hours of the initiating treatment
the patient was discharged after two weeks and prescribed augmentin syrup for another two weeks . at one month of follow - up , the condition of the neonate was much improved with no proptosis and full extra - ocular movements .
4a and b ] follow - up at one month ( a ) decrease in proptosis ( b ) neonatal tooth with no abscess
orbital cellulitis is a rare , but potentially lethal condition in children that has been occasionally reported in infants . at maximum ,
orbital cellulitis due to dental infection has been reported in children and adults , but only one case has been reported in a neonate .
laura et al . , reported a 24-day - old neonate with orbital cellulitis accompanied by a sub - periosteal abscess of the dental origin .
dolter et al . , reported a case of orbital cellulites in a one - month - old secondary to ethmoidal sinusitis .
harris reported a case of orbital cellulitis in a one - week - old secondary to septic thrombophlebitis from an intravenous line in a scalp vein .
, reported the same in a nine - day - old neonate with septic arthritis of the left ankle joint and ethmoidal sinusitis .
most of the previously reported cases presented with systemic features that were absent in our case .
the patient could have acquired the infection through a contaminated nipple during the breastfeeding , which then spread to a labial ulcer or prominent tooth bud on the left side .
the infection could have then tracked subperiosteally and entered the soft tissue , causing orbital cellulitis with abscess formation .
the management of orbital cellulitis in neonate and infants is challenging and must be treated urgently so as to prevent more serious complications . in the presence of a dental abscess , the treatment with medication alone is usually insufficient .
it is necessary to remove the source of infection , which can be achieved through abscess drainage .
antibiotic treatment of orbital cellulitis should include broad spectrum coverage against both aerobic and anerobic organisms , including oral pathogens . in this case ,
however , the patient showed significant improvement with medical management alone within 24 hours of initiating antibiotic treatment .
while conservative treatment was used in this case , harris protocol calls for emergent drainage of the abscess if vision is compromised and urgent drainage ( within 24 hours ) of large abscesses causing pain , superior or inferior extension , intracranial infection , or those of anaerobic or dental origin . in the case
reported by laura et al . , the condition of the baby worsened and proptosis increased with medical management alone , therefore surgical decompression and subsequent sinus drainage became necessary . fortunately , our patient showed improvement with medical management alone .
however , had our patient 's condition worsened , surgical removal of the abscess would have been necessary . in conclusion ,
a complete examination of the oral cavity to determine the dental origin of infection should be carried out .
regardless of the patient 's age , early diagnosis and prompt treatment with proper antibiotics coverage can result in a good prognosis for the patient . | orbital cellulitis is a serious , yet uncommon infection in neonates .
it can result in significant sight and life threatening complications .
most commonly , it occurs secondarily as the result of a spread of infection from the sinuses .
orbital cellulitis , secondary to dental infection is rare .
we hereby report a case of orbital cellulitis secondary to dental infection in a 15-day - old neonate without any systemic features . |
leukocyte adhesion deficiency type i ( lad - i ) is a rare autosomal recessive disorder with usual described incidence of 1 in 1 million .
overall about 300 cases have been reported until date with very few cases in newborn .
lad - i is characterized by delayed separation of the umbilical cord at birth , persistent leukocytosis and recurring bacterial and fungal infections involving skin , mucosa , impaired pus formation and wound healing .
we present a case of unusual presentation of lad - i in a neonate with normal separation of umbilical cord and absence of omphalitis .
the present case report is about a 23-day - old full term female infant , the first offspring of 3 degree consanguineous marriage was admitted with a history of multiple bullous lesions starting from day 5 of life over right upper arm progressing to neck , upper limb and groin over subsequent 10 days , which did not respond to the local treatment .
, there were multiple necrotic ulcerative skin lesions [ figure 1 ] with erythematous base over neck , right elbow , groin and left thigh predominantly with evolving bullous and papular skin lesions over chest and trunk .
investigations revealed hemoglobin 8.5 g / dl , total leucocyte count 85,800/mm , absolute neutrophil count 66,066/mm , band forms 11% , platelets 102,000 with peripheral smear showing anisopoikilocytosis and toxic granules .
infant was started on antibiotics as per sensitivity results . in view of high leucocyte counts in an apparently well grown child ,
screening tests done for primary immunodeficiency were quantification of immunoglobulin levels ( igm , igg , ige and iga ) , flow cytometry , ch50 levels and nitroblue tetrazolium test . apart from flow
flow cytometry analysis revealed that none of the neutrophils in the infant were positive for cd18 , cd11a and cd11c with normal levels noted in both parents [ figure 2 ] .
flow cytometry in leukocyte adhesion deficiency type i diagnosis was confirmed as lad - i severe form .
skin biopsy revealed epidermal necrosis with ulceration with inflammatory granular cell infiltrates composed largely by lymphocyte , plasma cell , histiocytes , macrophages and rarely eosinophils .
the baby responded to antibiotics clinically with resolution of pneumonia , healing of skin lesions and normalization of counts .
the parents were advised to avoid bacillus calmette - gurin , typhoid vaccines and were instructed to adhere to the national immunization schedule .
in addition , advice regarding human leukocyte antigen typing and subsequent bone marrow transplantation was given but due to financial reasons the parents refrained from further diagnostic and therapeutic work - up .
lad disorders are a group of rare diseases characterized by a lack of leukocyte cell surface expression of integrin molecules , which are essential for adhesion of leukocytes to the endothelial cells and chemotaxis .
three types of this disorder are described to date ; lad - i caused by the lack of cd 18 integrins on the neutrophils cell surface has the worst prognosis and most patients die within the 1 years of life .
the severity of infections and complications is related to the severity of cd18 deficiency ; cases with < 1% expression are clinically severe , whereas those with 2.5 - 10% expression are moderate to mild . in lad - ii ,
the neutrophils are rich in cd 11 and cd18 integrins , but lack the required surface membrane carbohydrate sialyl - lewis x which is essential for adherence to activated endothelial cells . a defect in activation of rap 1 protein
has recently been described , which results in lack of integrin activation regulation and a set of similar manifestations which are diagnosed as lad - iii .
lad - iii ( also called lad - i variant ) is characterized by both severe bacterial infections and a severe bleeding disorder .
lad - i is caused by mutations in the itgb2 gene ( 21q22.3 ) , encoding the beta-2-integrin cd18 .
lad - ii results from mutations in the slc35c1 gene ( 11p11.2 ) , encoding the guanosine 5'- diphosphate - fucose transporter .
lad - iii is caused by mutations in the fermt3 gene ( 11q13.1 ) , encoding kindlin-3 in hematopoietic cells .
lad - i is clinically characterized by delayed shedding of the umbilical stump , recurrent bacterial infections mainly of the skin and mucosal membranes , leukocytosis , periodontitis , absence of pus and poor wound healing .
the mean time of normal cord separation was reported between 5.8 and 10.9 days in different studies .
our patient presented with multiple skin lesions on day 23 with normal separation of cord on day 10 with absence of omphalitis .
. severe form of lad - i usually manifest in neonatal period but are diagnosed at a later age , require frequent hospital admissions for recurrent infection .
only 2 cases in literature have been diagnosed as lad - i in neonatal period while others have been diagnosed in infancy .
confirmation of the diagnosis requires demonstration of the absence of cd18 and the associated alpha subunit cd11a , cd11b and cd11c on the surface of the leucocytes , based on the flow cytometry using monoclonal antibodies cd11 and cd18 . in lad - iii
differential diagnoses include interleukin-1 receptor - associated kinase 4 deficiency , autosomal dominant hyper ige syndrome , chronic granulomatous disease , neutrophil dysfunction and a leukemoid reaction .
the treatment of the disease depends on the severity of the clinical picture . in the case of patients with the severe disease phenotype ,
the absence of lymphocyte function - associated antigen 1 in these patients can constitute an advantage for transplantation , since graft rejection appears to depend in part on the cd18 complex .
the largest series published to date describes 36 children in 14 centers subjected to transplantation between 1993 and 2007 and followed - up on for 5 years after transplantation .
these patients die in childhood if transplantation is not carried out as soon as possible .
if transplantation is performed before serious infections develop , the resulting prognosis is very good .
based on the above case report it can be concluded that lad - i is a rare form of congenital immune deficiency , it must be taken into account in neonates who present with this unusual clinical picture . | leukocyte adhesion deficiency type i is an extremely serious and rare form of congenital immunodeficiency with recurrent episodes of infection since neonatal period .
it is usually diagnosed in childhood , but if diagnosed early in neonatal period then definitive treatment can be instituted early with promising results .
high index of suspicion is necessary to diagnose this condition in neonates .
neonate may present with serious bacterial infections with a leukemoid reaction . |
insect samples were collected from preserved specimens of the insect museum at kangwon national university in chuncheon ( gangwon province , korea ) .
collected insects were surface sterilized in a 2% sodium hypochlorite solution for 3 min , rinsed with plenty of sterile distilled water , then dried using filter paper .
surface sterilized cadavers were plated onto potato dextrose agar ( pda ) containing 0.25 mg / ml chloramphenicol to inhibit bacterial growth and incubated at 25. hyphae of the fungi growing and sporulating on cadavers ( and on pda medium ) were cut , transferred to fresh pda plates and incubated at 25.
fungal genomic dna samples were extracted using instagene matrix ( bio - rad laboratories , hercules , ca , usa ) .
the primers its1 primer ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its5 ( 5'-ggaagtaaaagtcgtaacaagg-3 ' ) and its4 primer ( 5'-tcctccgcttattgatatgc-3 ' ) were used for the pcr . the pcr reaction was performed with 20 ng of genomic dna as the template in a 30 l reaction mixture by using ef - taq ( solgent , daejeon , korea ) as follows : activation of taq polymerase at 95 for 2 min , 35 cycles of 95 for 1 min , 55 , and 72 for 1 min each were performed , finishing with a 10-min step at 72. the amplification products were purified with a multiscreen filter plate ( millipore corp . ,
the dna samples containing the extension products were added to hi - di formamide ( applied biosystems , foster city , ca , usa ) .
the mixture was incubated at 95 for 5 min , followed by 5 min on ice , and then analyzed by the abi prism 3730xl dna analyzer ( applied biosystems , foster city , ca , usa ) .
the sequences were compared using the ncbi blast program ( http://www.ncbi.nlm.nih.gov/blast ) for identification of the isolates .
sequences used to calculate phylogeny were first determined using blast results from databases and a phylogenetic tree was subsequently prepared by the neighbor - joining method .
conidiophores are observed as two - stage branched ( terverticillate ) with all elements adpressed and stipes rough - walled .
morphologically , conidia and conidiophores of our strain were similar to those of penicillium polonicum ( fig .
1 ) . when the internal transcribed spacer ( its ) sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 3 - 2 was highly related to p. polonicum , with a 99% sequence similarity ( table 1 , fig .
conidiophores from surface hyphae , symmetrically biverticillate , stipes smooth , and width 2.2~3.0 m ; metulae in whorls of 3~6 , 13~18 2.5~3.3 m ; ampulliform phialides , 7.0~10 2.2~3.0 m ; conidia smooth - walled , broadly ellipsoidal , and slightly fusiform in some strains , 2.3~3.1 2.0~2.6 m ( fig .
when the its sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 1 - 8 was highly related to p. steckii with 99% sequence similarity ( table 1 , fig .
the mycelium typically consists of a highly branched network of multinucleate , septate , and usually colorless hyphae .
the conidiospores are the main dispersal route of the fungi , and are often greenish in color .
conidia are globose , ellipsoidal , cylindrical or fusiform , hyaline or greenish , and smooth or rough - walled ( fig .
when its sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 2 - 9 was highly related to penicillium sp . with 100% sequence similarity ( table 1 , fig .
conidiophores are observed as two - stage branched ( terverticillate ) with all elements adpressed and stipes rough - walled .
morphologically , conidia and conidiophores of our strain were similar to those of penicillium polonicum ( fig .
1 ) . when the internal transcribed spacer ( its ) sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 3 - 2 was highly related to p. polonicum , with a 99% sequence similarity ( table 1 , fig .
conidia observed were broadly ellipsoidal . conidiophores from surface hyphae , symmetrically biverticillate , stipes smooth , and width 2.2~3.0 m ; metulae in whorls of 3~6 , 13~18 2.5~3.3 m ; ampulliform phialides , 7.0~10 2.2~3.0 m ; conidia smooth - walled , broadly ellipsoidal , and slightly fusiform in some strains , 2.3~3.1 2.0~2.6 m ( fig .
when the its sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 1 - 8 was highly related to p. steckii with 99% sequence similarity ( table 1 , fig .
the mycelium typically consists of a highly branched network of multinucleate , septate , and usually colorless hyphae .
the conidiospores are the main dispersal route of the fungi , and are often greenish in color .
conidia are globose , ellipsoidal , cylindrical or fusiform , hyaline or greenish , and smooth or rough - walled ( fig .
when its sequences of the strain were compared with related species retrieved from genbank , sequence analysis by blast indicated that knu12 - 2 - 9 was highly related to penicillium sp . with 100% sequence similarity ( table 1 , fig . | three penicillium species have been isolated from insect specimens in korea ; penicillium sp . , p. steckii , and p. polonicum .
penicillium sp .
( knu12 - 3 - 2 ) was isolated from lixus imperessiventris , while p. polonicum ( knu12 - 1 - 8 ) and penicillium steckii ( knu12 - 2 - 9 ) were isolated from muljarus japonicas and meloe proscarabaeus , respectively .
the identification was based on the morphological characteristics of the fungi and in internal transcribed spacer analysis .
this is the first report on the isolation of these three species of penicillium from insects in korea . |
the protozoan pathogen , trypanosoma brucei , undergoes complex cycles of differentiation and multiplication in its vector , the tsetse fly , genus glossina . the infection is first established in the midgut and the trypanosomes migrate anteriorly from this stronghold on a tortuous journey to the paired salivary glands , where they mature into infective metacyclics secreted in the saliva .
the essential facts of the lifecycle were described by , but others have contributed important details missed in the early work , for example the role of the peritrophic membrane or matrix ( pm ) [ 2 - 5 ] .
it takes a minimum of 2 weeks for t. brucei to complete the cycle , but the fly then continues to produce metacyclics for the rest of its life , potentially infecting a new host every few days when it takes a bloodmeal or even just pierces the skin with its proboscis .
the cycle in the fly effectively amplifies the typically scanty parasitaemia found in the mammalian host , giving an immense advantage over simple mechanical transmission by contamination of the mouthparts with infected blood .
in addition , t. brucei has the opportunity for sexual reproduction in the fly . from the above
foremost of these is the poor efficiency of the process both in field and laboratory flies , leading some authors to conclude that the trypanosomes are poorly adapted to the tsetse vector , perhaps reflecting recent evolution , e.g. ref . .
typically far fewer than 1% of wild flies have salivary gland infections , e.g. , and even under laboratory conditions , flies are refractory to infection .
for example , in experimental transmissions of various clones of t. b. brucei through our laboratory colony of glossina morsitans morsitans , a reasonable proportion ( typically one third ) of flies will develop a midgut infection if given trypanosomes in their first bloodmeal , but only a proportion of these infections then go on to mature ( typically less than half ) .
the nature of the barriers firstly to establishment of trypanosomes in the midgut , and secondly , to onward progression to maturation in the salivary glands , are uncertain .
many factors are known to influence fly infection rates , including variables specific to the trypanosome , infected host or fly . in experimental transmissions in the laboratory , trypanosome and host - specific factors can be held constant , allowing the influence of environmental or fly factors , such as immunity , to be studied . here
we have used highly conspicuous green fluorescent trypanosomes to study the early events in establishment of infection in the fly midgut .
groups of flies were fed a single bloodmeal containing trypanosomes and were then dissected at intervals of approximately 24 hours .
the guts were examined immediately as live , wet preparations by fluorescence microscopy and the position and estimated density of fluorescent trypanosomes were recorded for each fly .
samples of the infected bloodmeal from two replicate experiments were cultured in vitro to follow the timing of appearance of fluorescent trypanosomes as the bloodstream forms differentiated to procyclics , with concomitant expression of green fluorescent protein ( gfp ) .
the trypanosomes were not visible at first , but faintly fluorescent trypanosomes were just visible after 2 hours of incubation and trypanosomes were brightly fluorescent after 4 hours in culture .
fluorescent trypanosomes therefore represent only the proportion of bloodstream form trypanosomes that were viable and capable of differentiation . between 24 and 72 hours ( 13 days ) after feeding , trypanosomes were found in every fly examined ( total 21 from 3 separate experiments ) .
the trypanosomes were always exclusively in the bloodmeal , which was held confined within the pm in the midgut ( fig 1 ) .
the trypanosomes were very active , swimming freely in the bloodmeal ( see additional file 1 : movie 1 ) , and appeared to increase in numbers over time . in later infections ( 4872 hours ) , some trypanosomes could be seen lying adjacent to the pm , with the length of their body apparently in contact with it ( fig 2 ; see additional file 1 : movie 1 ) .
trypanosomes in the midgut 48 hours after the infective feed . a , brightfield image showing top of bloodmeal within the anterior midgut 48 hours after the infective feed .
trypanosomes in the midgut 72 hours after the infective feed a , brightfield image showing part of the bloodmeal bounded by the pm within the midgut 72 hours after the infective feed .
some trypanosomes are in close proximity to the pm , but none are in the ectoperitrophic space ( es ) . by 96120 hours ( 45 days )
the bloodmeal was almost completely digested and the midguts of most flies were empty except for a brownish residue towards the posterior end , at the junction of the malpighian tubules . in some flies ( 4 ) , trypanosomes were only found in this region , sometimes just beyond the junction of the malpighian tubules in the hindgut ( fig 3 ) . although it is possible that small numbers of non - fluorescent or weakly fluorescent trypanosomes , especially sluggish ones , might have been missed in these flies ,
large numbers would have been seen . in flies where digestion was less advanced ( 3 ) ,
however , in two flies trypanosomes were found in the empty , middle portion of the midgut , well anterior to the digested bloodmeal .
no trypanosomes were detected in a proportion of flies dissected between 96 and 120 hours after infection ( 10 of 19 flies , or 53% , in 3 separate experiments ) .
trypanosomes in the residual bloodmeal at the midgut / hindgut junction a , brightfield image .
mg , posterior part of midgut ; hg , anterior end of hindgut ; mp , malpighian tubule . division between the mid- and hindgut is marked by the junction of the malpighian tubules .
a dramatically different picture was seen in flies dissected after 144 hours ( 6 days ) .
flies were either completely negative or contained large numbers of highly active trypanosomes in the anterior region of the midgut ; importantly , these trypanosomes were no longer associated with the residue of the bloodmeal . for flies dissected between 144 and 168 hours ( 67 days ) , 8 of 12 flies ( 67% ) from 2 separate experiments were negative .
144 hours was the earliest timepoint at which trypanosomes were found in the ectoperitrophic space , ie . between the pm and the gut wall ( fig 4 ; see additional file 2 : movie 2 ) .
trypanosomes in the ectoperitrophic space a , brightfield image of part of the anterior midgut 6 days after the infective feed .
the midgut is now empty of blood ; the dark line ( arrowed ) shows the boundary of the peritrophic matrix ( pm ) .
b , uv image revealing green fluorescent trypanosomes within the ectoperitrophic space ( es ) . two routes of migration into the ectoperitrophic space have been suggested previously : direct penetration of the pm or migration around the open , posterior end .
em analysis has provided convincing evidence of direct penetration through the pm in the middle region of the midgut for t. brucei . in agreement with this , we observed trypanosomes lying parallel to the pm in the middle portion of the midgut ( e.g. fig 2 ) , and sometimes found large numbers of trypanosomes associated with fragments of pm during counting of homogenized guts ( see below ) . on the other hand ,
the existence of a posterior route could be indicated by the flies dissected on days 45 after infection , in which trypanosomes were found only at the posterior end of the midgut ( e.g. fig 3 ) . in these flies , however , trypanosomes were present in small numbers and it is difficult to see how these scanty trypanosomes could build up to the great numbers seen in infected flies from day 6 onwards .
it seems more likely that the small numbers of trypanosomes found at the very end of the midgut represent a terminal stage of infection .
observations after day 7 had to be carried out on flies that had received a second bloodmeal .
of 12 flies dissected between 9 and 14 days after the infective feed in 2 separate experiments , 4 ( 33% ) were completely negative and 8 ( 67% ) had very heavy infections .
trypanosomes were seen in large numbers , extending from the proventriculus through the middle portion of the midgut and becoming less dense towards the posterior end .
the proventriculus was often packed with trypanosomes ( fig 5 ; see additional file 3 : movie 3 ) , as was the ectoperitrophic space ( fig 6 ) . the density of infection made it impossible to see whether trypanosomes were in the ectoperitrophic space only or were also in the endoperitrophic space .
infection of the ectoperitrophic space in the region of the midgut with columnar epithelium gave a tessellated or honeycomb appearance ( fig 7 ) . despite the very great numbers in the anterior midgut and proventriculus , trypanosomes
proventriculus packed with trypanosomes uv image of proventriculus from an infected fly 2 weeks after the infected feed . numerous green fluorescent trypanosomes swarm in the anterior midgut ( am ) and at the rim of the proventriculus , such that these organs appear bathed in fluorescence .
all the green fluorescence seen is from trypanosomes ; these tsetse tissues show little if any autofluorescence .
the oesophagus is faintly visible , emerging from the proventriculus ( arrowed ) ; no trypanosomes were observed within it .
ectoperitrophic space packed with trypanosomes a , brightfield image of part of the anterior midgut in a fly fed a second bloodmeal ; the dark line shows the boundary of the peritrophic matrix ( pm ) containing the bloodmeal .
b , uv image revealing large numbers of green fluorescent trypanosomes within the ectoperitrophic space ( es ) .
honeycomb appearence of midgut trypanosomes uv image of part of midgut showing the " honeycomb " appearance of trypanosomes confined between the peritrophic matrix and columnar cells of the midgut epithelium 9 days after the infective feed in some infections cyst - like forms were observed , apparently associated with or imbedded in the pm ( fig 8) .
such cyst - like forms were observed from day 5 onwards , but were particularly evident in flies dissected on days 911 after infection .
some cysts contained highly motile trypanosomes with flagella ; the internal structure of other cysts was indeterminate and they appeared to consist of aggregates of rounded - up trypanosomes .
similar cyst - like forms were first noted by robertson , who eloquently described 3 types of cyst ( " confused motile forms with several flagella , amoeboid forms , wriggling masses of half - fused trypanosomes " ) , and have been reported by several observers . whether these cyst - like forms represent degenerating forms , trypanosomes engulfed by phagocytic cells or a stage in the lifecycle is not known .
however , there is a striking similarity to the forms produced by treating procyclics with the lectin cona in vitro to induce cell death .
cyst - like bodies associated with the pm in the midgut uv images of cyst - like bodies found associated with the peritrophic matrix in flies dissected 1011 days after the infective feed .
images a and b show aggregations of rounded up trypanosomes ; c shows a cyst - like body containing highly motile trypanosomes .
the concentration of trypanosomes in the infective bloodmeal was estimated to be 4 10l , based on the level of parasitaemia in the blood before cryopreservation .
a fly taking a typical 20 l bloodmeal would therefore imbibe an estimated 8 10trypanosomes .
a sample of the infected bloodmeal from one experiment was cultured in vitro to establish the efficiency of procyclic transformation and growth rate ( fig 9 ) . the density of fluorescent trypanosomes after 24 hours in culture represented a concentration of approximately 10trypanosomes lin the infected bloodmeal , assuming that transformation to procyclics , but not division , had occurred by this time .
this indicates that less than 10% of the original population of bloodstream form trypanosomes were viable and able to transform into procyclics . the total number of fluorescent trypanosomes counted in individual fly guts after 24 hours in this experiment was in the range 1.9 10to 20.6 10 , which tallies with the number expected after a 20 l bloodmeal containing 10trypanosomes l .
this suggests that any immediate fall in trypanosome numbers on entering the midgut in this experiment is due to trypanosome rather than fly factors , ie .
growth of trypanosomes in in vitro culture compared to fly midgut graph showing the growth of trypanosomes in culture ( diamonds ; geometric mean of 2 separate cultures ) compared to the fly midgut ( circles ; geometric mean of pooled flies ) .
the numbers in culture reflect growth of 10 ul of the same trypanosome - containing bloodmeal used to infect the flies .
we do not routinely assess the recovery of bloodstream forms after cryopreservation , so reduced viability can not be ruled out .
neither can inability to differentiate , as it is assumed that only a proportion of bloodstream forms ( the short stumpy forms ) can readily transform into procyclics , because they are metabolically preadapted for transformation .
it is not unrealistic that a combination of these factors might have resulted in the initial tenfold drop in numbers observed .
similarly , a drastic initial drop in numbers within 48 hours of the infected feed was recorded for experimental infection of g. m. morsitans with t. b. brucei , and about 80% of bloodstream form t. b. rhodesiense had abnormal motility and/or morphology after 10 hours in the midgut of g. m. morsitans .
evans concluded from his successful attempts to infect flies using procyclics , that many infections were probably lost by failure of bloodstream forms to transform into procyclics .
after transformation , the trypanosomes grew exponentially in culture as expected ( fig 9 ) , and trypanosomes within the fly midgut also multiplied exponentially during the first 24 to 72 hours ( fig 10 ) . in some flies ,
trypanosome numbers continued to increase or remained at a high level up to day 6 when the experiment ended .
however , in other flies trypanosome numbers started to fall after 3 days of infection , leading to the complete elimination of infection in some flies .
numbers of trypanosomes in individual flies counts of trypanosomes in individual flies on days 1 to 6 following the infected feed .
infections have been divided into 5 categories according to the number of trypanosomes ; nd , not detectable .
statistical analysis showed that the frequency distribution of number of trypanosomes in the flies was essentially normal on days 1 and 2 , indicating that infected flies were a single population ( table 1 ) . by day 5 , however , the frequency distribution was significantly different from normal , showing a clear split into 2 populations of flies : those with heavy infections and those with undetectable infections ( fig 10 ; table 1 ) .
although not significant , evidence for this was also seen on days 3 and 4 ( table 1 ) ; a larger sample size might increase statistical support for this trend .
these results confirm previous observations also made using g. m. morsitans from the bristol colony infected with t. brucei , that the number of infected flies fell between days 3 and 5 after infection , and that the proportion of negative flies increased from 48 hours after infection .
frequency distribution of trypanosome numbers per fly p - values from kolmogorov - smirnov test .
the cause of elimination of some trypanosome infections from day 3 onwards remains to be determined .
there is indirect evidence that some infections are terminated by the action of trypanocidal lectins in the midgut , since the proportion of infected flies can be significantly increased by feeding glucosamine , a lectin - binding sugar .
however , glucosamine has other effects , notably the inhibition of tsetse midgut trypsin : the same concentration of glucosamine used to enhance midgut infection rates inhibited about 40% of the proteolytic action of trypsin in vivo . in preliminary experiments
, we found that feeding glucosamine in the first bloodmeal visibly slowed the rate of digestion ( unpublished results ) .
nonetheless , the timing of lectin secretion fits with the timecourse of trypanosome elimination determined here . a slow rise in lectin levels between days 0 and 8 after eclosion was demonstrated by haemagglutination assays , with significant amounts appearing after day 3 ; lectin levels rose exponentially after day 8 and reached very high levels by day 14 when the experiment terminated .
interestingly , the lectin was also found to be associated with the pm , which developed to fully line the midgut within 8090 hours of eclosion .
exploration of the interactions of trypanosomes with components of the tsetse anti - microbial immune system is at an early stage .
flies are able to detect the presence of pathogens , including trypanosomes , in the midgut , as shown by upregulation of transcription of the anti - microbial peptides , attacin and defensin , in the fat body within a few days of infection . in addition , flies that were immune - stimulated by prior injection with bacteria showed significantly reduced infection levels with trypanosomes compared to controls when dissected 20 days after infection .
although there is clearly molecular communication between the midgut and the immune organs of the fly , the nature of the effector molecules is as yet unknown .
the timecourse of trypanosome elimination also coincides with digestion and movement of the bloodmeal along the gut : by day 4 flies held at 25c have only a brown residue from the bloodmeal remaining at the posterior end of the midgut , and by day 5 , the midgut is completely empty of any residual bloodmeal .
digestive enzymes are largely absent from the anterior midgut , being secreted in the posterior half [ 22 - 24 ] , perhaps explaining why trypanosome survival is correlated with migration towards the anterior . for infection to endure after day 5 , trypanosomes apparently need to transfer to the ectoperitrophic space .
it is possible that conditions in the digesting bloodmeal become inimical to trypanosome survival and the ectoperitrophic space offers some kind of refuge .
the pm may serve to compartmentalize regions of high enzyme concentrations and intensive digestion , perhaps by binding certain molecules to its surface .
groups of flies were fed a single bloodmeal containing trypanosomes and were then dissected at intervals of approximately 24 hours .
the guts were examined immediately as live , wet preparations by fluorescence microscopy and the position and estimated density of fluorescent trypanosomes were recorded for each fly .
samples of the infected bloodmeal from two replicate experiments were cultured in vitro to follow the timing of appearance of fluorescent trypanosomes as the bloodstream forms differentiated to procyclics , with concomitant expression of green fluorescent protein ( gfp ) .
the trypanosomes were not visible at first , but faintly fluorescent trypanosomes were just visible after 2 hours of incubation and trypanosomes were brightly fluorescent after 4 hours in culture .
fluorescent trypanosomes therefore represent only the proportion of bloodstream form trypanosomes that were viable and capable of differentiation . between 24 and 72 hours ( 13 days ) after feeding , trypanosomes were found in every fly examined ( total 21 from 3 separate experiments ) .
the trypanosomes were always exclusively in the bloodmeal , which was held confined within the pm in the midgut ( fig 1 ) .
the trypanosomes were very active , swimming freely in the bloodmeal ( see additional file 1 : movie 1 ) , and appeared to increase in numbers over time . in later infections ( 4872 hours ) , some trypanosomes could be seen lying adjacent to the pm , with the length of their body apparently in contact with it ( fig 2 ; see additional file 1 : movie 1 ) .
trypanosomes in the midgut 48 hours after the infective feed . a , brightfield image showing top of bloodmeal within the anterior midgut 48 hours after the infective feed .
trypanosomes in the midgut 72 hours after the infective feed a , brightfield image showing part of the bloodmeal bounded by the pm within the midgut 72 hours after the infective feed .
some trypanosomes are in close proximity to the pm , but none are in the ectoperitrophic space ( es ) . by 96120 hours ( 45 days )
the bloodmeal was almost completely digested and the midguts of most flies were empty except for a brownish residue towards the posterior end , at the junction of the malpighian tubules . in some flies ( 4 ) , trypanosomes were only found in this region , sometimes just beyond the junction of the malpighian tubules in the hindgut ( fig 3 ) . although it is possible that small numbers of non - fluorescent or weakly fluorescent trypanosomes , especially sluggish ones , might have been missed in these flies ,
large numbers would have been seen . in flies where digestion was less advanced ( 3 ) ,
however , in two flies trypanosomes were found in the empty , middle portion of the midgut , well anterior to the digested bloodmeal .
no trypanosomes were detected in a proportion of flies dissected between 96 and 120 hours after infection ( 10 of 19 flies , or 53% , in 3 separate experiments ) .
trypanosomes in the residual bloodmeal at the midgut / hindgut junction a , brightfield image .
mg , posterior part of midgut ; hg , anterior end of hindgut ; mp , malpighian tubule . division between the mid- and hindgut is marked by the junction of the malpighian tubules .
a dramatically different picture was seen in flies dissected after 144 hours ( 6 days ) .
flies were either completely negative or contained large numbers of highly active trypanosomes in the anterior region of the midgut ; importantly , these trypanosomes were no longer associated with the residue of the bloodmeal . for flies dissected between 144 and 168 hours ( 67 days ) , 8 of 12 flies ( 67% ) from 2 separate experiments were negative .
144 hours was the earliest timepoint at which trypanosomes were found in the ectoperitrophic space , ie . between the pm and the gut wall ( fig 4 ; see additional file 2 : movie 2 ) .
trypanosomes in the ectoperitrophic space a , brightfield image of part of the anterior midgut 6 days after the infective feed .
the midgut is now empty of blood ; the dark line ( arrowed ) shows the boundary of the peritrophic matrix ( pm ) .
b , uv image revealing green fluorescent trypanosomes within the ectoperitrophic space ( es ) . two routes of migration into the ectoperitrophic space have been suggested previously : direct penetration of the pm or migration around the open , posterior end .
em analysis has provided convincing evidence of direct penetration through the pm in the middle region of the midgut for t. brucei . in agreement with this , we observed trypanosomes lying parallel to the pm in the middle portion of the midgut ( e.g. fig 2 ) , and sometimes found large numbers of trypanosomes associated with fragments of pm during counting of homogenized guts ( see below ) . on the other hand ,
the existence of a posterior route could be indicated by the flies dissected on days 45 after infection , in which trypanosomes were found only at the posterior end of the midgut ( e.g. fig 3 ) . in these flies , however , trypanosomes were present in small numbers and it is difficult to see how these scanty trypanosomes could build up to the great numbers seen in infected flies from day 6 onwards .
it seems more likely that the small numbers of trypanosomes found at the very end of the midgut represent a terminal stage of infection .
observations after day 7 had to be carried out on flies that had received a second bloodmeal .
of 12 flies dissected between 9 and 14 days after the infective feed in 2 separate experiments , 4 ( 33% ) were completely negative and 8 ( 67% ) had very heavy infections .
trypanosomes were seen in large numbers , extending from the proventriculus through the middle portion of the midgut and becoming less dense towards the posterior end .
the proventriculus was often packed with trypanosomes ( fig 5 ; see additional file 3 : movie 3 ) , as was the ectoperitrophic space ( fig 6 ) . the density of infection made it impossible to see whether trypanosomes were in the ectoperitrophic space only or were also in the endoperitrophic space .
infection of the ectoperitrophic space in the region of the midgut with columnar epithelium gave a tessellated or honeycomb appearance ( fig 7 ) . despite the very great numbers in the anterior midgut and proventriculus , trypanosomes
proventriculus packed with trypanosomes uv image of proventriculus from an infected fly 2 weeks after the infected feed . numerous green fluorescent trypanosomes swarm in the anterior midgut ( am ) and at the rim of the proventriculus , such that these organs appear bathed in fluorescence .
all the green fluorescence seen is from trypanosomes ; these tsetse tissues show little if any autofluorescence .
the oesophagus is faintly visible , emerging from the proventriculus ( arrowed ) ; no trypanosomes were observed within it .
ectoperitrophic space packed with trypanosomes a , brightfield image of part of the anterior midgut in a fly fed a second bloodmeal ; the dark line shows the boundary of the peritrophic matrix ( pm ) containing the bloodmeal .
b , uv image revealing large numbers of green fluorescent trypanosomes within the ectoperitrophic space ( es ) .
honeycomb appearence of midgut trypanosomes uv image of part of midgut showing the " honeycomb " appearance of trypanosomes confined between the peritrophic matrix and columnar cells of the midgut epithelium 9 days after the infective feed in some infections cyst - like forms were observed , apparently associated with or imbedded in the pm ( fig 8) .
such cyst - like forms were observed from day 5 onwards , but were particularly evident in flies dissected on days 911 after infection .
some cysts contained highly motile trypanosomes with flagella ; the internal structure of other cysts was indeterminate and they appeared to consist of aggregates of rounded - up trypanosomes .
similar cyst - like forms were first noted by robertson , who eloquently described 3 types of cyst ( " confused motile forms with several flagella , amoeboid forms , wriggling masses of half - fused trypanosomes " ) , and have been reported by several observers . whether these cyst - like forms represent degenerating forms , trypanosomes engulfed by phagocytic cells or a stage in the lifecycle is not known .
however , there is a striking similarity to the forms produced by treating procyclics with the lectin cona in vitro to induce cell death .
cyst - like bodies associated with the pm in the midgut uv images of cyst - like bodies found associated with the peritrophic matrix in flies dissected 1011 days after the infective feed .
images a and b show aggregations of rounded up trypanosomes ; c shows a cyst - like body containing highly motile trypanosomes .
the concentration of trypanosomes in the infective bloodmeal was estimated to be 4 10l , based on the level of parasitaemia in the blood before cryopreservation . a fly taking a typical 20 l bloodmeal
a sample of the infected bloodmeal from one experiment was cultured in vitro to establish the efficiency of procyclic transformation and growth rate ( fig 9 ) . the density of fluorescent trypanosomes after 24 hours in culture represented a concentration of approximately 10trypanosomes lin the infected bloodmeal , assuming that transformation to procyclics , but not division , had occurred by this time .
this indicates that less than 10% of the original population of bloodstream form trypanosomes were viable and able to transform into procyclics .
the total number of fluorescent trypanosomes counted in individual fly guts after 24 hours in this experiment was in the range 1.9 10to 20.6 10 , which tallies with the number expected after a 20 l bloodmeal containing 10trypanosomes l .
this suggests that any immediate fall in trypanosome numbers on entering the midgut in this experiment is due to trypanosome rather than fly factors , ie .
growth of trypanosomes in in vitro culture compared to fly midgut graph showing the growth of trypanosomes in culture ( diamonds ; geometric mean of 2 separate cultures ) compared to the fly midgut ( circles ; geometric mean of pooled flies ) .
the numbers in culture reflect growth of 10 ul of the same trypanosome - containing bloodmeal used to infect the flies .
we do not routinely assess the recovery of bloodstream forms after cryopreservation , so reduced viability can not be ruled out .
neither can inability to differentiate , as it is assumed that only a proportion of bloodstream forms ( the short stumpy forms ) can readily transform into procyclics , because they are metabolically preadapted for transformation .
it is not unrealistic that a combination of these factors might have resulted in the initial tenfold drop in numbers observed .
similarly , a drastic initial drop in numbers within 48 hours of the infected feed was recorded for experimental infection of g. m. morsitans with t. b. brucei , and about 80% of bloodstream form t. b. rhodesiense had abnormal motility and/or morphology after 10 hours in the midgut of g. m. morsitans .
evans concluded from his successful attempts to infect flies using procyclics , that many infections were probably lost by failure of bloodstream forms to transform into procyclics .
after transformation , the trypanosomes grew exponentially in culture as expected ( fig 9 ) , and trypanosomes within the fly midgut also multiplied exponentially during the first 24 to 72 hours ( fig 10 ) .
in some flies , trypanosome numbers continued to increase or remained at a high level up to day 6 when the experiment ended .
however , in other flies trypanosome numbers started to fall after 3 days of infection , leading to the complete elimination of infection in some flies .
numbers of trypanosomes in individual flies counts of trypanosomes in individual flies on days 1 to 6 following the infected feed .
infections have been divided into 5 categories according to the number of trypanosomes ; nd , not detectable . statistical analysis showed that the frequency distribution of number of trypanosomes in the flies was essentially normal on days 1 and 2 , indicating that infected flies were a single population ( table 1 ) . by day 5 , however , the frequency distribution was significantly different from normal , showing a clear split into 2 populations of flies : those with heavy infections and those with undetectable infections ( fig 10 ; table 1 ) .
although not significant , evidence for this was also seen on days 3 and 4 ( table 1 ) ; a larger sample size might increase statistical support for this trend .
these results confirm previous observations also made using g. m. morsitans from the bristol colony infected with t. brucei , that the number of infected flies fell between days 3 and 5 after infection , and that the proportion of negative flies increased from 48 hours after infection .
frequency distribution of trypanosome numbers per fly p - values from kolmogorov - smirnov test . * significantly different from normal distribution ( p < 0.05 ) .
the cause of elimination of some trypanosome infections from day 3 onwards remains to be determined .
there is indirect evidence that some infections are terminated by the action of trypanocidal lectins in the midgut , since the proportion of infected flies can be significantly increased by feeding glucosamine , a lectin - binding sugar .
however , glucosamine has other effects , notably the inhibition of tsetse midgut trypsin : the same concentration of glucosamine used to enhance midgut infection rates inhibited about 40% of the proteolytic action of trypsin in vivo . in preliminary experiments , we found that feeding glucosamine in the first bloodmeal visibly slowed the rate of digestion ( unpublished results ) .
nonetheless , the timing of lectin secretion fits with the timecourse of trypanosome elimination determined here . a slow rise in lectin levels between days 0 and 8 after eclosion was demonstrated by haemagglutination assays , with significant amounts appearing after day 3 ; lectin levels rose exponentially after day 8 and reached very high levels by day 14 when the experiment terminated .
interestingly , the lectin was also found to be associated with the pm , which developed to fully line the midgut within 8090 hours of eclosion .
exploration of the interactions of trypanosomes with components of the tsetse anti - microbial immune system is at an early stage .
flies are able to detect the presence of pathogens , including trypanosomes , in the midgut , as shown by upregulation of transcription of the anti - microbial peptides , attacin and defensin , in the fat body within a few days of infection . in addition
, flies that were immune - stimulated by prior injection with bacteria showed significantly reduced infection levels with trypanosomes compared to controls when dissected 20 days after infection . although there is clearly molecular communication between the midgut and the immune organs of the fly , the nature of the effector molecules is as yet unknown .
the timecourse of trypanosome elimination also coincides with digestion and movement of the bloodmeal along the gut : by day 4 flies held at 25c have only a brown residue from the bloodmeal remaining at the posterior end of the midgut , and by day 5 , the midgut is completely empty of any residual bloodmeal .
digestive enzymes are largely absent from the anterior midgut , being secreted in the posterior half [ 22 - 24 ] , perhaps explaining why trypanosome survival is correlated with migration towards the anterior . for infection to endure after day 5 , trypanosomes apparently need to transfer to the ectoperitrophic space .
it is possible that conditions in the digesting bloodmeal become inimical to trypanosome survival and the ectoperitrophic space offers some kind of refuge .
the pm may serve to compartmentalize regions of high enzyme concentrations and intensive digestion , perhaps by binding certain molecules to its surface .
we now have a clearer picture of the early events in establishment of t. brucei in the tsetse midgut . by using gfp expression as an indicator of transformation
, we were able to record a greater than tenfold disparity between the estimated number of bloodstream form trypanosomes in the infective bloodmeal compared to the number of procyclics in the midgut or in vitro culture 24 hours after infection .
therefore the initial factors inhibiting establishment of infection in the midgut are trypanosome - specific , depending on viability and ability to differentiate .
the trypanosomes that have undergone successful differentiation appear to experience an environment within the midgut suited to their unrestricted growth for the first 3 days . after this time , a process of attrition is evident , which leads to the complete elimination of infection in some flies . by day 5 , flies fall into 2 groups according to the level of infection : high or undetectable .
this timecourse coincides with lectin secretion , development of the pm and the digestion and movement of the bloodmeal along the gut .
the earliest timepoint that trypanosomes were found in the ectoperitrophic space was day 6 and they were then present in great numbers .
infection at this level was maintained until the end of observations ( day 14 ) , with trypanosomes swarming in the proventriculus and midgut , but becoming less dense towards the posterior end .
the group 2 trypanosoma brucei gambiense stock th2 ( mhom / ci/78/th2 [ 78e ] , ) was transfected with a construct containing the gene for green fluorescent protein under the control of the procyclin promotor as described by .
aliquots of e21 bloodstream forms in log phase growth were cryopreserved for fly infection . e21 bloodstream forms were not visibly fluorescent , but became fluorescent on transformation to procyclics due to the concomitant upregulation of the procyclin promotor .
the experimental flies were from the long - established bristol laboratory colony of glossina morsitans morsitans , originally from zimbabwe .
male flies , approximately 1824 hours post - eclosion , were fed a single bloodmeal of sterile horse blood containing a thawed aliquot of bloodstream form e21 trypanosomes ; the final concentration of trypanosomes in the bloodmeal was estimated as 4 10l .
flies were subsequently maintained in small cages containing 3 5 flies without feeding for up to 6 days and longer if given a second bloodmeal of sterile horse blood .
flies were briefly chilled before dissection of the whole posterior gut from the proventriculus to the rectum .
whole tsetse midguts were dissected into a drop of pbs and viewed as wet mounts . a dmrb microscope ( leica ) equipped with either a 24 bit colour coolview camera ( photonic science ) or 10 bit monochrome megaview ii camera ( norfolk analytical ) was used for fluorescence and brightfield microscopy , with imagepro plus software ( photonic science ) or analysis software ( norfolk analytical ) respectively .
multiple images captured with a minimum timelapse were assembled to create video sequences ( imagepro plus ) .
results were compiled from 5 batches of flies infected in separate experiments . for counting trypanosomes in individual flies , the whole abdomen , including the posterior gut with the proventriculus ,
was placed in 50 l of pbs in a microcentrifuge tube and thoroughly disrupted using a teflon pestle .
the trypanosomes were fixed by adding paraformaldehyde to a final concentration of 0.1 % w / v in pbs and counted immediately using a haemocytometer . only fluorescent trypanosomes were counted .
the lower limit of detection was 18 trypanosomes per fly , based on the number of trypanosomes present if only 1 trypanosome was recorded in 6 haemocytometer counts .
seven replicate experiments were carried out to give adequate numbers of flies at each time point .
the frequency distribution of trypanosome numbers in flies was analysed for conformity to a normal distribution using a single - sample kolmogorov - smirnov test ( spss ) .
the group 2 trypanosoma brucei gambiense stock th2 ( mhom / ci/78/th2 [ 78e ] , ) was transfected with a construct containing the gene for green fluorescent protein under the control of the procyclin promotor as described by .
aliquots of e21 bloodstream forms in log phase growth were cryopreserved for fly infection . e21 bloodstream forms were not visibly fluorescent , but became fluorescent on transformation to procyclics due to the concomitant upregulation of the procyclin promotor .
the experimental flies were from the long - established bristol laboratory colony of glossina morsitans morsitans , originally from zimbabwe .
male flies , approximately 1824 hours post - eclosion , were fed a single bloodmeal of sterile horse blood containing a thawed aliquot of bloodstream form e21 trypanosomes ; the final concentration of trypanosomes in the bloodmeal was estimated as 4 10l .
flies were subsequently maintained in small cages containing 3 5 flies without feeding for up to 6 days and longer if given a second bloodmeal of sterile horse blood .
flies were briefly chilled before dissection of the whole posterior gut from the proventriculus to the rectum .
whole tsetse midguts were dissected into a drop of pbs and viewed as wet mounts . a dmrb microscope ( leica ) equipped with either a 24 bit colour coolview camera ( photonic science ) or 10 bit monochrome megaview ii camera ( norfolk analytical ) was used for fluorescence and brightfield microscopy , with imagepro plus software ( photonic science ) or analysis software ( norfolk analytical ) respectively .
multiple images captured with a minimum timelapse were assembled to create video sequences ( imagepro plus ) .
for counting trypanosomes in individual flies , the whole abdomen , including the posterior gut with the proventriculus , was placed in 50 l of pbs in a microcentrifuge tube and thoroughly disrupted using a teflon pestle .
the trypanosomes were fixed by adding paraformaldehyde to a final concentration of 0.1 % w / v in pbs and counted immediately using a haemocytometer . only fluorescent trypanosomes were counted .
the lower limit of detection was 18 trypanosomes per fly , based on the number of trypanosomes present if only 1 trypanosome was recorded in 6 haemocytometer counts .
seven replicate experiments were carried out to give adequate numbers of flies at each time point .
the frequency distribution of trypanosome numbers in flies was analysed for conformity to a normal distribution using a single - sample kolmogorov - smirnov test ( spss ) .
wg carried out the tsetse transmission experiments and drafted the manuscript ; mb carried out the imaging and statistical analyis .
both authors contributed to the design of the study , read and approved the final manuscript .
movie showing live green fluorescent trypanosomes in bloodmeal in tsetse midgut movie showing live green fluorescent trypanosomes in ectoperitrophic space in tsetse midgut movie showing live green fluorescent trypanosomes in tsetse proventriculus and anterior midgut .
all the green fluorescence seen is from trypanosomes ; these tsetse tissues show little if any autofluorescence .
we thank mrs jenny berry and the international atomic energy agency , vienna , for providing tsetse flies and dr lewis bingle for generating the recombinant trypanosome clone . | backgroundthe protozoan pathogen , trypanosoma brucei , undergoes complex cycles of differentiation and multiplication in its vector , the tsetse fly , genus glossina .
flies are refractory to infection and resistance mechanisms operate at a number of levels and timepoints . here
we have used highly conspicuous green fluorescent trypanosomes to study the early events in establishment of infection in the fly midgut.resultsless than 10% of the bloodstream form trypanosomes in the infected feed differentiated into viable procyclics . up to day 3 , trypanosomes were found in the bloodmeal in every fly examined , and increased in number between days 1 and 3 .
flies dissected on days 5 and 6 fell into 2 clearly distinct groups : those with high numbers of trypanosomes and those with undetectable infection .
trypanosomes were found in the ectoperitrophic space and proventriculus from 6 days following the infective feed.conclusiontrypanosomes that have undergone successful differentiation appear to experience an environment within the midgut suited to their unrestricted growth for the first 3 days . after this time , a process of attrition is evident in some flies , which leads to the complete elimination of infection . by day 5 , flies fall into 2 groups according to the level of infection : high or undetectable .
this timecourse coincides with lectin secretion , development of the pm and the digestion and movement of the bloodmeal along the gut .
further experiments are needed to discriminate between these factors . |
patient 6 is a 62-year - old man from the town of mandal who was bitten by a tick ; onset of symptoms began 2 weeks later . at the end of may ,
the patient was dizzy and weak , had a headache , chills , and fever .
the antibody from serum sample profiles showed previous infection with herpes simplex and varicella zoster viruses .
nucleic acids from herpes simplex virus , varicella - zoster virus , or enterovirus were not detected in cerebrospinal fluid ( csf ) .
tbev igm and igg antibodies were detected in serum samples , with high levels of igm ( optical density [ od ] 1.580 on june 13 to od 0.899 on july 18 ) and high igg levels ( 13.06 , od 1.235 on june 13 to od 1.742 on july 18 ) .
cut - off values were 0.250 for igm and 0.263 for igg on june 13 and 0.271 for igm and 0.278 for igg on june 18 .
neutralization test antibodies in serum samples rose from < 5 in samples taken on june 13 to 10 in samples from july 18 .
patient 7 is a 53-year - old man who was visiting a cabin in the coastal area near mandal .
symptoms began at the end of june , with fever , increasing headache , nausea , and vomiting .
nucleic acids from herpes simplex virus , varicella - zoster virus , or enterovirus were not detected in csf .
tbev igm and igg antibodies were detected in serum samples , with high levels of igm ( od 2.064 on july 22 ; od 1.916 on july 30 ; and od 1.499 on august 8) and rising igg levels ( od 0.597 on july 22 ; od 0.876 on july 30 ; and od 1.993 on august 8) .
neutralization test antibody levels rose from < 5 in serum taken july 21 to 10 in serum from november 25 .
borderline values of tbev antibodies were found in spinal fluid . during the first several months after illness onset , the patient had cognitive dysfunction but gradually returned to work .
patient 8 is a 74-year - old man , who lives in kristiansand and has a camper in sgne .
since august 2003 , he had increasing headache and from october 3 , 2003 the headache was intense and accompanied by nausea and vomiting .
his personality was altered during these weeks , with reduced memory about recent events in particular , irritability , and verbal aggressiveness .
high levels of tbev igm ( od 1.461 on october 6 and od 1.200 on november 5 ) were detected in sera together with rising igg levels ( od 0.652 on october 6 and od 1.475 on november 11 ) .
in spinal fluid from october 3 , intrathecal production of tbev antibodies could not be detected , but one month later , intrathecal igm was produced . during hospitalization , the patient recovered well .
after 10 to 11 days , he was aware , and his mental situation improved considerably .
the agder counties have the highest incidence of borrelia infections in norway ( 33 cases/100,000 persons , 19972003 ) .
the previously published clinical signs and symptoms and results from these five patients ( 1,2 ) are summarized as case 15 , while the three new patients are presented as patients 68 ( table 1 and table 2 ) .
the eight patients included seven men and one woman from 42 years to 74 years of age .
all patients had intense headache , seven had vertigo and nausea , and six had vomiting .
seven patients were hospitalized , three with reduced consciousness , two with mental disturbances ; all seven had more or less severe neurologic abnormalities .
three had ataxia ; one had diplopia ; and one had speech difficulties , bilateral ptosis , paresis of eye and pharynx muscles , and paresis of muscles in the left shoulder .
serum samples were obtained from all eight patients and had signs of inflammation with c - reactive protein level of 10105
seven patients underwent lumbar puncture ; csf pleocytosis and elevated protein levels were found in all patients .
nucleic acids from herpes simplex virus , varicella - zoster virus , or enterovirus were not detected in the spinal fluid specimens , which excludes the most common differential diagnostic causes of encephalitis . in all patients ,
high serum levels of tbev igm and igg antibodies were detected with enzyme - linked immunosorbent assay methods . in neutralization tests ,
serum antibody titers increased from < 5 to 10 in five of the patients , 10 to 20 in one , and 10 to 40 in one patient ( 1,2 ) .
although the diagnostic tests are not absolutely specific for tbe compared to the closely related louping ill virus , no cases of louping ill virus in livestock ( snorre stuen , pers . comm . ) or human infections have been reported in norway since 1991 ; none of the eight patients lived close to or worked with sheep or goats .
the clinical characteristics of the norwegian patients are similar to those of swedish patients ( 5 ) . in sweden ,
all eight patients with tbe in norway became ill after being bitten by a tick in the coastal areas of the agder counties .
four had been on tromy island in aust - agder county before becoming ill , while one had been in lyngdal and three in mandal and sgne in vest - agder county .
thus , all patients with suspicious cases of tbe may not have been tested for antibodies to tbev . in agder
, we have tested for tbe since 1999 , but the disease may still be underdiagnosed .
tbev igg antibodies were detected in 0.3% to 0.4% of the serum samples from persons in agder counties . from persons on tromy island ,
antibodies were found in 2.4% of serum samples , and in other coastal districts , the seroprevalence was 0%11% ( 1,6 ) .
the number of human serum samples tested is limited , and the vaccination status is unknown .
however , vaccination is unlikely because norwegians are only vaccinated against flaviviruses on special travel indications . in sweden ,
the incidence of human cases of tbe has risen during the last few years , and new tbe foci have been reported ( 7 ) . during the last 2 decades
changes in the distribution of tbev have been indicated , and the norwegian cases are from areas where new foci have been predicted ( 8) .
in norway , 02 cases of tbe were diagnosed per year from 1997 to 2003 .
all patients have been bitten by a tick in the agder counties in southern norway .
of the first eight norwegian patients , four had been on tromy island in aust - agder county before becoming ill .
the four most recent patients were bitten by ticks in lyngdal , mandal , and sgne in the coastal areas of vest - agder county .
the seroprevalence studies indicate that tromy and some spots along the coast in the southernmost part of vest - agder county may have a higher incidence of tbe than the rest of the agder counties .
although tbe is a rare disease in norway , the situation has to be monitored carefully . | the first reported case of tickborne encephalitis ( tbe ) in norway occurred in 1997 . from 1997 to 2003 , from zero to two cases of human tbe have been diagnosed per year in norway , for a total of eight cases .
clinical tbe cases in dogs are not reported in norway . |
communicable disease control is a public health priority at the international level to prevent the spread of contagious diseases ( 1 , 2 ) .
the increase of emerging and re - emerging communicable diseases such as sars , multi drug resistance tuberculosis ( mdr tb ) , ebola and increasing information needs causes increasing interest in communicable disease reporting and surveillance system(36 ) .
therefore , part of the task of public health agencies in the national level is an efficient and effective policy making on reporting and control of infectious diseases ( 7 , 8) .
policymaking and control of communicable disease requires an accurate and efficient surveillance and reporting system at all levels ( 7 , 9 ) .
disease reporting has been implemented traditionally with poor infrastructure and support in most of the countries ; therefore , its quality improving has been a challenge ( 10 , 11 ) . because of the heterogeneity about disease and lack of suitable financial resources ,
setting the standard for disease reporting and surveillance system is extremely difficult ( 7 ) .
thus to achieve a new and simple model , there is a need to design , process and facilitate the flow of information and reporting systems ( 12 , 13 ) . in iran , reporting system of communicable diseases has been integrated into the health system for decades ( 1416 ) . in the recent years , it has also been tried to strengthen this reporting and surveillance system . however , there is a need to translate global evidence , to localize and convert them into effective action .
knowledge of global reporting systems may help to find and repair defects and gaps in the country`s reporting system such as communicable disease reports from hospitals and private sector .
reform and repair of the iranian communicable diseases reporting system require a study to assess the world evidence and experience of other countries , and assess the general characteristics of these reporting systems and determines the solutions for reducing the problems of the disease reporting system .
this systematic review aimed at gathering experiences of other countries in disease reporting system in order to improve iran`s share of diseases reporting system .
data were collected using the following databases and search engines ; proquest , science direct , pub med , scopes , springer , ebesco . for persian databases , we used ; sid , iranmedex and magiran .
the search was performed using keywords such as communicable diseases , notifiable disease , disease notification , reporting system , surveillance systems and evaluation regardless of their date and range .
boolean operators ; and , or and not were also used during the search . in addition , paper and printed information sources were searched manually at the iranian center for disease management .
farsi and english articles about at least one of important characteristic of communicable surveillance diseases according to who guideline ( 17 , 18 ) . contains ; priority diseases for surveillance , surveillance system structure , core function of surveillance systems , support functions of surveillance systems , surveillance quality were included in the study since 1980 .
these criteria were used for reviewing and choosing the studies . from all those articles , only one disease surveillance and reporting system were excluded .
time and space limitations of this study were from 27 september to 7 october 2014 .
retrieved resources considering above - mentioned points were investigated by two reviewers and the rejection of each of the studies were expressed . in case of disagreement , the third party reviewer did the reviews . finally , all the resources for our study were controlled and confirmed by an expert .
the quality of the study was evaluated by a researcher according to the main purpose of our study .
a total of 1856 articles and 33 print sources , including booklets and instructions relating to our study were found .
after the elimination of duplicate recourses and considering exclusion criteria , we had only 236 resources . from 236 remaining cases ,
179 were excluded from the study by literature review , and 57 articles remained in the study . by reviewing the references of these articles , 9 other articles were found and finally 66 resources were entered in this systematic review ( table 1 ) .
selected studies were reviewed carefully by two independent reviewers , and basic concepts and themes were extracted .
findings were categorized in the 3 main categories : 1-specifications , objectives and duties of diseases reporting system , 2-problems related to the disease reporting system and 3-strategies for improving disease reporting system .
three subcategories were considered for data classification in each category ( level of reporting ) .
data were collected using the following databases and search engines ; proquest , science direct , pub med , scopes , springer , ebesco . for persian databases , we used ; sid , iranmedex and magiran .
the search was performed using keywords such as communicable diseases , notifiable disease , disease notification , reporting system , surveillance systems and evaluation regardless of their date and range .
boolean operators ; and , or and not were also used during the search . in addition , paper and printed information sources were searched manually at the iranian center for disease management .
farsi and english articles about at least one of important characteristic of communicable surveillance diseases according to who guideline ( 17 , 18 ) . contains ; priority diseases for surveillance , surveillance system structure , core function of surveillance systems , support functions of surveillance systems , surveillance quality were included in the study since 1980 .
these criteria were used for reviewing and choosing the studies . from all those articles ,
time and space limitations of this study were from 27 september to 7 october 2014 .
retrieved resources considering above - mentioned points were investigated by two reviewers and the rejection of each of the studies were expressed . in case of disagreement , the third party reviewer did the reviews . finally , all the resources for our study were controlled and confirmed by an expert .
the quality of the study was evaluated by a researcher according to the main purpose of our study .
a total of 1856 articles and 33 print sources , including booklets and instructions relating to our study were found .
oversell , 1889 resources had inclusion criteria . after the elimination of duplicate recourses and considering exclusion criteria
179 were excluded from the study by literature review , and 57 articles remained in the study . by reviewing the references of these articles , 9 other articles were found and finally 66 resources were entered in this systematic review ( table 1 ) .
selected studies were reviewed carefully by two independent reviewers , and basic concepts and themes were extracted .
findings were categorized in the 3 main categories : 1-specifications , objectives and duties of diseases reporting system , 2-problems related to the disease reporting system and 3-strategies for improving disease reporting system .
three subcategories were considered for data classification in each category ( level of reporting ) .
several items were evaluated in the studies which we had found . concerning priority diseases for surveillance , unfortunately , no study had mentioned the priorities of diseases .
nineteen studies ( 28.8% ) discussed the surveillance system structure . in addition , 34 studies ( 51.5% ) had some comments and texts on the core function of surveillance systems and support functions of surveillance systems . finally , 26 studies ( 39.4% ) presented the features of surveillance quality .
people who were responsible for reporting disease in reporting systems include communicable diseases nurse , infection control nurses , physician , and nurse , health workers that reported their data using paper cards , paper forms , phone , short message service ( sms ) , fax , email , internet , and software .
characteristics of disease reporting system were noted . some of these criteria included : determining the policies and legislation , specific budget allocation , assessing the needs of reporting and surveillance systems , quantitative and qualitative assessment , specifying responsibilities and duties , preparing booklets and guidelines , analysis of data based on the required variables and giving feedback to the reporting levels ( table 2 ) .
the most important problems of the reporting system in reporter level , reporting process and recipient level included shortages in human resources , high workload of the person who is responsible for disease reporting and staff unconsciousness about the disease reporting system were problems at reporter level .
lack of standard processes for reporting diseases , lack of obligation to report , not analyzing reporting of diseases , lack of proper training to personnel about disease reporting and high costs for training and maintenance of electronic reporting systems are among problems in reporting process . not giving feedback to the reporter levels , not having access to the private sector data and limited budget for disease reporting system
some of the strategies presented in these articles include ; determining a specific process for disease reporting , simplifying the reporting forms , appoint a substitute for a person who is responsible for reporting using and developing electronic technology , operational guideline formulation for laboratories , private sectors and hospitals , using bulletins to publish information about the health care and reporting systems ( table 4 ) .
people who were responsible for reporting disease in reporting systems include communicable diseases nurse , infection control nurses , physician , and nurse , health workers that reported their data using paper cards , paper forms , phone , short message service ( sms ) , fax , email , internet , and software . characteristics of disease reporting system were noted . some of these criteria included : determining the policies and legislation , specific budget allocation , assessing the needs of reporting and surveillance systems , quantitative and qualitative assessment , specifying responsibilities and duties , preparing booklets and guidelines , analysis of data based on the required variables and giving feedback to the reporting levels ( table 2 ) .
the most important problems of the reporting system in reporter level , reporting process and recipient level included shortages in human resources , high workload of the person who is responsible for disease reporting and staff unconsciousness about the disease reporting system were problems at reporter level .
lack of standard processes for reporting diseases , lack of obligation to report , not analyzing reporting of diseases , lack of proper training to personnel about disease reporting and high costs for training and maintenance of electronic reporting systems are among problems in reporting process . not giving feedback to the reporter levels , not having access to the private sector data and limited budget for disease reporting system
some of the strategies presented in these articles include ; determining a specific process for disease reporting , simplifying the reporting forms , appoint a substitute for a person who is responsible for reporting using and developing electronic technology , operational guideline formulation for laboratories , private sectors and hospitals , using bulletins to publish information about the health care and reporting systems ( table 4 ) .
given that some items were incomplete in the literature , it seems that the procedure of reporting communicable diseases with high priority is based on the local guidelines presented to the health care system .
these guidelines are not available online , and based on the experiences of the authors , many countries , for example , have a list of reportable diseases that is not mentioned in any article .
therefore , there is a need to publish these documents online ; additionally , the articles about disease reporting systems should cover these issues more comprehensively .
the main purpose of reporting communicable diseases is to prevent the spread of the disease , epidemics , death or disability resulting from the disease .
the best communicable disease reporting and surveillance system should provide rapid identification , timely response and information on the incidence and prevalence of disease ( 7 ) .
completeness and timeliness of data are principles of disease reporting system ( 7 , 1921 ) and all attempts to reform disease reporting system must be done in order to achieve these two goals . based on the results , disease reporting is performed in health centers , hospitals , clinics , private offices and laboratories ( 22 , 23 ) . physicians and primary care staff are the most qualified people for reporting communicable diseases and efforts should be made to obtain the report from these individuals ( 13 , 24 ) .
these individuals transfer the data during the reporting process to data collecting levels in different ways , such as paper forms , phone , mobile , sms , wireless , fax , email , internet and electronic software .
diversity in disease reporting systems is due to differences in regulatory requirements , reporting date , reporting process and available financial resources ( 7 ) .
however , this diversity must not result in the loss of two important principles namely completeness and timeliness .
considering low sources and problems related to the use of traditional techniques such as paper forms and missing data , to overcome ineffectiveness in transferring information , there is a need for simple and efficient methods for communicable disease reporting and surveillance system ( 2527 ) . increasing tendency to use email , mobile phones and electronic systems
, these methods can be fast , effective , efficient and cost - effective tool for collecting data ( 2830 ) . however , there are challenges using electronic methods ( 27 , 31 ) .
the use of electronic systems requires resources , funding to set up , and maintenance .
a partnership between the health sectors , personnel training and maintaining the confidentiality of patient information ( 20 , 27 , 31 ) should be considered in disease reporting system of iran as well .
considering epidemiology of the diseases , reporting the diseases based on laboratory confirmation ( 32 ) or without laboratory confirmation ( 33 ) is different . given the important role of laboratories , specially private laboratories , in addition to clinical assessment in the diagnosis of many diseases , laboratory reporting development , organizing , training , and coordination in laboratory procedures an also the inclusion of mandatory reporting laboratories alongside the medical report as a contributory mechanism is required .
it can improve the communicable disease reporting and surveillance system ( 14
, 20 , 34 , 35 ) which is important in iran . in order to provide the right decisions and achieving goals , communicable disease surveillance and reporting systems should be assessed in terms of quality , efficiency and effectiveness ( 18 , 3638 ) .
non - adherence to timely and completeness of data in surveillance and reporting systems , causes problems in data analyzing .
this happens because of slowness in non - electronic reporting system , long time answering the laboratory tests , reporting overlap with the responsibilities of other individual`s and assuming that the disease been reported by anybody else ( 39 ) .
preparing manuals , guidelines , standards and protocols for reporting diseases and updating them , specifying the responsibilities and identifying duties and skills of staff about disease reporting and surveillance system can assist the staff in carrying out assigned tasks and make better accuracy of data in the system ( 24 , 29 , 36 ) .
data collection , data analysis , and feedback to the reporting levels are the main items and functions of the disease reporting and surveillance system ( 40 , 41 ) . due to the sudden outbreak of some communicable diseases ,
creating warning and quick response teams to design appropriate intervention programs and simulation exercises of epidemics is disease reporting and surveillance system requirements .
shortages in human resources , high workload of the person who is responsible for disease reporting and not appointing a substitute leads to lack of reporting motivation . in some cases , there are no standard and defined processes for communicable disease reporting system from high level reporting sources or they are complex and have little flexibility to change ( 30 , 37 ) .
appoint a trained person for reporting , determining the specific process , using harmonized and simplified reporting methods and create incentives ( material or spiritual ) could improve the reporting system .
lack of knowledge and skills of service providers is one of the main problems and obstacles .
there is always a need to educate others to interact with the objectives of public health programs ( 9 , 13 , 42 , 43 ) . in most countries , disease - reporting systems
has a weak infrastructure and rules and the data on this are not used in planning and program prioritization ( 10
, 30 , 37 ) .
therefore , policies and rules related to disease reporting and surveillance system should be revised in all parts of the health system including private sector and military , which should be involved in the disease , and reporting system .
in addition , using appropriate technologies and planning matrix this system should be strengthened ( 3 , 10 , 35 , 44 ,
45 ) .
bulletins can be used for dissemination and exchange of information and sharing the best practices for combating communicable diseases in both paper and electronic forms ( 35 ) .
one of the main problems that the various studies are not considered enough is monitoring and evaluation of the disease reporting system .
a good reporting system should have a proper monitoring program that could quickly identify problems of the system and show the process of removing the problems .
although the disease reporting process in different regions can be different ; however , timeliness and completeness are two major principles in system design
. therefore , detailed explanations of duties and providing appropriate instructions are important points in integrating an efficient reporting system .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication , redundancy , etc . ) were strictly controlled by the authors . | background : communicable disease reporting and surveillance system has poor infrastructure and supporters in most of countries . its quality improvement is a challenge and requires an accurate and efficient care and reporting systems at all levels to achieve new and simple models .
this study evaluates reporting systems of communicable diseases using systematic review.methods:this was a systematic review study . for data collection ,
we used the following database and search engines : proquest , science direct , pub med , scopes , springer , and ebesco . for persian databases , we used sid , iranmedex and magiran .
our key words were communicable diseases , notifiable disease , disease notification , reporting system , surveillance systems and evaluation .
two independent researchers reviewed the resources and the results were classified in different domains.results:from 1889 cases , only 66 resources were studied .
the results were classified in several domains , including those who were reporting , reporting methods and procedures , responsibilities and reporting system characteristics , problems and solutions of the report , the reporting process , and receptor level.conclusion:disease-reporting system has similar problems in all parts of the world .
change , improve , update and continuous monitoring of the reporting system are very important .
although the reporting process can vary in different regions , but being perfect and timely are important principles in system design .
detailed explanations of tasks and providing appropriate instructions are the most important points to integrate an efficient reporting system . |
dry eye syndrome is a disorder of the ocular surface that is characterized by loss of natural function of the corneal and conjunctival epithelial barrier . moreover ,
cytomorphological studies have revealed microchanges in the epithelium comprising changes in size of surface cells and number of goblet cells as well as occurrence of snake - like chromatin [ 13 ] .
the resulting deficit in wetting is compensated by use of tear substitutes that also should protect the mucosa against desiccation [ 1,47 ] .
there are three categories of artificial tears according to the product description table : artificial tears with preservative , artificial tears with disappearing preservative and artificial tears without preservative . despite the fact that various products improve the situation of dry eye - patients , examinations demonstrating objectively the protective effects are rare [ 811 ] .
recently we have tested in vitro the effectiveness of different agents to prevent desiccation ( eg . artelac edo , vidisic edo , vidisic fluid edo , acuolens , viscofresh and hyal drops sdu ) . for this purpose we used human epithelial cells in a standardised fashion . in
the present study we studied the protective effect of another set of lubricating eye drops ( e.g. arufil uno , arufil , lacrimal o.k . , lacophtal sine , siccaprotect , tears again , vidisept edo , vistil , wet - comod ) that were also widely applied in drye eye disease in order to compare the results with established data previously .
different pharmacological substances were tested for their capability to prevent desiccation of the conjunctival epithelial cell line chang 1 - 5c-4 ( ccl-20.2 american type culture collection ; series 1 ) and corneal cell culture line 2.040 prsv - t ( 2.040prsv - t american type culture collection ; series 2 ) as described previously .
on confluent cell growth , cells were incubated ( 20 min ) with the artificial tears ( see below ) .
incubation with pbs ( 100 l ) served as a negative control and positive control was incubation with unsupplemented medium ( 100 l ) . after incubation artificial eye drops were discarded and the cell cultures were exposed to a constant air flow for 0 , 15 , 30 and 45 minutes . to assess the amount of vital cells , cultures were incubated with the vital dye alamarblue ( biosource , camarillo , usa ) .
absorption of the oxidised form of the dye was measured using an elisa - reader , in order to detect the amount of live epithelial cells still present .
the following medicines were used : arufil uno povidone 20 mg , arufil povidone 20 mg , lacrimal o.k .
polyvinyl alcohol 14 mg , povidone 6 mg , lacophtal sine povidone 20 mg , siccaprotect dexpanthenol 30 mg , polyvinyl alcohol 14 mg , tears again soy lecithine 10 mg , vidisept edo povidone 20 mg , vistil polyvinyl alcohol 14 mg , wet - comod povidone 20 mg ) ; pbs as negative control ( 100 l ) . cells ( 1.510 cells/100 l
culture medium / well ) were cultured in 96-well plates ( nunc , wiesbaden ) overnight ( 37c , respective culture conditions ) . for experiments involving the 2.040.prsv-t cells the 96-well plates were coated 2 hours before use with 1 ml solution [ 0.01 mg / ml fibronectin ( sigma , st .
louis , usa ) and 0.03 mg / ml vitrogen 100 ( invitrogen , karlsruhe ) ] . when cells were confluent
the medium was removed and 1 to 2 drops test solution were added to the cells , followed by incubation at 37c for 20 min . per test solution 4 wells were used .
after removal of respective agents , cells were dried ( with continuous air flow ) for 0 min , 15 min , 30 min and 45 min , respectively . after washing [ 3 times with 100 l pbs ( 1pbs ) ]
the cells were incubated for 4 hours at 37c with respective medium and 10% alamar blue ( biosource ) .
the absorption of the oxidised dye was measured using an elisa - reader ( anthos , eugendorf , austria ) at 570 nm and 630 nm .
the survival rate was assessed with the following formula ( alamarblue assay booklet by biosource page 17 ) : ( ox)1 = 80,586 ( molar extinction coefficient of alamar blue ( oxidised ) at wave length 570 nm ) ( ox)2 = 34,798 ( molar extinction coefficient of alamar blue ( oxidised ) at wave length 630 nm ) a1 = absorption of the sample at 570 nm a2 = absorption of the sample at 630 nm a1 = absorption of the positive control at 570 nm a2 = absorption of the positive control at 630 nm the medians for all substances tested were depicted in the graph in dependence of exposure times for the respective cell line ( overall means ) allowing a rapid comparison of the different cell survival rates and the protective effect of the substances tested .
the following medicines were used : arufil uno povidone 20 mg , arufil povidone 20 mg , lacrimal o.k .
polyvinyl alcohol 14 mg , povidone 6 mg , lacophtal sine povidone 20 mg , siccaprotect dexpanthenol 30 mg , polyvinyl alcohol 14 mg , tears again soy lecithine 10 mg , vidisept edo povidone 20 mg , vistil polyvinyl alcohol 14 mg , wet - comod povidone 20 mg ) ; pbs as negative control ( 100 l ) .
culture medium / well ) were cultured in 96-well plates ( nunc , wiesbaden ) overnight ( 37c , respective culture conditions ) . for experiments involving the 2.040.prsv-t cells the 96-well plates were coated 2 hours before use with 1 ml solution [ 0.01 mg / ml fibronectin ( sigma , st .
louis , usa ) and 0.03 mg / ml vitrogen 100 ( invitrogen , karlsruhe ) ] . when cells were confluent
the medium was removed and 1 to 2 drops test solution were added to the cells , followed by incubation at 37c for 20 min . per test solution 4 wells were used .
after removal of respective agents , cells were dried ( with continuous air flow ) for 0 min , 15 min , 30 min and 45 min , respectively . after washing [ 3 times with 100 l pbs ( 1pbs ) ]
the cells were incubated for 4 hours at 37c with respective medium and 10% alamar blue ( biosource ) .
the absorption of the oxidised dye was measured using an elisa - reader ( anthos , eugendorf , austria ) at 570 nm and 630 nm .
the survival rate was assessed with the following formula ( alamarblue assay booklet by biosource page 17 ) : ( ox)1 = 80,586 ( molar extinction coefficient of alamar blue ( oxidised ) at wave length 570 nm ) ( ox)2 = 34,798 ( molar extinction coefficient of alamar blue ( oxidised ) at wave length 630 nm ) a1 = absorption of the sample at 570 nm a2 = absorption of the sample at 630 nm a1 = absorption of the positive control at 570 nm a2 = absorption of the positive control at 630 nm
the medians for all substances tested were depicted in the graph in dependence of exposure times for the respective cell line ( overall means ) allowing a rapid comparison of the different cell survival rates and the protective effect of the substances tested .
all artificial tears reduced the quantity of vital cells in the examined cell cultures with increased drying time periods .
the baseline values ( after 0 min ) for live prsv - t cells were better compared to the chang cells . in addition , protection against desiccation was more effective in the prsv - t cells than in the conjunctival chang cells that is in accordance to previous studies .
after 30 or 45 min of desiccation significant protective effects were seen only for 2 substances , namely lacrimal and lacophthal sine .
after the maximum exposure time of 45 min , the overall means from the 3 sets of experiments were very close , with a very high proportion of living cells compared to the other test substances .
in detail , cell survival rates in series 1 after 0 , 15 30 , 45 min were ( 0.62;0.58;0.52;0.34 ) for arufil uno , ( 0.41;0.35;0.33;0.33 ) for arufil , ( 0.97;0.89;0.70;0.46 ) for lacrimal , ( 0.84;0.75;0.55;0.36 ) for lacophthal sine , ( 0.35;0.33;0.32;0.34 ) for siccaprotect , ( 0.84;0.43;0.33;0.34 ) for tears again , ( 0.90;0.80;0.37;0.35 ) for vidisept edo , ( 0.78;0.74;0.50;0.34 ) for vistil , ( 0.90;0.84;0.46;0.34 ) for wet comod , ( 0.94;0.66;0.45;0.33 ) for pbs and in series 2 ( 0.55;0.26;0.03;0.16 ) for arufil uno , ( 0.32;0.13;0.20;0.22 ) for arufil , ( 1.0;0.56;0.34;0.06 ) for lacrimal , ( 1.02;0.43;0.02;0.22 ) for lacophthal sine , ( 0.06;0.16;0.20;0.21 ) for siccaprotect , ( 1.0;0.06;0.02;0.20 ) for tears again , ( 1.0;0.57;0.12 ; 0.23 ) for vidisept edo , ( 0.82;0.49;0.08;0.21 ) for vistil , ( 0.86;0.53;0.01;0.18 ) for wet comod and ( 0.98;0.01;0.21;0.23 ) for pbs .
a similar decrease of cell viability in both cell lines was seen after treatment with pbs , arufil uno , arufil , lacrimal o.k . ,
lacophtal sine , siccaprotect , tears again , vidisept edo , vistil , and wet - comod .
moreover , in comparison to other test substances and the negative controls the overall means for lacrimal were not above average after exposure to air .
a decrease of survival rate was assessed during increasing drying time for the individual substances tested in both cell cultures . except lacrimal o.k .
both cell cultures showed an initially good tolerability of the test substances ( figure 1a , b ) .
the graph demonstrates the substantial protective effect of lacrimal o.k . on chang cells exposed to the air before drying .
after the maximum exposure time of 45 min , the overall means from the 3 sets of experiments were very close , with a very high proportion of living cells compared to the other test substances . in the chang cell culture
when the proportion of living cells after a drying time of 0 to 45 minutes is taken into account , tears again in the chang cell culture tends to show a lower protective effect on the cell culture to compared pbs .
and lacophtal sine have a significantly better protective effect compared to the other preparations before the drying of both cell lines tested . both artificial tears with benzalconiumchloride ( bac )
vistil with oxyd as a biodegradable preservative system showed statistical significant difference , whereas arufil and siccaprotect with bac caused higher rate of apoptocic cells ( after desiccation time 15 min and 30 min ) .
combined artificial tears from polyvinyl alcohol and povidone without preservative system had the highest rates of cell viability after 45 min desiccation time .
we have tested the efficacy of another series of pharmacological substances to prevent desiccation of cultured human conjunctival and corneal cell lines .
lacrimal and wet comod were most effective on chang 1 - 5c-4 cells ( series 1 ) while vidisept edo ( 0.57 ) and lacrimal showed the best protective effect on 2.040 prsv - t corneal cell line after 115 min of desiccation .
after 45 min highest survival was seen in series 1 for lacophthal sine ( 0.36 ) and lacrimal ( 0.46 ) and in series 2 for lacrimal ( 0.06 ) and arufil ( 0.16 ) compared to pbs ( 0.23 ) . the cell culture system was previously described and shown to be suitable to assess the protective effect of artificial tears in vitro .
the former study investigated artelac edo , vidisic edo , vidisic fluid edo , hyal drops sdu , artelac 10 ml with preservative , systane 10 ml , aculens edo , viscofresh edo ) and it turned out that vidisic fluid edo and vidisic edo showed significantly higher survival rates or markedly lower cell loss on epithelial cells .
for instance cell viability decreased progressively after constant air flow exposure during few minutes . when drying time increased ( more than 15 min ) no or only slight increase in cell loss was seen .
although it is to assume that cells die by necrosis the underlying mechanism of cell death especially after 1 or 15 min of drying remains to be explored . in the present study
lacrimal o.k . was defined in both cell culture lines by the highest survival rates of epithelial cells . when drying time increased up to 45 minutes , no or only slight increase in cell loss could be observed .
substantial cytotoxic effects on the cultured cells were observed when preparations with established preservatives were used .
to compare with bac preservative substances , artificial tears with oxyd led to maintain better cell viability and barrier function of human conjunctival and corneal epithelial cells .
though , these decomposing preservatives also prevent the potential protective effect against drying , the effect demonstrated by preservative - free substance such as lacrimal o.k . accordingly
preservative - free wetting agents offer , as expected , the best possible preconditions for an effective protection against drying in the highly differentiated epithelium at the ocular surface , whether normal or damaged .
previously , cell viability in an immortalized corneal epithelial cell line ( t - hec ) showed a 4% to 11% increase in apoptotic cells after treatment with 3 different contact lens multipurpose solutions .
moreover the same solutions led to disturbed expression of tight junction proteins zo-1 and occludin .
otherwise another multipurpose solution did not affect cell viability or expression of tight junction proteins .
although the in vitro test systems yield interesting results on cytotoxicity of different medicines , the results have to be interpreted carefully since cultured cells are quite different from the normal ocular surface epithelium .
for example , the cell culture model does not consider the stratified character of the conjunctival barrier , drug diffusion , conjunctival blood supply , mucin production and composition and tear fluid .
therefore , in vitro studies can not exactly predict the properties of pharmaceuticals during in vivo use [ 1516,22 ] .
however the present study is in line to previous studies employing cell culture models for in vitro ocular toxicological studies in order to understand mechanisms of some external eye diseases [ 6,1416 ] as a major disadvantage artificial tears often contain potentially toxic preservatives , stabilizers , and other additives that can cause further problems to the compromised cornea in the dry eye condition .
although the concentration of preservatives is usually low , high frequency of use may result in a cumulative effect and damage of the ocular surface .
this problem can be prevented by using preservative - free unit - dose artificial tears [ 30,24,3133 ] .
therefore , it is useful to objectively assess corneal - protective effects of artificial tears and to compare the effects of products that contain different components .
in conclusion , the results of the present study suggest that the in vitro fluorometric system comprising resazurin ( alamar blue ) microplate assay with human corneal and conjunctival cell culture would be a valuable potential in vitro screening approach in the product development of artificial eye drops .
arufil uno : povidone 20 mg , 2h2o disodium edetate , disodium phosphate 2h2o , sodium 2h2o , sodium chloride , water f. inj - purposes ; arufil : povidone 20 mg , 2h2o disodium edetate , disodium phosphate 12h2o , sodium 2h2o , sodium chloride , water f. inj - purposes , benzalkonium chloride 0.03 mg ; lacrimal o.k . : polyvinyl alcohol 14 mg , povidone 6 mg , natriumchloride ; lacophtal sine : povidone 20 mg , natriumchloride , natriumhydroxide , boric acid , water f. inj - purposes ; siccaprotect : dexpanthenol 30 mg , polyvinyl alcohol 14 mg , benzalkoniumchloride , kaliumdihydrogenphosphate , kaliummonohydrogenphosphate ; tears again : soy lecithine 10 mg , natriumchloride 8 mg , ethanol 8 mg , phenoxyethanol 5 mg , retinyl palmitate 0.25 mg , -tocopherol 0.02 mg ; vidisept edo : povidone 20 mg , boric acid , natriumchloride , natriumhydroxide ; vistil : polyvinyl alcohol 14 mg , oxyd , edetinacid , dinatriumsaline ; wet - comod : povidone 20 mg , natriumhydroxide , citrate buffer , sorbitol ; pbs as negative control ( 100 l ) ; unsupplemented medium as a positive control ( 100 l ) .
arufil uno : povidone 20 mg , 2h2o disodium edetate , disodium phosphate 2h2o , sodium 2h2o , sodium chloride , water f. inj - purposes ; arufil : povidone 20 mg , 2h2o disodium edetate , disodium phosphate 12h2o , sodium 2h2o , sodium chloride , water f. inj - purposes , benzalkonium chloride 0.03 mg ; lacrimal o.k . :
polyvinyl alcohol 14 mg , povidone 6 mg , natriumchloride ; lacophtal sine : povidone 20 mg , natriumchloride , natriumhydroxide , boric acid , water f. inj - purposes ; siccaprotect : dexpanthenol 30 mg , polyvinyl alcohol 14 mg , benzalkoniumchloride , kaliumdihydrogenphosphate , kaliummonohydrogenphosphate ; tears again : soy lecithine 10 mg , natriumchloride 8 mg , ethanol 8 mg , phenoxyethanol 5 mg , retinyl palmitate 0.25 mg , -tocopherol 0.02 mg ; vidisept edo : povidone 20 mg , boric acid , natriumchloride , natriumhydroxide ; vistil : polyvinyl alcohol 14 mg , oxyd , edetinacid , dinatriumsaline ; wet - comod : povidone 20 mg , natriumhydroxide , citrate buffer , sorbitol ; pbs as negative control ( 100 l ) ; unsupplemented medium as a positive control ( 100 l ) . | summarybackgrounda large number of artificial tears is widely used to treat dry eye symptoms . to test the efficacy of these drugs independent of individual parameters in vitro models are required . as described previously , we employed a reproducible in vitro cell culture system to evaluate the desiccation protection capability of some artificial tears . in the present paper data
is presented of another set of pharmaceutical agents.material/methodsconjunctival epithelial cell line chang 1 - 5c-4 ( series 1 ) and the corneal cell line 2.040 prsv - t ( series 2 ) were cultured under standard conditions .
confluent cells were wetted for 20 min with artificial tears ( arufil uno , arufil , lacrimal , lacophthal sine , siccaprotect , tears again , vidisept edo , vistil , wet comod ) or pbs as a control . after exposure to a constant air flow for 0 , 15 , 30 and 45 minutes respectively
, cells were incubated with the vital dye alamarblue .
subsequently , absorption of the oxidised form of the dye was assessed using an elisa-reader.resultscell best survival rates in series 1 after 15 min were found for lacrimal ( 0.89 ) , wet comod ( 0.84 ) compared to pbs ( 0.66 ) and in series 2 for vidisept edo ( 0.57 ) and lacrimal ( 0.56 ) compared to pbs ( 0.01 ) .
after 45 min highest survival was seen in series 1 for lacrimal ( 0.46 ) and lacophthal sine ( 0.36 ) compared to pbs ( 0.33 ) and in series 2 for lacrimal ( 0.06 ) and arufil ( 0.16 ) compared to pbs ( 0.23).conclusionsboth cell lines tested showed different susceptibility towards desiccation and the artificial tears showed differences in preventing cells from desiccation . |
a variety of bacterial pathogens are responsible for causing urinary tract infections ( utis ) , but the most prominent are escherichia coli , enterococcus spp . ,
pseudomonas aeruginosa and mirabilis , klebsiella pneumoniae , candida albicans , enterobacter spp . , and coagulase - negative staphylococci .
however , it is the uropathogenic e. coli ( upec ) strains that are the primary causative agents of up to 90% of utis . formally , upec is defined traditionally , as other e. coli strains , by the presence of somatic , capsular polysaccharide , and flagellar antigens ( o , k , and h respectively ) . among upec variants ,
the o antigens 1 , 2 , 4 , 6 , 7 , 8 , 16 , 25 , and 75 occur more often than others .
while the k and h antigens appear to have no significant trends , the k1 antigen , typically associated with expec ( extraintestinal pathogenic e. coli ) strains causing neonatal meningitis [ nmec ] , can be found among the more virulent strains of upec .
upec strains are similar to other pathogenic e. coli strains in that they generally carry larger genomes than k12 or commensal e. coli isolates , most likely due to the survival needs outside the human intestinal tract .
to date , the genomes of three upec isolates have been fully sequenced : two pyelonephritis isolates , cft073 ( o6:k2:h1 ) and 536 ( o6:k15:h31 ) and one cystitis isolate , uti89 ( o18:k1:h7 ) .
while no unique genetic factors have yet been found specific to upec , the sequenced isolates indicate that upec encodes more virulence factors than k12/commensal strains .
these include -hemolysin , cytotoxic necrotizing factor 1 ( cnf1 ) , lipopolysaccharide ( lps ) modification systems , virulent capsule antigens , iron acquisition systems including the sidereophores aerobactin and enterobactin , proteases , and a variety of pili including type 1 , p , s , and f1c .
for instance , the rapid identification and treatment of patients with complicated utis remains an important problem .
fortunately , certain anatomic factors contributing to classification of a uti as complicated can be remedied quickly and effectively to minimize the morbidity associated with the uti .
for example , anatomic urinary tract obstruction ( i.e. stone , stricture ) can be alleviated with internal or external drainage during the acute infectious period prior to definitive management of the etiology causing the obstruction .
however , ultimately the efficacy of future therapy in patients with complicated utis will be impaired unless the underlying complicating factor(s ) are identified and appropriately managed .
another common etiology of complicated utis is functional disturbances of the urinary tract resulting in abnormal micturition that can be neurologic or non - neurologic in origin .
a frequently utilized modality to treat the abnormal bladder function in patients with functional disturbances of the urinary tract is clean intermittent catheterization ( cic ) .
this has been shown to be safe and effective since its first introduction by lapides in 1972 .
cic has revolutionized the approach to lower urinary tract disease states . however , an unfortunate consequence of cic is a high incidence of bacteriuria , with rates ranging from 1585% .
the clinical sequela of the bacteriuria is unclear as the incidence of uti in patients performing cic varies widely .
the incidence of symptomatic uti developing in patients with normal lower urinary tract function and untreated bacteriuria can also vary dependent upon patient population . specifically , in pregnant women , the rate of development of symptomatic uti with untreated bacteriuria can be as high as 30% , which can then precipitate preterm labor .
therefore , nearly every pregnant woman diagnosed with bacteriuria on screening urine cultures will be offered treatment to avoid the complications associated with preterm labor .
similarly , untreated bacteriuria detected on screening urine cultures in school - age children with normal urinary tracts has the potential to develop into a symptomatic uti in approximately 10% .
lastly , in one study , untreated bacteriuria detected on weekly screening urine cultures in 14 patients over a six - month period who performed cic with a normal upper urinary tract resulted in five symptomatic utis developed during the 323 week follow - up period .
clearly , the treatment of bacteriuria is highly dependent upon patient demographics and better descriptors are needed to identify those patients that have a need for continuous antibiotic therapy to prevent sequelae .
utis are the result of a complex series of interactions between the uropathogen and the host that can result either in asymptomatic disease ( commensalism ) or symptomatic disease 3 . of the bladder , kidney , or both .
important factors that influence the outcome of utis include : virulence factors of the uropathogen , functional and anatomical status of the urinary tract , inoculum size , genetic factors , and the competency of the host immune system .
the bladder itself is a hostile environment for bacteria in general . except for p. mirabilis which produces a urease , there are no nutrients available in the urine .
furthermore , bacteria that come into contact with the epithelial cells of the bladder can trigger innate immune responses initiated by toll - like receptor 4 ( tlr4 ) , among others , which is responsive to the lps produced by many bacteria , and upec specifically .
this immune cascade results in the production of various proinflammatory cytokines and chemoattractants , including il-8 , important in neutrophil recruitment .
the host can also respond with other innate immune phagocytes such as macrophages , or by producing antimicrobial peptides , and , the most drastic response , total exfoliation of the superficial layer of bladder epithelial cells ( recently reviewed in:12 , 13 ) . once thought to be strictly an extracellular pathogen ,
recent studies from the murine cystitis model[1520 ] have established a new intracellular paradigm of upec infection within superficial bladder epithelial cells .
intracellular growth within superficial epithelial cells of the bladder provides many advantages to upec including : access to nutrients enabling robust intracellular growth , evasion of professional phagocytic cells,[2224 ] avoidance of expulsion , and protection from the antibacterial properties of urea , ammonium , and osmolarity of urine .
time - lapse fluorescence video microscopy of infected murine bladders revealed that upec proceed through a complex developmental pathway forming biofilm - like communities termed intracellular bacterial communities ( ibcs ) .
this study was instrumental in that it unified previously described pathogenic events from single time points into a comprehensive model of uti pathogenesis that elucidates four separate developmental stages of the ibc pathway .
after invasion , early ibcs appear as loose collections of non - motile rods that rapidly divide within the cytoplasm of the bladder epithelial cells .
middle ibcs occur approximately 6 - 8 h following infection as colonies of tightly packed , coccoid bacteria exhibiting biofilm - like traits with slower growth rates .
concurrent with maturation into the coccoid morphology , a subpopulation of the intracellular bacteria becomes filamentous . during late stage ibcs ,
coccoid bacteria differentiate into motile rods that disperse from the ibc in a flagellar - mediated process called fluxing .
finally , egressing out of the infected epithelial cells enables upec to re - enter the ibc cascade through multiple rounds , albeit with slower kinetics to eventually establish a latent infection .
additionally , the subpopulation of elongated , filamentous bacteria inhibit killing by polymorphonuclear neutrophils pmns , as well as promoting adhesion to the underlying layer of cells in the bladder after exfoliation , allowing the bacteria to enter the latent phase of the infection cascade [ figure 1 ] .
the cartoon depicts the stages through which upec ( blue and green ) proceeds during infection of superficial bladder epithelial cells ( sbecs ) .
see text for details studies have demonstrated that the ibc pathway is neither limited to a single upec isolate nor to a single mouse strain .
for instance , ibc formation was found to occur in five genetic backgrounds of inbred mice strains . additionally , the majority of upec strains isolated from women with different uti syndromes [ asymptomatic bacteriuria , recurrent cystitis , pyelonephritis , and acute cystitis ] were competent for ibc formation in the murine cystitis model . although each of the upec strains from the uti syndromes were proficient at ibc formation differences were observed in the size and number of ibcs as well as the kinetics of infection .
intriguingly , ibcs derived from acute cystitis isolates were both significantly smaller and less numerous when compared to the other uti syndromes .
these results suggest that ibc formation is a common attribute of upec strains and that the propensity of a given strain to form ibcs may be associated with persistence in the urinary tract .
strikingly , evidence of the ibc pathogenic cycle was found in the urines of women with acute cystitis .
rosen et al . , observed that ibcs shed into the urine of women with cystitis were indistinguishable from exfoliated ibcs found in mouse urine upon subsequent histologic examination .
furthermore , filamentous uropathogens 20 m in length were found in the urines from several other gram - negative species including upec , proteus mirabilis , klebsiella pneumoniae , and enterobacter aerogenes .
however , neither evidence of exfoliated ibcs nor filaments were found in the urines of women with an asymptomatic infection and women infected by gram - positive bacteria .
this evidence is consistent with recent reports that intracellular bacteria were not observed in biopsies of patients with neurogenic bladder who experience numerous episodes of asymptomatic bacteriuria .
indeed , when biopsies were performed on women with recurrent utis it was determined that 88% of biopsies contained bacteria upon the urothelium by scanning electron microscopy sem analysis and bacteria could be recovered from 50% of the biopsies of women with sterile urine cultures .
the evidence that an intracellular bacterial reservoir is established in both mice[1520 ] and humans has several important clinical considerations for the management of utis .
prophylactic antibiotic therapy has been a mainstay for treatment of recurrent urinary tract infections . with the rise in antibiotic - resistant pathogens and the discussions of whether all patients require antibiotic therapies , it is important to identify new algorithms for treatment built upon evidence - based investigations . to fully evaluate the effectiveness of antibiotic treatment against utis , mulvey and
colleagues recently evaluated the efficacy of eight different classifications of antibiotics on the eradication of intracellular upec in the mouse model for human uti .
these investigators determined that three - day oral treatment with any of the antibiotics , including those capable of intracellular accumulation , had no effect on the latent bacterial burden in the bladder .
importantly , the quiescent intracellular reservoirs avoided elimination despite the presence of antibiotic concentrations in the urine that greatly exceeded the minimal inhibitory concentration . when the bacteria were liberated from the bladder epithelium , the antibiotic sensitivity was enhanced , suggesting that the intracellular compartment , as well as the ibc structure , provide significant protection from antibiotic therapies .
these observations further complicate evaluation of treatments given that sterile urine does not correlate with sterile bladder tissue .
these results could provide an explanation to the epidemiological findings that upwards of 68% of recurrences are caused by bacteria that are isogenic to the original strain.[3339 ] furthermore , each ibc is clonal , that is , invasion of a single upec is sufficient to support all of the growth observed within a single epithelial cell .
in addition , a single epithelial cell can support at least 10 upec individuals , indicating that invasion of a single bacterium is sufficient to initiate an acute infection . this new paradigm for recurrence parallels clinical observations and changes the management for treatment of these infections from a hygienic problem to an antibiotic - insensitive latent infection . in summary
specifically , new diagnostic and therapeutic approaches based on the combination of host genetic factors , innate immunity , and bacterial virulence factors are needed to identify the patients most prone to utis to avoid the cost and potential side - effects of treatment .
fortunately , advances being made in both basic and clinical scientific research of the urinary tract in patients and animal models are providing some explanations and insight into the clinical problems that remain with the management of utis .
improved knowledge of the genetics , uropathogen virulence factors , and host immune responses to utis will enhance the ability of clinicians to more readily distinguish high - risk patients from uncomplicated patients , which is necessary to prevent major sequelae in these high - risk patients .
, the rapid identification and treatment of patients with complicated utis remains an important problem .
fortunately , certain anatomic factors contributing to classification of a uti as complicated can be remedied quickly and effectively to minimize the morbidity associated with the uti .
for example , anatomic urinary tract obstruction ( i.e. stone , stricture ) can be alleviated with internal or external drainage during the acute infectious period prior to definitive management of the etiology causing the obstruction .
however , ultimately the efficacy of future therapy in patients with complicated utis will be impaired unless the underlying complicating factor(s ) are identified and appropriately managed .
another common etiology of complicated utis is functional disturbances of the urinary tract resulting in abnormal micturition that can be neurologic or non - neurologic in origin .
a frequently utilized modality to treat the abnormal bladder function in patients with functional disturbances of the urinary tract is clean intermittent catheterization ( cic ) .
this has been shown to be safe and effective since its first introduction by lapides in 1972 .
cic has revolutionized the approach to lower urinary tract disease states . however , an unfortunate consequence of cic is a high incidence of bacteriuria , with rates ranging from 1585% .
the clinical sequela of the bacteriuria is unclear as the incidence of uti in patients performing cic varies widely .
the incidence of symptomatic uti developing in patients with normal lower urinary tract function and untreated bacteriuria can also vary dependent upon patient population . specifically , in pregnant women , the rate of development of symptomatic uti with untreated bacteriuria can be as high as 30% , which can then precipitate preterm labor .
therefore , nearly every pregnant woman diagnosed with bacteriuria on screening urine cultures will be offered treatment to avoid the complications associated with preterm labor .
similarly , untreated bacteriuria detected on screening urine cultures in school - age children with normal urinary tracts has the potential to develop into a symptomatic uti in approximately 10% .
lastly , in one study , untreated bacteriuria detected on weekly screening urine cultures in 14 patients over a six - month period who performed cic with a normal upper urinary tract resulted in five symptomatic utis developed during the 323 week follow - up period .
clearly , the treatment of bacteriuria is highly dependent upon patient demographics and better descriptors are needed to identify those patients that have a need for continuous antibiotic therapy to prevent sequelae .
utis are the result of a complex series of interactions between the uropathogen and the host that can result either in asymptomatic disease ( commensalism ) or symptomatic disease 3 . of the bladder , kidney , or both .
important factors that influence the outcome of utis include : virulence factors of the uropathogen , functional and anatomical status of the urinary tract , inoculum size , genetic factors , and the competency of the host immune system .
the bladder itself is a hostile environment for bacteria in general . except for p. mirabilis which produces a urease ,
furthermore , bacteria that come into contact with the epithelial cells of the bladder can trigger innate immune responses initiated by toll - like receptor 4 ( tlr4 ) , among others , which is responsive to the lps produced by many bacteria , and upec specifically .
this immune cascade results in the production of various proinflammatory cytokines and chemoattractants , including il-8 , important in neutrophil recruitment .
the host can also respond with other innate immune phagocytes such as macrophages , or by producing antimicrobial peptides , and , the most drastic response , total exfoliation of the superficial layer of bladder epithelial cells ( recently reviewed in:12 , 13 ) . once thought to be strictly an extracellular pathogen , recent studies from the murine cystitis model[1520 ]
intracellular growth within superficial epithelial cells of the bladder provides many advantages to upec including : access to nutrients enabling robust intracellular growth , evasion of professional phagocytic cells,[2224 ] avoidance of expulsion , and protection from the antibacterial properties of urea , ammonium , and osmolarity of urine .
time - lapse fluorescence video microscopy of infected murine bladders revealed that upec proceed through a complex developmental pathway forming biofilm - like communities termed intracellular bacterial communities ( ibcs ) .
this study was instrumental in that it unified previously described pathogenic events from single time points into a comprehensive model of uti pathogenesis that elucidates four separate developmental stages of the ibc pathway .
after invasion , early ibcs appear as loose collections of non - motile rods that rapidly divide within the cytoplasm of the bladder epithelial cells .
middle ibcs occur approximately 6 - 8 h following infection as colonies of tightly packed , coccoid bacteria exhibiting biofilm - like traits with slower growth rates .
concurrent with maturation into the coccoid morphology , a subpopulation of the intracellular bacteria becomes filamentous . during late stage ibcs ,
coccoid bacteria differentiate into motile rods that disperse from the ibc in a flagellar - mediated process called fluxing .
finally , egressing out of the infected epithelial cells enables upec to re - enter the ibc cascade through multiple rounds , albeit with slower kinetics to eventually establish a latent infection . additionally , the subpopulation of elongated , filamentous bacteria inhibit killing by polymorphonuclear neutrophils pmns , as well as promoting adhesion to the underlying layer of cells in the bladder after exfoliation , allowing the bacteria to enter the latent phase of the infection cascade [ figure 1 ]
the cartoon depicts the stages through which upec ( blue and green ) proceeds during infection of superficial bladder epithelial cells ( sbecs ) .
see text for details studies have demonstrated that the ibc pathway is neither limited to a single upec isolate nor to a single mouse strain .
for instance , ibc formation was found to occur in five genetic backgrounds of inbred mice strains . additionally , the majority of upec strains isolated from women with different uti syndromes [ asymptomatic bacteriuria , recurrent cystitis , pyelonephritis , and acute cystitis ] were competent for ibc formation in the murine cystitis model . although each of the upec strains from the uti syndromes were proficient at ibc formation differences were observed in the size and number of ibcs as well as the kinetics of infection .
intriguingly , ibcs derived from acute cystitis isolates were both significantly smaller and less numerous when compared to the other uti syndromes .
these results suggest that ibc formation is a common attribute of upec strains and that the propensity of a given strain to form ibcs may be associated with persistence in the urinary tract . strikingly , evidence of the ibc pathogenic cycle was found in the urines of women with acute cystitis .
rosen et al . , observed that ibcs shed into the urine of women with cystitis were indistinguishable from exfoliated ibcs found in mouse urine upon subsequent histologic examination .
furthermore , filamentous uropathogens 20 m in length were found in the urines from several other gram - negative species including upec , proteus mirabilis , klebsiella pneumoniae , and enterobacter aerogenes .
however , neither evidence of exfoliated ibcs nor filaments were found in the urines of women with an asymptomatic infection and women infected by gram - positive bacteria .
this evidence is consistent with recent reports that intracellular bacteria were not observed in biopsies of patients with neurogenic bladder who experience numerous episodes of asymptomatic bacteriuria .
indeed , when biopsies were performed on women with recurrent utis it was determined that 88% of biopsies contained bacteria upon the urothelium by scanning electron microscopy sem analysis and bacteria could be recovered from 50% of the biopsies of women with sterile urine cultures .
the evidence that an intracellular bacterial reservoir is established in both mice[1520 ] and humans has several important clinical considerations for the management of utis .
prophylactic antibiotic therapy has been a mainstay for treatment of recurrent urinary tract infections . with the rise in antibiotic - resistant pathogens and the discussions of whether all patients require antibiotic therapies , it is important to identify new algorithms for treatment built upon evidence - based investigations . to fully evaluate the effectiveness of antibiotic treatment against utis , mulvey and
colleagues recently evaluated the efficacy of eight different classifications of antibiotics on the eradication of intracellular upec in the mouse model for human uti .
these investigators determined that three - day oral treatment with any of the antibiotics , including those capable of intracellular accumulation , had no effect on the latent bacterial burden in the bladder .
importantly , the quiescent intracellular reservoirs avoided elimination despite the presence of antibiotic concentrations in the urine that greatly exceeded the minimal inhibitory concentration . when the bacteria were liberated from the bladder epithelium , the antibiotic sensitivity was enhanced , suggesting that the intracellular compartment , as well as the ibc structure , provide significant protection from antibiotic therapies .
these observations further complicate evaluation of treatments given that sterile urine does not correlate with sterile bladder tissue .
these results could provide an explanation to the epidemiological findings that upwards of 68% of recurrences are caused by bacteria that are isogenic to the original strain.[3339 ] furthermore , each ibc is clonal , that is , invasion of a single upec is sufficient to support all of the growth observed within a single epithelial cell .
in addition , a single epithelial cell can support at least 10 upec individuals , indicating that invasion of a single bacterium is sufficient to initiate an acute infection . this new paradigm for recurrence parallels clinical observations and changes the management for treatment of these infections from a hygienic problem to an antibiotic - insensitive latent infection . in summary , researchers are beginning to uncover the molecular details that underlie utis .
specifically , new diagnostic and therapeutic approaches based on the combination of host genetic factors , innate immunity , and bacterial virulence factors are needed to identify the patients most prone to utis to avoid the cost and potential side - effects of treatment .
fortunately , advances being made in both basic and clinical scientific research of the urinary tract in patients and animal models are providing some explanations and insight into the clinical problems that remain with the management of utis .
improved knowledge of the genetics , uropathogen virulence factors , and host immune responses to utis will enhance the ability of clinicians to more readily distinguish high - risk patients from uncomplicated patients , which is necessary to prevent major sequelae in these high - risk patients . | urinary tract infections ( utis ) represent one of the most commonly acquired diseases among the general population as well as hospital in - patients , yet remain difficult to effectively and consistently treat .
high rates of recurrence , anatomic abnormalities , and functional disturbances of the urinary tract all contribute to the difficulty in management of these infections .
however , recent advances reveal important molecular and genetic factors that contribute to bacterial invasion and persistence in the urinary tract , particularly for the most common causative agent , uropathogenic escherichia coli .
recent studies using animal models of experimental utis have recently provided mechanistic insight into the clinical observations that question the effectiveness of antibiotic therapy in treatment .
ultimately , continuing research will be necessary to identify the best targets for effective treatment of this costly and widespread infectious disease . |
however , it is also associated with variable post - operative consequences like pain , swelling and trismus .
corticosteroids are some of the most common pharmaceutical agents employed to manage these sequelae . of these
, methylprednisolone is a highly selective , intermediate acting , synthetic glucocorticosteroid , more potent than hydrocortisone and dexamethasone .
surprisingly , few studies have evaluated the use of oral methylprednisolone alone or in combination with popular non - steroidal anti - inflammatory drugs ( nsaids ) , in spite of obvious benefits .
this study was conducted as part of a dissertation , after approval by the ethical committee of the concerned institution and university , and with detailed , written , informed consent from each patient .
thirty consenting patients in the age group of 18 - 50 years , with clinical and radiographic presence of bony impaction of the third molar , were included in this study . only impacted teeth , asymptomatic at the time of surgery and requiring surgical removal ( including bone removal ) ,
these 30 patients were randomly allotted to one of the two experimental groups of 15 each , namely the methylprednisolone and the diclofenac sodium group a and the diclofenac sodium and the placebo group b. patients who were excluded from the study were those with any systemic disease , with clinical symptoms in the third molar at the time of presentation , pre - existing chronic infection , giving a history of use of corticosteroids over the previous 3 months or allergy to the drug .
pre - operative investigations were performed after taking consent of the patient and their case histories .
the details recorded pre - operatively included the tooth to be removed , type of impaction , inter - incisal distance ( iid ) and facial baseline measurement ( fm ) .
fm was determined by a modification of the tape measuring method described by gabka and matsumara .
three measurements were made between five reference points : mandibular angle ( g ) , tragus ( t ) , lateral canthus of the eye ( l ) , outer corner of the mouth ( a ) and soft tissue pogonion ( p ) by using a measuring tape to follow the contour of the face [ figure 1 ] .
iid was evaluated by measuring the distance on maximal opening between the right maxillary and mandibular incisor with a vernier caliper [ figure 2 ] .
three facial measurements between five reference points : mandibular angle ( g ) , tragus ( t ) , lateral canthus of the eye ( l ) , outer corner of the mouth ( a ) and soft tissue pogonion ( p ) using a measuring tape measuring maximal distance between the right maxillary and the mandibular incisor with a vernier caliper all surgeries were carried out by the same surgeon . under aseptic condition and under local anesthesia ,
bone buccal and distal to the tooth was removed using a bur and the tooth was delivered out of the socket .
after wound closure with a 3 - 0 black silk suture material , a wet gauze pack was placed .
all the patients were given tablets of diclofenac sodium 50 mg orally three times a day for 3 days and amoxicillin 500 mg three times a day orally for 5 days .
patients in the group a were additionally prescribed tablets of methylprednisolone 8 mg orally three times a day for 3 days , whereas patients in group b were given placebo tablets ( sugar pills ) in similar dosage .
patients were recalled after 24 and 72 h and on the seventh post - operative day for follow - up .
pain , iid and swelling ( fm ) were recorded after 24 and 72 h and the on seventh post - operative day .
pain was recorded using a pre - calibrated 100 mm visual analogue scale ( vas ) .
the end points of the scale were no pain and pain could not be worse .
the means from the vas were classified as none / no pain ( 0 - 10 mm ) , mild pain ( 11 - 30 mm ) , moderate pain ( 3160 mm ) and severe pain ( 61 - 100 mm ) .
all patients were given a questionnaire to evaluate the post - operative morbidity and their quality of life following the surgical procedure , and it was correlated with the clinical presentation .
the results were evaluated on the basis of the chi - square test ( for possible association between two categorical variables ) and the independent sample t - test ( to test the equality of the two samples ) ( p = 0.05 ) .
thirty consenting patients in the age group of 18 - 50 years , with clinical and radiographic presence of bony impaction of the third molar , were included in this study . only impacted teeth , asymptomatic at the time of surgery and requiring surgical removal ( including bone removal ) , were included .
these 30 patients were randomly allotted to one of the two experimental groups of 15 each , namely the methylprednisolone and the diclofenac sodium group a and the diclofenac sodium and the placebo group b.
patients who were excluded from the study were those with any systemic disease , with clinical symptoms in the third molar at the time of presentation , pre - existing chronic infection , giving a history of use of corticosteroids over the previous 3 months or allergy to the drug .
pre - operative investigations were performed after taking consent of the patient and their case histories .
the details recorded pre - operatively included the tooth to be removed , type of impaction , inter - incisal distance ( iid ) and facial baseline measurement ( fm ) .
fm was determined by a modification of the tape measuring method described by gabka and matsumara .
three measurements were made between five reference points : mandibular angle ( g ) , tragus ( t ) , lateral canthus of the eye ( l ) , outer corner of the mouth ( a ) and soft tissue pogonion ( p ) by using a measuring tape to follow the contour of the face [ figure 1 ] .
iid was evaluated by measuring the distance on maximal opening between the right maxillary and mandibular incisor with a vernier caliper [ figure 2 ] .
three facial measurements between five reference points : mandibular angle ( g ) , tragus ( t ) , lateral canthus of the eye ( l ) , outer corner of the mouth ( a ) and soft tissue pogonion ( p ) using a measuring tape measuring maximal distance between the right maxillary and the mandibular incisor with a vernier caliper all surgeries were carried out by the same surgeon . under aseptic condition and under local anesthesia , a modified terrance ward 's incision was placed and the mucoperiosteal flap was raised .
bone buccal and distal to the tooth was removed using a bur and the tooth was delivered out of the socket .
after wound closure with a 3 - 0 black silk suture material , a wet gauze pack was placed .
all the patients were given tablets of diclofenac sodium 50 mg orally three times a day for 3 days and amoxicillin 500 mg three times a day orally for 5 days .
patients in the group a were additionally prescribed tablets of methylprednisolone 8 mg orally three times a day for 3 days , whereas patients in group b were given placebo tablets ( sugar pills ) in similar dosage .
patients were recalled after 24 and 72 h and on the seventh post - operative day for follow - up .
pain , iid and swelling ( fm ) were recorded after 24 and 72 h and the on seventh post - operative day .
pain was recorded using a pre - calibrated 100 mm visual analogue scale ( vas ) .
the end points of the scale were no pain and pain could not be worse .
the means from the vas were classified as none / no pain ( 0 - 10 mm ) , mild pain ( 11 - 30 mm ) , moderate pain ( 3160 mm ) and severe pain ( 61 - 100 mm ) . fm and iid were measured as done previously .
all patients were given a questionnaire to evaluate the post - operative morbidity and their quality of life following the surgical procedure , and it was correlated with the clinical presentation .
the results were evaluated on the basis of the chi - square test ( for possible association between two categorical variables ) and the independent sample t - test ( to test the equality of the two samples ) ( p = 0.05 ) .
the methylprednisolone diclofenac sodium group ( group a ) and the nsaid only ( diclofenac sodium ) group ( group b ) consisted of 15 patients each .
the age range of the patients in group a was 19 - 40 years , with a mean age of 25.9 years whereas in group b , it was 19 - 35 years , with a mean age of 25.4 years , with no significant difference ( p = 0.781 ) in both groups as found through the t - test . nor was any significant difference ( p = 0.456 ) found in the number of patients in each sex between the two groups , based on a chi - squared statistic . in both
the groups , seven patients ( 46.7% ) had their left and eight patients ( 53.3% ) had their right mandibular third molars extracted , with an insignificant mean difference ( p = 0.587 ) on applying the chi - square test . in group a [ graph 1 ] , the mean pain on the vas was 26.2614.64 mm after 24 h , which lies in the range of mild pain . after 72 h , it was 11.06 10.27 mm ( no pain to mild pain ) , with 0.6 1.45 mm ( no pain ) on the seventh day .
in group b , mean pain seen was 50 18.92 mm ( moderate pain ) after 24 h , 28.46 13.42 mm ( mild pain ) after 72 h and 9.53 9.79 mm ( i.e. no pain ) on the seventh day .
after applying the chi - square test , it was statistically analyzed that the mean difference in pain experienced between the two groups was significant ( p = 0.015 ) at 24 h , whereas it was highly significant at 72 h ( p = 0.001 ) and on the seventh day ( p = 0.005 ) . comparing mean pain experienced on the vas by patient group a and patients group b in group a [ graph 2 ] , the mean inter - incisal distance ( miid ) pre - operatively was 47.26 4.62 mm , 37.333.97 mm after 24 h , 43.934.008 ) mm after 72 h and 46.864.79 mm on the seventh post - operative day . in group b ,
miid measured pre - operatively was 45.334.169 mm , 32.06 6.67 mm after 24 h , 36.866.446 ) mm after 72 h and 42.535.488 mm on the seventh post - operative day .
the mean difference in inter - incisal distance between the two groups after applying the independent sample t - test was statistically insignificant ( p = 0.239 ) pre - operatively , but was statistically significant after 24 ( p = 0.014 ) and 72 h ( p = 0.001 ) and on the seventh post - operative day ( p = 0.001 ) . comparing the mean inter - incisal distance between group a and group b in group a [ graph 3 ] , mean fm measured pre - operatively was 379.623.36 mm , 391.224.119 mm after 24 h , 382.8623.18 ) mm after 72 h and 380.223.32 on the seventh post - operative day . in group
b , the mean fm measured pre - operatively was 355.424.49 mm , 383.325.93 mm after 24 h , 371.5323.76 mm after 72 h and 357.7324.38 mm on the seventh post - operative day .
the mean difference in change in fm between the two groups after applying the independent sample t - test was statistically highly significant after 24 h ( p = 0.001 ) and 72 h ( p = 0.0001 ) , and still significant on the seventh post - operative day ( p = 0.047 ) . comparing the mean facial measurement between group a and group b no post - operative complications were found in any of the two groups .
in group a [ graph 1 ] , the mean pain on the vas was 26.2614.64 mm after 24 h , which lies in the range of mild pain . after 72 h , it was 11.06 10.27 mm ( no pain to mild pain ) , with 0.6 1.45 mm ( no pain ) on the seventh day . in group
b , mean pain seen was 50 18.92 mm ( moderate pain ) after 24 h , 28.46 13.42 mm ( mild pain ) after 72 h and 9.53 9.79 mm ( i.e. no pain ) on the seventh day .
after applying the chi - square test , it was statistically analyzed that the mean difference in pain experienced between the two groups was significant ( p = 0.015 ) at 24 h , whereas it was highly significant at 72 h ( p = 0.001 ) and on the seventh day ( p = 0.005 ) . comparing mean pain experienced on the vas by patient group a and patients group b
in group a [ graph 2 ] , the mean inter - incisal distance ( miid ) pre - operatively was 47.26 4.62 mm , 37.333.97 mm after 24 h , 43.934.008 ) mm after 72 h and 46.864.79 mm on the seventh post - operative day . in group
b , miid measured pre - operatively was 45.334.169 mm , 32.06 6.67 mm after 24 h , 36.866.446 ) mm after 72 h and 42.535.488 mm on the seventh post - operative day .
the mean difference in inter - incisal distance between the two groups after applying the independent sample t - test was statistically insignificant ( p = 0.239 ) pre - operatively , but was statistically significant after 24 ( p = 0.014 ) and 72 h ( p = 0.001 ) and on the seventh post - operative day ( p = 0.001 ) . comparing the mean inter - incisal distance between group a and group b
in group a [ graph 3 ] , mean fm measured pre - operatively was 379.623.36 mm , 391.224.119 mm after 24 h , 382.8623.18 ) mm after 72 h and 380.223.32 on the seventh post - operative day . in group
b , the mean fm measured pre - operatively was 355.424.49 mm , 383.325.93 mm after 24 h , 371.5323.76 mm after 72 h and 357.7324.38 mm on the seventh post - operative day .
the mean difference in change in fm between the two groups after applying the independent sample t - test was statistically highly significant after 24 h ( p = 0.001 ) and 72 h ( p = 0.0001 ) , and still significant on the seventh post - operative day ( p = 0.047 ) . comparing the mean facial measurement between group a and group b no post - operative complications were found in any of the two groups .
extraction of the third molar , as with any surgical procedure , results in an intense inflammatory response that consists of edema , erythema , pain , rise in temperature and loss of function .
pain reaches its peak at 6 h post - operatively and swelling reaches its maximum on the first post - operative evening and remains at a high level until the fourth day . extending a surgical procedure more than 10 min results in more post - operative sequelae . an ideal drug for administration
after the surgical removal of an impacted lower third molar should alleviate pain , reduce inflammation , trismus , facilitate healing and cause no undesirable side - effects .
it is now universal knowledge that steroids reduce post - operative complications . in spite of their obvious numerous benefits ,
their use is mostly limited by most surgeons due to their association with a host of potential side - effects , most of which are relative to mineralocorticoid activity and and/or chronic dosing regimens .
hypothalmic pituitary - adrenal ( hpa ) axis suppression , which is a significant phenomenon only with chronic dosing , should not , however , be a contraindication to the use of steroids in third molar surgery .
corticosteroid doses for less than 5 - 7 days result in insignificant hpa suppression and cortisol levels return to the normal value in 5 - 7 days .
such short - term steroid therapy is safe in the absence of contraindications to its use , as listed by pederson et al . in this study ,
glucocorticosteroids for only 3 days were given to minimize potential side - effects and true contraindications .
methylprednisolone is five times more potent than hydrocortisone and has non - existent mineral - corticoid action .
al - khateeb et al . conducted a randomized study to determine the efficacy of dexamethasone versus methylprednisolone , and concluded that pain and trismus was more reduced in the methylprednisolone group .
several other research articles have also shown that swelling and pain that developed after third molar removal were significantly reduced by methylprednisolone , therefore , methylprednisolone was the drug of choice among steroids . for steroids , different modes of administration
the iv route offers instantaneous blood levels , but while high - dosing iv studies have demonstrated significant short - term improvements , the effects were not sustained .
rebound swelling can occur if the duration of use is inadequate ; therefore , it is important to maintain the levels of short - duration steroid formulations for more than 1 day .
the i m route provides an anti - inflammatory effect for long , but it may cause a higher risk of adrenaline suppression .
the convenience of oral dosing , in contrast , has general appeal , with the oral route , glucocorticosteroids are rapidly and almost completely absorbed , making them as effective as parenteral avenues .
therefore , in the present study , the oral route and post - operative dosing were selected .
methyprednisolone has an intermediate half - life of 18 - 36 h. when glucocorticosteroids are orally administered , repeated dosing is required to maintain adequate blood levels throughout the immediate post - operative period .
there is a need to continue corticosteroid therapy for a minimum of 3 days because swelling in patients treated with steroids does not appear to peak until the third day after surgery .
previous investigations , however , are not convincing regarding clinical improvement following oral administration because of low dosing schedules and short observation periods .
corticosteroid doses to be significantly effective should range from 80 to 625 mg hydrocortisone equivalent anti - inflammatory dosage , which in case of methylprednisolone is 16125 mg .
it has also been corroborated that a dosage of methylprednisolone of less than or equal to 40 mg does not produce any adverse systemic effects .
therefore , in the present study , 24 mg of methylprednisolone was given in three divided doses for three days and no side - effects were observed following administration of the steroid .
it has been established that tapering off doses is not mandatory in short - term dosing . in this research too , steroid was terminated without tapering dose .
a few authors believe that corticosteroids are the drug of choice in preventing edema ; however , nsaids may be required for better pain relief .
previous studies have proven that diclofenac sodium reduces pain and swelling more , if not equally effective , when compared with other single anti - inflammatory drugs and/or fixed drug combinations after impacted third molar removal .
it can therefore be used as an agent to control post - operative sequelae and was the nsaid of choice for this study . of its three dispensable forms , namely intravenous infusion , soft gel and tablets ,
per - oral tablets were favored because of the limited duration of action and higher cost of treatment with infusions and gel .
further , pre - operative use of nsaids may not offer a pre - emptive analgesic effect in patients who have had adequate analgesia during the surgery .
continued use of analgesic drugs during the post - operative period is therefore recommended by most authors , and was followed in the present study too . while the sample size for this study is not large by any means ,
it was nevertheless adequate for statistical purposes , given the implied limitation of time period in a dissertation .
a placebo group comprising of patients not being given nsaids was thought implausible and corrupt on moral and ethical grounds , because pain follows this kind of surgery , even if the severe post - operative pain is of a short duration .
calibrating and measuring pain is always a difficult proposition . of the various pain scales available for adults , namely , vas , verbal numerical rating scale and verbal descriptor scale , the vas was chosen for this study .
it is a pain scale where each patient indicates his / her perceived pain intensity along a 100 mm horizontal line . whether it is a linear scale or not , or whether the data are ratio or ordinal , is still debatable .
but , it is undoubtedly the most widely used and popular pain scale , which correlates well with acute pain and with minimal error , and easily lends itself to statistical analysis as well .
simultaneous administration of glucocorticosteroid may synergize the anti - inflammatory effect of nsaids and contribute to the reduction of inflammatory exudates as well as edema and pain .
therefore , the co - administration of diclofenac and methylprednisolone may be expected to reduce post - operative pain more than that achieved with either of them alone .
, buyukkurt et al . , hyrkas et al . , schultze - mosgau et al . ( 1995 ) and others , who found the nsaid steroid combination to be better than nsaid alone in the treatment of post - operative pain , trismus and swelling after dental surgical procedures .
it is well known that steroids suppress immune function , increasing the potential risk of infection .
however , use of usual dosages of corticosteroids over 3 - 4 days has no significant adverse effects on wound healing , as further emphasized by our study .
we did not encounter any cases of infection , especially in the combination group , because glucocorticosteroids were used only for 3 days and also because antibiotics were administered to both the groups .
it was concluded that a single class of drugs is not maximally effective in controlling both post - operative pain and swelling and trismus . the combination of diclofenac sodium ( a nsaid ) and methylprednisolone ( a corticosteroid ) is more effective than diclofenac sodium alone in the sequelae of oral surgical procedures .
therefore , this pharmaceutical combination should be considered for the attenuation of post - operative sequelae in healthy patients undergoing surgical removal of the impacted third molar .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | aim and objectives : this study evaluated the efficacy of oral methylprednisolone and diclofenac sodium on post - operative sequelae after third molar surgery.settings and design : a randomized double - blind clinical trial was conducted ( with institutional and university approval for dissertation ) to evaluate the effect of methylprednisolone with diclofenac sodium ( group a ) as compared with diclofenac sodium and placebo ( group b ) on three variables : pain , swelling and trismus , after third molar surgery.materials and methods : thirty consecutive consenting patients for surgical removal of mandibular impacted third molar were randomly placed into two groups of 15 each ( groups a and b ) .
pain , swelling and trismus were observed by visual analog scale , facial measurements and inter - incisal opening .
scores were recorded after 24 and 72 h and on the seventh post - operative day .
results were subjected to the chi - square test and independent sample t - test ( p = 0.05).results : mean difference in pain experienced between the two groups was statistically significant at 24 h ( p = 0.015 ) and 72 h ( p = 0.001 ) and on the seventh day ( p = 0.005 ) .
difference in inter - incisal distance was insignificant ( p = 0.239 ) pre - operatively , but significant after 24 h ( p = 0.014 ) and 72 h ( p = 0.001 ) and on the seventh post - operative day ( p = 0.001 ) .
mean difference in swelling was highly significant after 24 h ( p = 0.001 ) and 72 h ( p = 0.0001 ) and on the seventh post - operative day ( p = 0.047).conclusions : the combination of oral dose of methylprednisolone ( a corticosteroid ) diclofenac sodium ( a non - steroidal anti - inflammatory drug ) was found to be more effective than diclofenac sodium alone on the sequelae of surgical removal of impacted mandibular third molar . |
we present a case that occurred at bunbury hospital where a kirschner wire ( k - wire ) has been propelled distally down the intramedullary canal of the ulna when it made contact with the drill . in both cases ,
ms r is a 68-year - old female who had a fall off a push bike and presented to the emergency department with a comminuted fracture of the left olecranon .
she was taken to theatre for an open reduction and internal fixation with a tension band wire construct . the fracture was reduced and held with two intramedullary k - wires .
a hole was drilled in the proximal ulna to secure the cerclage wire distal to the fracture . during this process ,
the drill made contact with the k - wire and it was propelled distally within the intramedullary canal .
the wire was secured and removed with a set of artery clips ( fig . 1 ) .
once removed , the fracture was re - reduced with two bicortical k - wires and a set of artery clips were used to secure them ( fig . 1 ) .
figure 1:left k - wire secured and removed with a pair of artery clips . right
there is currently no published literature on avoiding intra - operative k - wires migration into the distal intramedullary canal of the ulna during olecranon open reduction and internal fixation .
the literature on k - wire migration in the context of olecranon orif is limited to outward migration of the k - wires post - operatively [ 15 ] .
there is also literature on post - operative intra - thoracic migration of k - wires post - clavicle orif . in all cases ,
migration of the k - wire has been attributed to its smooth circular surface , which is prone to loose fixation on the surrounding bone . to prevent migration of the k - wires ,
the rates of post - operative k - wire migration in olecranon tension band wiring are noted to be between 20 and 45% [ 13 ] .
the techniques discussed in the literature are to prevent the k - wires from backing out post - operatively . in the context of upper extremity and thoracic trauma ,
sharma et al . found that post - operative migration was eliminated through bending the end of the k - wire . in this situation , with the k - wire
being pushed into the distal ulna , bending the end would not be appropriate . in the first case , the cerclage wire needs to be positioned prior to bending . in the second case , where the k - wires are used to temporarily hold the fracture , it would make repositioning and removal of the wires difficult .
some studies have delineated methods that can be employed to minimize the risk of intra - operative migration of the k - wire into the ulna .
larsen et al . suggested the use of non - sliding pins that have a sharp k - wire on one end and a bulb with a hole for the cerclage wire on the other end .
the smaller diameter of the k - wire compared with the bulb stops it from migrating distally .
non - sliding pins however are more expensive than standard k - wires and removing them , if required , is also technically more challenging .
for these reasons , although good results have been produced since the mid-1980s , they are not widely used . in both
described instances of distal intra - operative migration of k - wires , the wires were inserted unicortically , within the ulna intramedullary canal .
the modified technique of bi - cortical wire placement has shown benefits in terms of lower complication profile : with lower rates of wire migration - post - operatively and also improved stability [ 5 , 8 , 9 ] .
critics have argued that bi - cortical positioning of k - wires could cause iatrogenic damage to the ulna artery and the median nerve .
cadaveric studies have demonstrated that this could be avoided by ensuring that the wire end does not protrude beyond the anterior ulnar cortex by > 1 cm at a distance of 1.5 cm distal to the coronoid .
as well as offering superior biomechanical fixation , bi - cortical positioning of the k - wires offers greater resistance to migration of the wire both intra - operatively and post - operatively .
it may be employed as a strategy to avoid the wire from migrating through the medullary canal of the ulna when the drill makes contact with it .
it may , however , cause the wire to be propelled out of the ulna .
firstly , we suggest drilling the distal hole prior to placement of the k - wires .
secondly , the k - wires can be secured with artery clips to ensure that the wires remain in position . | at bunbury regional hospital , we recently saw a case where kirschner wires ( k - wires ) , used during open reduction and internal fixation of olecranon , were propelled down the intramedullary canal intraoperatively .
the k - wire was only secured unicortically . from our experience
, we have developed steps to prevent this complication .
first , where the wires are being used as part of a tension band wire construct , we suggest drilling the distal hole prior to the placement of the k - wires and , secondly , where k - wires are being used to secure the fracture fragment , the k - wires can be secured with artery clips to ensure that the wires remain in position . |
df of the bone is an extremely rare , lytic , locally aggressive but non - metastatic primary bone tumor .
histologically , df consists of wavy fibroblasts and abundant collagenous tissue , bears a close resemblance to a desmoid tumor of the soft tissues and exhibits a characteristic production of collagen fibers ( 1 ) .
df may involve any bone , but is most often found in the mandible ( 22% ) and less frequently in the femur ( 15% ) , pelvic bones ( 13% ) , radius ( 12% ) and tibia ( 9% ) ( 2,3 ) .
the incidence of df is estimated to be 0.06% among all bone tumors and 0.3% among benign bone tumors ( 1,35 ) .
df of the bone most often occurs during the first three decades of life and exhibits no gender predilection ( 6 ) .
the most common symptoms include pain and swelling , and 12% of the patients present with pathological fractures . however , a number of patients may be asymptomatic ; thus , the tumor is often incidentally identified ( 7 ) .
on radiographs , the lesion is lytic , occasionally with poorly defined borders , while on computed tomography ( ct ) it is mildly hyperdense , with thin sclerotic margins , with or without destruction of the contiguous cortex , and exhibits low signal intensity on t2-weighted magnetic resonance imaging ( mri ) .
the recurrence rates in patients treated with and without resection are 17 and 5572% , respectively ( 7 ) .
this disease was first identified by jaffe in 1958 ( 8) . to date , ~200 cases of desmoplastic fibroma of the bone have been reported in the literature ; of these , 13 have been reported in the femur ( 920 ) , with only 2 in the proximal femur ( 9,14 ) and the remaining in the distal femur ( 1013,1518,20,21 ) .
image - guided percutaneous biopsy is becoming an increasingly accepted modality for initial diagnosis in the majority of musculoskeletal tumors .
however , despite its well - documented specificity and sensitivity ( 22,23 ) , there has been continued reluctance on the part of orthopedic oncologists to accept this technique as the modality of choice for initial biopsy in primary bone tumors .
detractors of percutaneous needle biopsy suggest that sufficient material can not be obtained to ensure an accurate diagnosis , particularly in patients with sclerotic bone lesions ( 24 ) .
historically , all biopsies to date were performed via an open surgical biopsy , which remains the gold standard for a proportion of physicians .
the reported accuracy of an open surgical biopsy , whether incisional or excisional , is 98% ( 23,25 ) , whereas needle biopsy under ct guidance has a reported accuracy estimated between 78 and 98.4% ( 26 ) .
the needle pathway is determined on the basis of the expected definitive treatment , in order to avoid intercompartmental contamination , minimize the amount of biopsy tract to be removed at the time of definitive surgery in case of malignancy , and avoid neurovascular structures .
the choice of ct vs. fluoroscopy is generally determined on the basis of three factors , namely size , location and attenuation ( 27 ) . to the best of our knowledge ,
this study presents the only published case of minimally invasive diagnosis of df of the bone .
written informed consent was obtained from the patient for the publication of the case details .
in august 2015 , a 36-year - old african female patient presented to the emergency room of arcispedale santa maria nuova - irccs ( reggio emilia , italy ) , which is a medium - sized general hospital , complaining of pain in the rib cage , knees and lower thighs following a mild trauma she had suffered a few days earlier after a fall , for which she had not been hospitalized . there were no evident abnormalities on physical examination , with the exception of pain on percussion .
radiography showed a well - defined , eccentric , low - density lesion in the distal metaphysis of the left tibia ( fig .
the contiguous cortex was apparently not eroded , but minimally thinned due to compression by the expanding tumor .
the patient was advised on an outpatient basis to undergo a magnetic resonance imaging ( mri ) scan and a subsequent orthopedic evaluation . due to the x - ray and bone mri results , the orthopedic surgeon suggested an image - guided bone biopsy . after a ct scan confirmed the location and imaging characteristics of the lesion ( solid , well - circumscribed , no cortex erosions ) , local anesthesia with lidocaine
was administered , a 17-gauge bone biopsy needle ( osteo - site ratchet ; cook medical , limerick , ireland ) was inserted , passing through the anterolateral face of the skin overlying the tibia , the tibialis anterior muscle and , finally , the cortex , into the core of the lesion ( fig .
2 ) and a single specimen was extracted . since no peri - procedural complications occurred , the patient was discharged after a few hours in good health and without the need for oral pain medication , with the recommendation to refer to her general practitioner in case of any discomfort .
histological examination of the bone specimen revealed focal peripheral proliferation of spindle - shaped cells resembling fibroblasts , showing strong reactivity for anti - vimentin antibody ; the appearance was in accordance with a fibroblastic desmoid tumor of the bone .
percutaneous needle bone biopsy is a safe and accurate method ( 22,25,2729 ) for obtaining a tissue diagnosis . in general , better results are obtained with lesions of the extremities or pelvis compared with those in the spine ( 30 ) .
percutaneous needle biopsies have a very low complication rate ( 1.1% ) , whereas open biopsy has a complication rate of 16% ( 31 ) .
a serious complication of percutaneous biopsy is the risk of seeding malignant cells along the needle track , particularly if the lesion is a sarcoma , which would necessitate resection of the needle track en bloc with the tumor in limb - sparing reconstructive surgery ( 32 ) .
fine - needle aspiration ( fna ) may differentiate between a metastasis and a benign lesion ; however , core biopsy is superior to fna in determining cell type and tumor grade , which is necessary for the diagnosis of primary bone tumor . in our experience , of the various options that are available for bone biopsy ( 33 ) ,
the osteo - site ratchet ( cook medical ) 13-gauge coaxial bone biopsy system is effective in obtaining tissue from sclerotic lesions where normal bone cortex needs to be traversed . in general
, all bone biopsies are performed under local anesthesia ; however , conscious sedation may be required in children or uncooperative patients .
an adequate biopsy sample is considered to be that providing adequate abnormal tissue for the pathologist to reliably make a diagnosis .
we have found that , despite the low incidence of df , a single long core of abnormal tissue obtained with a 13-gauge needle is sufficient . in conclusion , this study reported the case of a 36-year - old african female patient with a df of the bone incidentally detected by an x - ray and diagnosed by means of percutaneous ct - guided bone biopsy .
this case differs from other published studies due to the minimally invasive , preoperative , image - guided histological diagnosis .
this type of diagnosis is commonly made based on a resection sample obtained through incisional biopsy under general anesthesia .
the results of image - guided biopsy in terms of cost - to - benefit ratio are similar to those of open biopsy , with reduced comparable morbidity . | desmoplastic fibroma ( df ) is a benign , rare fibroblastic intraosseous neoplasm histologically resembling a desmoid soft tissue tumor .
although classified as benign , df frequently exhibits an aggressive behavior , has a moderate - to - high recurrence rate , and often causes pathological fractures and extensive bone destruction .
this case report presents an incidentally detected df of the tibia , which was diagnosed using a minimally invasive approach .
a 36-year - old african female patient was referred to the department of diagnostic imaging of arcispedale santa maria nuova - irccs ( reggio emilia , italy ) , to be examined by a computed tomography scan on an outpatient basis , after an x - ray examination of the tibia , which was performed after an injury to exclude the presence of a fracture , revealed a hyperlucency of unknown origin .
the aim of this study was to discuss the clinical , histological , immunohistochemical and radiographic characteristics of this rare neoplasm , with a focus on image - guided bone biopsy . |
transurethral resection of the prostate ( turp ) is considered the best option for treating benign prostatic enlargement ( bpe ) ; however , it is associated with significant bleeding . in recent years
, we have seen a gradual increase in life expectancy and , particularly in more developed countries , an increased incidence of cardiovascular diseases , metabolic syndrome , and cerebrovascular stroke , all of which are conditions that require oral antiplatelet or anticoagulation ( oap / oac ) therapy . owing to the severe bleeding risk in these patients ,
turp is contraindicated . furthermore , suspending oap / oac therapy is also not recommended .
for example , the use of laser technology has advantages in patients receiving oap / oac therapy who have severe comorbidities and bleeding disorders .
thulium vaporesection of the prostate ( thuvarp ) with a 2-m continuous - wave ( cw ) thulium - doped yttrium - aluminum - garnet ( tm : yag ) laser has been established as a new approach for bpe treatment .
this laser has a peak absorption spectrum closer to that of water than the holmium laser , which causes increased tissue vaporization and results in reduced penetration depth .
however , because the thulium laser operates in cw mode , it permits smaller and more precise cutting and consequently decreased risk of bleeding .
the aim of this study was to demonstrate the efficacy and safety of vaporesection with the 120-w tm : yag laser ( revolix duo ) in patients with bpe receiving systemic oap / oac therapy and to evaluate medium - term functional outcomes .
between october 2011 and december 2013 , a total of 103 patients with lower urinary tract symptoms due to bpe were enrolled in the study , and data were retrospectively analyzed .
a , oap / oac therapy was interrupted for 10 days before the operation and patients were switched to low molecular weight heparin ( lmwh ) until 2 weeks after the procedure . in group
group b patients were also divided into subgroups according to the oap / oac therapy that they were using : warfarin , acetyl salicylic acid ( asa ) , clopidogrel , ticlopidine , or asa+clopidogrel . according to the standard protocol of the university hospital in which the study was carried out ,
each patient was informed of the procedure upon admission and signed an informed consent allowing data collection for research purposes .
the study design was in accordance with the helsinki declaration , conformed to the committee on publication ethics guidelines , and was approved by the institutional review board of the department of human pathology , section of urology , university of messina , messina , italy in which it was performed .
all the design , analysis , data interpretation , drafting , and revisions followed the strobe ( strengthening the reporting of observational studies in epidemiology ) statement as well as the guidelines for reporting observational studies , available through the equator ( enhancing the quality and transparency of health research ) network .
all operations were performed by a single surgeon ( cm ) with extensive experience in laser prostatectomy and prostate resection .
the thuvarp technique used in this study was the same described in our previous publication .
the procedure was performed by using the 120-w 2-m cw tm : yag laser ( revolix duo ) .
the revolix duo has 2 laser generators : ( 1 ) a 120-w cw mode for soft tissue use and ( 2 ) a 20-w pulsed head to holmium for lithotripsy .
a 550-m core nude - ended fiber ( rigifib ; ohg , lisa laser , katlenburg - lindau , germany ) was used in combination with a 26-f continuous - flow laser resectoscope ( karl storz gmbh & co. kg , tuttlingen , germany ) .
incisions were made on the bladder neck at the 5 and 7 o'clock positions and vaporesection was performed on the median lobe .
finally , vaporesection of the lateral lobe was performed until the prostatic capsule was reached . by performing this technique ,
small tissue chips are obtained , which were evacuated through the resectoscope sheath . at the end of every procedure ,
a 20-f foley catheter was placed and bladder irrigation was performed in the case of hematuria .
the catheter was routinely removed 48 to 72 hours after the procedure regardless of preoperative prostate size when the urine was clear .
cefazolin ( 2 g ) was administered as a short protocol 1 hour before the procedure .
data collected included operative time , hospital stay , catheterization time , and hemoglobin values pre- and postoperatively .
in addition , maximal flow rate ( qmax ) , postmicturition residual urine ( pmr ) , international prostate symptom score ( ipss ) , and quality of life ( qol ) scores were assessed immediately before surgery ( baseline ) and at 3 , 6 , 12 , 18 , and 24 months after treatment .
prostate - specific antigen ( psa ) values were assessed preoperatively and at 12 and 24 months .
baseline variables were described by using mean and standard deviation or percentages , as appropriate .
mann - whitney u - tests were used to evaluate the difference between quantitative measurements that had nonparametric distributions .
a total of 103 patients who underwent laser prostatectomy were included in the study . in group
a , anticoagulant therapy was interrupted and switched for lmwh in 47 patients , whereas in group b , 56 patients underwent surgery while still on their anticoagulant therapy regimen . the mean ages of groups a and b were 68.56 and 69.66.8 years , respectively .
the mean operation , hospitalization , and catheterization durations are given in table 1 for each group .
the mean preoperative prostate volumes were 66.814.1 and 65.39.5 ml for groups a and b , respectively .
the mean amounts of resected prostate tissue were 43.810.9 and 43.16.7 ml for groups a and b , respectively .
in group b , 7 patients were on warfarin , 23 patients were on asa , 14 were on ticlopidine , 8 were on clopidogrel , and 4 patients were on asa + clopidogrel combination treatment .
the mean preoperative hemoglobin level of patients in group a was 13.71 and that of patients in group b was 13.51.5 .
the mean postoperative hemoglobin level of patients in group a was 131 and that of patients in group b was 13.21.5 . neither pre- nor postoperative hemoglobin values were statistically significantly different between groups a and b. however , the drop in hemoglobin values in the pre- and postoperative periods was significantly higher in group a : 0.70.5 and 0.30.5 in groups a and b , respectively ( p<0.005 ) .
when subgroup analyses were performed within group b , the mean drop in hemoglobin was significantly lower in patients who were on warfarin and ticlopidine compared with patients in group a ( p<0.005 , p<0.005 , respectively ) .
the mean pre- and postoperative hemoglobin values along with the mean drop in hemoglobin are given for each group in fig .
when the ipss values were compared between groups a and b , group a had significantly lower ipss values at 3 , 12 , 18 , and 24 months after surgery ( p<0.005 , p<0.05 , p<0.005 , p<0.001 , respectively ) .
however , in the preoperative period , the ipss was 15.9 in group a and 18.6 in group b and the difference was not statistically significant .
no statistically significant differences were found between groups a and b or subgroups of group b in qol scores , qmax values , or pmr volumes ( ml ) . during the whole follow - up period ,
the mean ipss , qmax , and pmr values along with qol scores for groups a and b and the subgroups of group b are given in table 2 and fig .
the mean psa values in the preoperative period were 3.11.5 and 3.11.9 in groups a and b , respectively . at 12 months after the operation , the values were 2.30.9 and 2.41.2 , and at 24 months , the values were 2.20.8 and 2.30.9 for groups
a , 38 patients had no complications , whereas 4 had transient urge incontinence , 4 had urinary tract infection ( uti ) , and 1 patient needed recatheterization for slight hematuria for 5 days which resolved without further intervention .
no complications occurred in 41 patients in group b ; however , in 6 patients , transient urge incontinence occurred ; 7 patients had a uti ; 1 patient needed recatheterization for 4 days due to hematuria ; and 1 patient needed transfusion due to intraoperative bleeding .
according to the latest guidelines of the european association of urology , turp is the best treatment option for patients with nonneurogenic lower urinary tract symptoms affected by bpe .
however , morbidity with turp remains fairly high , with associated bleeding risk and electrolytic disorders .
in particular , patients receiving oap / oac treatment carry a major risk for hemorrhage , and discontinuation of these treatments carries the risk of thromboembolic events .
this may be considered a double - edged sword : the management of bpe is problematic for the urologist owing to the increased use of oap / oac for primary or secondary prevention of cardiovascular complications . in their study , capodanno et al
. expressed that this commonly used bridging practice is potentially harmful in patients undergoing noncardiac surgeries and can result in worse ischemic outcomes , especially in the first 30 days , along with significant risk of bleeding .
. pointed out the importance of these risks and proposed an immediate change in daily practice to abandon bridging therapy in most of these patients .
to clarify this issue for urological procedures , several studies were conducted on turp in patients receiving oap / oac therapy regimens .
analyzed 163 cases and found a higher rate of perioperative bleeding in patients who continued oap / oac therapy than in those who were not on an oap / oac therapy . also , cerebrovascular and cardiovascular events occurred in patients who had discontinued their oap / oac treatments .
this study demonstrates the importance of studies in which patients are evaluated while they are still receiving oap / oac treatments . to question the safety of bridging therapy with lmwh and the discontinuation of prophylactic anticoagulants , dotan et al .
compared the bleeding rates of patients who were on chronic oap / oac treatment and switched to lmwh perioperatively with those of non warfarin - treated patients .
they found a bleeding rate of 20% in patients who switched to perioperative lmwh , and this situation is presumed to result in longer hospitalization duration .
high rates of bleeding were also reported by parr et al . in a cohort of 12 patients with more than 30% of them having transfusions .
however , they reported that several such patients who switched from warfarin to heparin before surgery presented to the emergency department with major thromboembolic events and some even required open - heart surgery .
thus , they concluded that there is no need for bridging therapy and that bleeding problems can be easier to manage than thromboembolic events .
ong et al . reported that in a population of 293 patients on chronic oap / oac therapies who underwent turp , bleeding complications occurred in 10% of patients , of whom 8% required prolonged bladder irrigation , 2% required re - catheterization , 2% required reoperation , and 2% needed blood transfusion .
although turp has been the gold standard for the surgical management of bpe for the past several decades , in recent years , a new series of lasers and techniques have become well established as an alternative treatment for bpe . in particular , holmium and thulium lasers have shown safety and efficacy in numerous studies throughout the past decade .
studies reporting the safety of laser prostatectomy in patients on oap / oac therapies are also becoming more common . in their study in 2007 , ruszat et al . reported that in 116 patients on oap / oac therapy who were compared with a group of 92 control cases , none of the patients required transfusions . owing to slight hematuria , 17% of these patients required prolonged bladder irrigation ; however , this frequency was 5% among the control cases . in our patient groups ,
one patient in each group was catheterized as a result of bleeding after the retrieval of the urinary catheter and required prolonged irrigation .
reported no statistically significant differences among the 2 groups in terms of ipss , qol scores , or qmax .
these findings agree with our study . in our patient cohort , although these variables all improved after surgery , there were no statistically significant differences between the groups showing that bridging oap / oac with lmwh or continuing oap / oac affected functional outcomes . in various studies ,
for example , fu et al . compared thuvarp and monopolar turp and stated that the efficacy of the two modalities was similar , although thuvarp resulted in less blood loss and a shorter hospital stay .
bach et al . compared the 120-w and 70-w tm : yag lasers in terms of hemostatic properties .
the device that we used , the 120-w tm : yag laser , provided better ablation rates and fewer bleeding problems compared with the 70-w device .
the 120-w thulium laser has a wavelength of 2013 nm , which is close to the absorption peak of water .
thus , the effects are independent of tissue vascularization , which provides good hemostasis , comparable to that of the holmium : yag laser . additionally , the cw laser beam in tm : yag lasers provides precise incisions and sufficient vaporization . in the study by bach et al . in 2010 and the study by fu et al . in 2010 about thuvarp , the authors did not indicate the vaporized prostate volume . in our study written by netsch et al .
in 2015 comparing thuvarp and thuvep , the amount of vaporized tissue was 42.5 ml .
the amount of vaporized tissue in our current study was 43.1 and 43.8 ml in the different groups .
psa is a tool that can be used to predict prostate volume and prostate growth . additionally , according to the pless ( proscar long - term efficacy and safety ) study , psa is valuable in predicting changes in symptoms , qol , and qmax . in our study ,
psa values were lower in the postoperative period compared with preoperative values in both groups a and b. this drop was due to tissue vaporization and the decrease in prostate volume , which also supports the amelioration of symptoms and improved qol after surgery . as expressed earlier , the bleeding complications during turp in patients receiving oap / oac therapies are high . with technological advancements and new laser devices ,
the bleeding rate is expected to decrease as a result of the high ablative and coagulative power of these modalities . in our study , apart from showing low bleeding rates similar to those reported in fu et al .
's study , we also compared patients on continuous oap / oac therapy and patients who underwent lmwh bridging .
although the subgroup comparisons showed statistically significant differences , the drop in hemoglobin levels was similar and the differences were not clinically relevant .
these results show that bridging therapy with lmwh has a small impact on bleeding complications that have clinical relevance .
although no cardiopulmonary adverse events occurred in our patient cohort , the high level of evidence in the literature about the seriousness of these events draws great attention to patients undergoing bridging therapy with lmwh .
when the low risk due to bleeding complications in our patient group is combined with the high risk of potential cardiopulmonary adverse events in the case of lmwh bridging , continuing oap / oac therapy during thuvarp seems to be the better way of patient management .
apart from bleeding complications , we did not encounter any bladder neck contracture or urethral stricture in the long - term follow - up .
fu et al . reported one case of urethral stricture and no cases of bladder neck contracture in their study .
in a recent study by yu et al . , the authors reported a uti rate of 10% . in our study , 4 of 47 patients in group a ( 8.5% ) and 7 of 56 patients in group b ( 12.5% ) had uti .
in fu et al . 's study , the rate of uti in thuvarp group was 6.9% .
another minor limitation was the lack of comparison with a group of patients undergoing turp , which is considered the gold standard operation for bpe .
however , in the absence of good prospective trials , the results obtained from well - analyzed retrospective studies can play significant roles in clinical practice and can set up new ideas for future randomized prospective studies .
continuing oap / oac therapy regimens for certain endoscopic procedures has been strongly encouraged in recent years owing to the high risk of cardiopulmonary thromboembolic events in these patients .
our study showed the safety profile of continuing different oap / oac regimens in terms of bleeding problems in patients undergoing thuvarp .
we strongly recommend abandoning lmwh bridging and maintaining the patient 's current oap / oac therapy regimen
. a larger cohort of patients and a longer follow - up period are needed to add more valuable contributions to our results . | purposethulium vaporesection of the prostate ( thuvarp ) is a new and safe approach for patients receiving anticoagulant therapy in whom transurethral resection of the prostate ( turp ) may possess a high bleeding risk .
we aimed to demonstrate the efficacy and safety of thuvarp in patients receiving oral antiplatelet / anticoagulant ( oap / oac ) therapy.materials and methodsa total of 103 patients who underwent thuvarp between 2011 and 2013 were enrolled in the study .
patients were divided into 2 groups .
group a consisted of 47 patients who underwent low molecular weight heparin ( lmwh ) bridging and group b consisted of 56 patients who were operated on while receiving oap / oac therapy.resultsthe drop in hemoglobin levels in the pre- and postoperative periods was significantly higher in group a than in group b. when subgroups were analyzed , the mean drop in hemoglobin was significantly lower in the warfarin and ticlopidine subgroups of group b than in group a. international prostate symptom scores were significantly lower 3 , 12 , 18 , and 24 months after surgery in group a than in group b. quality of life scores , maximal flow rate values , and postmicturition residual urine volumes ( ml ) were similar between the 2 groups .
a total of 38 and 41 patients in groups a and b , respectively , had no complications.conclusionsour study showed the safety profile of continuing different oap / oac therapies in terms of bleeding problems in patients undergoing thuvarp .
we strongly recommend abandoning lmwh bridging and maintaining the oap / oac regimen patients are already receiving . |
all subjects were recruited from patients ages 5 years to 17 years presenting to the pediatric clinic .
informed consent from parents was taken . to be enrolled , subjects had to have no ocular problems other than refractive error .
exclusion criteria were strabismus or amblyopia , any abnormalities of the disc or the retinal nerve fiber layer , family history of glaucoma , or any other hereditary eye disease , history of intraocular surgery or any kind of laser therapy mentally challenged , children with neurological , metabolic , vascular disorders , other systemic disease possibly affecting the eye , presence of a media opacity , best - corrected visual acuity of less than 20/30 , hypermetropia more
than + 3d , myopia more than 5d , or astigmatism more than 2d were also excluded .
normal - appearing disc , cup , and neuroretinal rim on careful examination of the optic nerve head with 90-d aided stereoscopic slit - lamp indirect ophthalmoscopy , with a cup - disc ratio of less than 0.7 or cup - disc ratio asymmetry of less than 0.2 being the selection criteria for further evaluation .
all subjects received a full ophthalmic examination including cycloplegic refraction , assessment of intraocular pressure ( goldmann applanation tonometry where feasible ) , assessment of ocular motility and alignment , slit - lamp biomicroscopic evaluation , and dilated fundus examination .
pupils were dilated with tropicamide 1% and cyclopentolate 1% or 2% drops , depending on age of the subject .
rnfl measurements were obtained through dilated pupils using a 3-generation optical coherence tomographer ( stratus oct , software version 4.0.4 , carl zeiss , dublin , ca ) .
this oct uses partial coherence interferometry technology ( wavelength , 820 nm ) to obtain cross - sectional images ( equivalent to b - scan ultrasound ) and achieves an axial resolution of approximately 10 um .
oct also has been shown to have good intra - observer and inter - observer reproducibility , and generally is not affected by optical aberrations or pupil diameter .
peripapillary measurements were performed using the fast scan protocol ( fast rnfl thickness scan [ 3.4 protocol ] ; this consists of a circular scan pattern in which the diameter is 3.46 mm for eyes with standard axial length [ 24.46 mm ] and refraction 0 diopters [ d ] ) .
three such circular scans were performed successively , with a total acquisition time of 1.92 s. the average of the 3 scans was used in the analysis .
an internal fixation target was used in all scans , and the location of each scan on the retina was monitored on the built - in infrared - sensitive video camera .
scans were accepted only if they were free of artifacts and had signal strengths of at least 6 .
mean rnfl thickness in micrometers along the whole circle circumference , four quadrants , 12o clock hours , and at 256 a - scan lengths were obtained .
12 30 sectors were also defined in clockwise order for the right eye and in counterclockwise order for the left eye : 1-superior - nasal , 2-nasal - superior , 3-nasal , 4-nasal - inferior , 5-inferior - nasal , 6-inferior , 7-inferior - temporal , 8-temporal - inferior , 9-temporal , 10-temporal - superior , 11-superior temporal , and 12-superior ) .
one eye of each subject was selected randomly for statistical analysis by selecting right and left eyes in an alternating fashion from the randomly ordered sample . for statistical comparison of variables between right and left eyes ,
all eyes were used . for purposes of analysis , 30-segment measurements of left eyes were mirror imaged ( i.e. , 3o clock was nasal for all eyes ) .
univariate and multivariate regression analyses were used to analyze the effect of age , gender , and refraction on rnfl thickness .
statistical comparisons of variables between right and left eyes were performed using the paired student 's t test . for comparisons of variables between genders ,
all statistics were done using the stata : data analysis and statistical software ( version 8.1 , texas , usa ) .
all subjects were recruited from patients ages 5 years to 17 years presenting to the pediatric clinic .
informed consent from parents was taken . to be enrolled , subjects had to have no ocular problems other than refractive error .
exclusion criteria were strabismus or amblyopia , any abnormalities of the disc or the retinal nerve fiber layer , family history of glaucoma , or any other hereditary eye disease , history of intraocular surgery or any kind of laser therapy mentally challenged , children with neurological , metabolic , vascular disorders , other systemic disease possibly affecting the eye , presence of a media opacity , best - corrected visual acuity of less than 20/30 , hypermetropia more
than + 3d , myopia more than 5d , or astigmatism more than 2d were also excluded .
normal - appearing disc , cup , and neuroretinal rim on careful examination of the optic nerve head with 90-d aided stereoscopic slit - lamp indirect ophthalmoscopy , with a cup - disc ratio of less than 0.7 or cup - disc ratio asymmetry of less than 0.2 being the selection criteria for further evaluation .
all subjects received a full ophthalmic examination including cycloplegic refraction , assessment of intraocular pressure ( goldmann applanation tonometry where feasible ) , assessment of ocular motility and alignment , slit - lamp biomicroscopic evaluation , and dilated fundus examination .
pupils were dilated with tropicamide 1% and cyclopentolate 1% or 2% drops , depending on age of the subject .
rnfl measurements were obtained through dilated pupils using a 3-generation optical coherence tomographer ( stratus oct , software version 4.0.4 , carl zeiss , dublin , ca ) .
this oct uses partial coherence interferometry technology ( wavelength , 820 nm ) to obtain cross - sectional images ( equivalent to b - scan ultrasound ) and achieves an axial resolution of approximately 10 um .
oct also has been shown to have good intra - observer and inter - observer reproducibility , and generally is not affected by optical aberrations or pupil diameter .
peripapillary measurements were performed using the fast scan protocol ( fast rnfl thickness scan [ 3.4 protocol ] ; this consists of a circular scan pattern in which the diameter is 3.46 mm for eyes with standard axial length [ 24.46 mm ] and refraction 0 diopters [ d ] ) .
three such circular scans were performed successively , with a total acquisition time of 1.92 s. the average of the 3 scans was used in the analysis .
an internal fixation target was used in all scans , and the location of each scan on the retina was monitored on the built - in infrared - sensitive video camera .
scans were accepted only if they were free of artifacts and had signal strengths of at least 6 .
mean rnfl thickness in micrometers along the whole circle circumference , four quadrants , 12o clock hours , and at 256 a - scan lengths were obtained .
12 30 sectors were also defined in clockwise order for the right eye and in counterclockwise order for the left eye : 1-superior - nasal , 2-nasal - superior , 3-nasal , 4-nasal - inferior , 5-inferior - nasal , 6-inferior , 7-inferior - temporal , 8-temporal - inferior , 9-temporal , 10-temporal - superior , 11-superior temporal , and 12-superior ) .
one eye of each subject was selected randomly for statistical analysis by selecting right and left eyes in an alternating fashion from the randomly ordered sample . for statistical comparison of variables between right and left eyes ,
all eyes were used . for purposes of analysis , 30-segment measurements of left eyes were mirror imaged ( i.e. , 3o clock was nasal for all eyes ) .
univariate and multivariate regression analyses were used to analyze the effect of age , gender , and refraction on rnfl thickness .
statistical comparisons of variables between right and left eyes were performed using the paired student 's t test . for comparisons of variables between genders ,
all statistics were done using the stata : data analysis and statistical software ( version 8.1 , texas , usa ) .
3 ( 3.3% ) did not co - operate for the oct imaging ( 3 were 5 years old ) . of the remaining 127 subjects ,
after random selection of one eye of each subject , 60 right and 60 left eyes entered statistical analysis .
there was no statistically significant difference between male and female subjects for refraction ( p = 0.26 ) and age ( p = 0.959 ) .
demographics of study subjects included in the analysis ( n=120 ) mean global rnfl thickness was 106.11 9.50 m ( range , 82.26 - 146.25 ) 105.64 8.87 m ( range , 84.68 - 129.75 ) in right eyes and 107.54 8.96 m ( range 91.35 - 140.33 ) in left eyes ; the difference was not statistically significant ( p = 0.453 ) . on average , the rnfl was thicker inferiorly and superiorly , thinner nasally and thinnest temporally [ table 2 ] . the distribution of global rnfl thickness for all eyes is shown in fig .
1 . the rnfl thickness in different peripapillary locations for all eyes is shown in fig .
2 . rnfl thickness for all quadrants and sectors in study subjects the distribution of global rnfl thickness for all eyes ( n = 120 ) retinal nerve fiber layer thickness in normal children as a function of peripapillary location ( n = 120 eyes ) .
peripapillary location is given in clock hours ( 3 = nasal , 6 = inferior , 9 = temporal , 12 = superior ) .
mean , boxplot gives five number summaries- the smallest observation ( sample minimum ) lower quartile ( q1 ) median ( q2 ) upper quartile ( q3 ) and largest observation ( sample maximum ) the mean rnfl thickness in four quadrants and for all clock hours is provided in [ table 2 ] .
the mean rnfl thickness was highest in the inferior quadrant followed by superior , nasal , and temporal quadrants [ table 2 ] .
the rnfl was thickest inferiorly ( 134.10 16.16 m ) and superiorly ( 133.44 15.50 m ) , thinner nasally ( 84.26 16.43 m ) , and thinnest temporally ( 70.72 14.80 m ) .
the rnfl thickness was not statistically different in males and females ( p = 0.342 ) .
in univariate regression analysis , age had a no statistically significant ( p = 0.7249 , r = 0.0011 ) effect on rnfl thickness [ fig .
3a ] while it had statistically significant effect on only in temporal quadrant when analyzed for individual quadrant rnfl thickness [ fig .
3a ] was y = 105.06 + 0.0915 x ( 95% confidence interval [ ci ] for the coefficient 422 to .
( a ) scatter plot showing average global retinal nerve fiber layer ( rnfl ) thickness in normal children as a function of age ( n = 120 eyes ) .
( b ) quadrant thickness as function of age shows no significant relation with age except in temporal quadrant refraction had a statistically significant ( p = 0.0008 , r = 0.2102 ) effect on rnfl thickness [ fig .
= 107.38 + 3.205 x ( 95% ci for the coefficient , 1.40 to 5.01 ) ( y = global rnfl thickness , x = refraction ) .
after removing outlier , ( n = 119 eyes ) refraction still had statistically significant effect on rnfl thickness .
the equation for the regression line after removing outlier was y = 106 + 2.35 x ( 95% ci for the coefficient , 0.70 to 3.99 ) ( y = global rnfl thickness , x = refraction ) .
refraction had statistically significant effect only in nasal quadrant when analyzed for individual quadrant rnfl thickness [ fig .
( a ) average global retinal nerve fiber layer ( rnfl ) thickness in normal children as a function of refraction ( n = 120eyes ) .
( b ) quadrant thickness as function of refraction shows no significant relation except in nasal quadrant gender did not have a statistically significant effect on global rnfl thickness ( p = 0.342 ) .
when age was controlled for in multivariate regression analysis , refraction still had a statistically significant effect on rnfl thickness ( p = 0.002 ) ; however , when refraction was controlled for , age no longer did ( p = 0.820 ) .
3 ( 3.3% ) did not co - operate for the oct imaging ( 3 were 5 years old ) . of the remaining 127 subjects ,
after random selection of one eye of each subject , 60 right and 60 left eyes entered statistical analysis .
there was no statistically significant difference between male and female subjects for refraction ( p = 0.26 ) and age ( p = 0.959 ) .
mean global rnfl thickness was 106.11 9.50 m ( range , 82.26 - 146.25 ) 105.64 8.87 m ( range , 84.68 - 129.75 ) in right eyes and 107.54 8.96 m ( range 91.35 - 140.33 ) in left eyes ; the difference was not statistically significant ( p = 0.453 ) . on average ,
the rnfl was thicker inferiorly and superiorly , thinner nasally and thinnest temporally [ table 2 ] .
1 . the rnfl thickness in different peripapillary locations for all eyes is shown in fig .
2 . rnfl thickness for all quadrants and sectors in study subjects the distribution of global rnfl thickness for all eyes ( n = 120 ) retinal nerve fiber layer thickness in normal children as a function of peripapillary location ( n = 120 eyes ) .
peripapillary location is given in clock hours ( 3 = nasal , 6 = inferior , 9 = temporal , 12 = superior ) .
mean , boxplot gives five number summaries- the smallest observation ( sample minimum ) lower quartile ( q1 ) median ( q2 ) upper quartile ( q3 ) and largest observation ( sample maximum ) the mean rnfl thickness in four quadrants and for all clock hours is provided in [ table 2 ] .
the mean rnfl thickness was highest in the inferior quadrant followed by superior , nasal , and temporal quadrants [ table 2 ] .
the rnfl was thickest inferiorly ( 134.10 16.16 m ) and superiorly ( 133.44 15.50 m ) , thinner nasally ( 84.26 16.43 m ) , and thinnest temporally ( 70.72 14.80 m ) .
the rnfl thickness was not statistically different in males and females ( p = 0.342 ) .
in univariate regression analysis , age had a no statistically significant ( p = 0.7249 , r = 0.0011 ) effect on rnfl thickness [ fig .
3a ] while it had statistically significant effect on only in temporal quadrant when analyzed for individual quadrant rnfl thickness [ fig .
3a ] was y = 105.06 + 0.0915 x ( 95% confidence interval [ ci ] for the coefficient 422 to . 6052 ) ( y = global rnfl thickness , x = age ) .
( a ) scatter plot showing average global retinal nerve fiber layer ( rnfl ) thickness in normal children as a function of age ( n = 120 eyes ) . ( b )
quadrant thickness as function of age shows no significant relation with age except in temporal quadrant refraction had a statistically significant ( p = 0.0008 , r = 0.2102 ) effect on rnfl thickness [ fig .
= 107.38 + 3.205 x ( 95% ci for the coefficient , 1.40 to 5.01 ) ( y = global rnfl thickness , x = refraction ) .
there was a linear relationship between refraction and global thickness . after removing outlier , ( n = 119 eyes ) refraction still had statistically significant effect on rnfl thickness .
the equation for the regression line after removing outlier was y = 106 + 2.35 x ( 95% ci for the coefficient , 0.70 to 3.99 ) ( y = global rnfl thickness , x = refraction ) .
refraction had statistically significant effect only in nasal quadrant when analyzed for individual quadrant rnfl thickness [ fig .
( a ) average global retinal nerve fiber layer ( rnfl ) thickness in normal children as a function of refraction ( n = 120eyes ) .
( b ) quadrant thickness as function of refraction shows no significant relation except in nasal quadrant gender did not have a statistically significant effect on global rnfl thickness ( p = 0.342 ) .
when age was controlled for in multivariate regression analysis , refraction still had a statistically significant effect on rnfl thickness ( p = 0.002 ) ; however , when refraction was controlled for , age no longer did ( p = 0.820 ) .
normative data are provided automatically by oct , but the database only includes individuals 18 years and older , limiting its use in children .
the average rnfl thickness in our study was determined 106.11 9.50 m which is consistent with findings reported in the literature .
table 3 shows oct values of previous studies of children and our present study as well .
average rnfl thickness values for our total sample were comparable with those of salchow et al . and
( population largely hispanic and all korean , respectively ) but higher than those of the younger subjects reported by parikh et al .
comparison of studies reporting oct - measured global rnfl thickness ( m ) in normal pediatric subjects compared with the large study of australian 6-year - olds by huynh et al .
average rnfl thickness of our study was lower with those of mrugacz and bakunowicz - lazarczyk , leung et al . , and qian et al .
by contrast , pediatric oct values reported by gupta et al . differed from all other reports , possibly owing to technical differences in the protocols used , the authors in this small study did not specify which generation of oct machine they used . in a recent study by turk et al .
the average rnfl thickness by spectral - domain oct ( sd - oct ) for turkish pediatric patients with ages between 6 years and 16 years was determined as 106.45 9.41 ( 83.33141.17 ) similar to our study though our study measured rnfl on time domain stratus oct .
possible explanations of difference from various studies include our stricter optic nerve and refractive criteria , and the existence of variation between distinct geographic areas and ethnic groups .
the differences between different versions of the device have also been found in adults and may be due to the use of different algorithms between spectral and time domain oct devices .
the distribution of rnfl thickness ( thickest inferiorly and superiorly , thinner nasally , and thinnest temporally ) in our study is in agreement with the normal distribution of rnfl around the optic nerve as has been previously reported for oct data from the normal children and adults .
the oct measurements of rnfl thickness have been previously described as dependent on race , age , axial length , and disc area in adult eyes , with a reported average rnfl thickness decrease of 2.2 m for every 1-mm increase in axial length , and an average rnfl thickness decrease of 2 m for every decade of aging .
these findings are consistent with pathology studies by dolman et al . suggesting that change due to age - related decay of the optic nerves is not significant until after age 60 years .
compared with adult studies , average rnfl thickness in our population was comparable with but slightly higher than that reported in the younger adults ( age , < 40 years ) by budenz et al . and leung et al . measurements of rnfl thickness were not dependent on age in our population of children when controlling for refraction . similarly , leung et al .
salchow et al . concluded that age did not show a significant effect on rnfl thickness after controlling for the effect of refractive error . in our study , mean rnfl thicknesses were similar in boys and girls , in accordance with the studies by salchow et al .
the effects of refraction on rnfl thickness have been debated ; some authors have found no difference ( mrugacz and bakunowicz - lazarczyk 2005 ) and others have described thinning of the rnfl with greater negative refraction ( huynh et al .
, there was no significant correlation between age , refraction defect , axial length values , and rnfl thickness . in the present study when age was controlled for in multivariate regression analysis ,
refraction still had a statistically significant effect on rnfl thickness ( p = 0.002 ) ; however , when refraction was controlled for , age no longer did ( p = 0.820 ) .
furthermore , this difference may also occur among different models of certain sd - oct devices .
taken together , these findings suggest that differences in oct technology may be responsible for the differences between our results and previously published data on rnfl thickness .
the reason why different versions of oct yield different values for rnfl thickness is not clear .
it is possible that different software algorithms used to delineate the rnfl may be among the factors responsible for this observation .
rnfl thickness is significantly correlated with age and axial length . however , in this study we did not consider potential influential factors for rnfl measurements , including axial length , optic disc size . secondly axial length and refraction are usually correlated which is not addressed by the present study . limited numbers and the exclusion of eyes with high refractive errors may have limited our ability to identify additional relationships between rnfl thickness and axial length . in conclusion , this study is the first to present normative stratus oct data for indian pediatric patients .
this study demonstrates normative values of retinal thickness and rnfl thickness in the indian pediatric age group .
this information should facilitate evaluation of oct assessments performed in children with diagnosed or suspected glaucoma as well as those with other optic neuropathies . | purpose : to measure the peripapillary retinal nerve fiber layer ( rnfl ) thickness in normal indian pediatric population.subjects and methods:120 normal indian children ages 5 - 17 years presenting to the pediatric clinic were included in this observational cross - sectional study .
rnfl thickness was measured with stratus optical coherence tomography ( oct ) .
children with strabismus or amblyopia , with neurological , metabolic , vascular , or other disorders and those with abnormal optic discs were excluded .
one eye of each subject was randomly selected for statistical analysis .
the effect of age , refraction and gender on rnfl thickness was investigated statistically.result:oct measurements were obtained in 120 of 130 ( 92.3% ) subjects .
mean age was 10.8 3.24 years ( range 5 - 17 ) .
average rnfl thickness was ( sd ) 106.11 9.5 m ( range 82.26 - 146.25 ) .
the rnfl was thickest inferiorly ( 134.10 16.16 m ) and superiorly ( 133.44 15.50 m ) , thinner nasally ( 84.26 16.43 m ) , and thinnest temporally ( 70.72 14.80 m ) .
in univariate regression analysis , age had no statistical significant effect on rnfl thickness ( p = 0.7249 ) and refraction had a significant effect on rnfl thickness ( p = 0.0008).conclusion : oct can be used to measure rnfl thickness in children .
refraction had an effect on rnfl thickness . in normal children ,
variation in rnfl thickness is large .
the normative data provided by this study may assist in identifying changes in rnfl thickness in indian children . |
for henry , the common presupposition that links classical philosophy with historical phenomenology from husserl and heidegger to merleau - ponty and levinas consists in the fact that they think the logos of phenomena as the logos of the world . to put it another way ,
for this reason , henry believes , they are not capable of explaining how intentionality brings itself forth , that is , how transcendence is able to transcend itself.10 in order to solve this problem it is necessary to go back to the immemorial ground of experience , in which , as henry puts it , intentionality takes possession of itself , or , rather ,
( sprouve soi - meme ) . only in such a way of primarily giving itself to itself
for henry , the task of a radical , i.e. , material phenomenology thus consists in returning to pure immanence and its inner structure and dynamic . against the ontological monism11 of western philosophy , i.e. , the presupposition that phenomenological distance is the ontological power which gives us access to things,12 such a phenomenology thematizes the duplicity of appearing.13 this implies that against manifestation which is henry s term for all transcendent , ecstatic , or worldly appearing is pitted a pure appearing or , to be more precise , a self - appearing or auto - revelation .
the concept of duplicity underscores the fact that this distinction is no way meant as a dichotomous comparison , but rather that what should be thought is a foundational relationship : ultimately , henry postulates , the essence of manifestation is founded in nothing other than precisely this self - appearing .
this does not takes place , however , within thought , representation or reflection , but rather in the mode of affectivity , or , stated more precisely , in the mode of auto - affection on the part of pure phenomenological life .
for henry the decisive presupposition of classical phenomenology thus lies in the fact that the life of consciousness is to be realized within the horizon of exteriority , visibility , or simply the world , i.e. , in the domain of a living subjectivity that in - tentionally exceeds itself . in such exteriority
it is through intentionality that the subject does not coincide with itself inasmuch as it always differs from itself , fractured by the difference of the world itself . in henry s view
it was husserl who first contemplated this insight about intentionality , which has been radicalized by post - husserl phenomenology , and has also influenced deconstruction .
however , when it is a question of thinking the proper essence of selfhood , henry takes all of these positions to be entirely insufficient . according to him , husserl s theory of self - constitution puts us on an aporetic path .
it does this by privileging an intentionality that constitutes objects within its temporal flow at the expense of a more primal self - presencing prior to the streaming of time.14 the question that arises here about a primordial selfhood had always remained a problem for husserl and classical phenomenology ( and , of course , it was in no way only a phenomenological problem ) .
according to henry , the task of rendering intelligible this primal self - presencing in its passive / affective foundation was an impossible one for husserl to achieve because he emphasized the cognitive structure of consciousness and its various intentional faculties ( e.g. , presentation , representation , imagination).15 henry goes back to husserl , however , in order to pose this question in a manner that is adequate to the task .
central for him is an early insight suggested by husserl in his lecture , the idea of phenomenology , one that is quickly forgotten and never developed in his subsequent writings .
husserl s insight is that nothing other than pure appearance as such , hence phenomenality , is the basic theme of phenomenology , and therefore not the primacy of the phenomenological
gaze ( schau ) , nor , more generally speaking , that of theory.16 phenomenality here means nothing other than the condition under which something in general is initially capable of attaining the status of a phenomenon .
we must add , however , that we can no longer appeal to a horizon , in which the staging of the thing itself would unfold ( i.e. , objectivity in husserl , being in heidegger , or the flesh of the world in late merleau - ponty , etc . ) .
rather , if one considers appearing in the dimension of its origin , particularly in regards to such a horizon , a problem arises , namely , that as a method phenomenology immediately loses sight of this pure appearing .
this is , one might say , its fate , insofar as it takes itself to be a process of making evident , as a mode of making - visible ( sehen - lassen ) , ex - plication or a mode of rendering - manifest . to put it in connection with traditional concepts
: henry argues that intentionality , by virtue of its constitutive character whereby it sets phenomena within a horizon , can not initiate us into the proper domain of phenomenological inquiry
i.e . , that which bears the distinction of being conscious of something as something is thus not primordially responsible for making the world open to us . as a method of beholding and grasping ( schauen und fassen ) , intentional phenomenology in fact conceals the genuine phenomenological register that henry has in mind .
thus , in contrast to husserl , who orders the original relation to the world by way of intentional reflection or consciousness of , henry does not . this is to say that husserl insists that intentional life relates the self to something different than itself . and
access to being is possible , which renders intentionality , as fink aptly puts it , the central hypothesis of husserl s phenomenology.17 this husserlian hypothesis , which posits consciousness , understood as originarily intentional , as the genuine mode of accessing being yields an extraordinary phenomenological situation : if the primordial how of givenness or of the thing itself is identified with intentional display , then the appearing of the being is substituted for the appearing as such : still expressed in a language that demonstrates the close affinity between this historical phenomenology and classical philosophy , every consciousness is a consciousness of something. thus we have , on the one hand , the appearing ( consciousness ) and , on the other hand , the something , the being . in itself
the being is foreign to appearing and thus unable to phenomenalize itself through itself . for its part ,
the appearing is such that it is necessarily the appearance of something other , of the being .
appearing turns away from itself in such a radical and violent way that it is directed entirely to something other than itself , namely , the outside
because appearing qua intentionality finds itself thus essentially displaced in what it allows to appear , appearing no longer appears , but only that which appearing lets appear within itself : the being . as a result , the object of phenomenology , the thing itself , is distorted in such a way that the object of phenomenology is no longer the appearing but rather the appearing of the being and ultimately the being itself , insofar as it appears.18 in contrast , what henry provides is the re - inscription of intentionality in the non - intentional .
he determines the non - intentional as a ground that is older than intentionality .
henry puts into play a radical reduction that reduces or disqualifies the world as the horizon of intentional exhibition and site of any and all givenness .
horizon - inscription and reduction to the ego are hereby suspended as constituents of the phenomenon s genesis . in regards to
this methodological approach henry speaks of a counter - reduction.19 this brings us back to the passive and affective foundation of the self , its pure self - appearing or its pure self - revelation , which are carried out in the night of subjectivity , in the
invisible life of our affectivity .
still expressed in a language that demonstrates the close affinity between this historical phenomenology and classical philosophy ,
every consciousness is a consciousness of something. thus we have , on the one hand , the appearing ( consciousness ) and , on the other hand , the something , the being . in itself the being is foreign to appearing and thus unable to phenomenalize itself through itself . for its part ,
the appearing is such that it is necessarily the appearance of something other , of the being .
appearing turns away from itself in such a radical and violent way that it is directed entirely to something other than itself , namely , the outside
because appearing qua intentionality finds itself thus essentially displaced in what it allows to appear , appearing no longer appears , but only that which appearing lets appear within itself : the being . as a result , the object of phenomenology , the thing itself , is distorted in such a way that the object of phenomenology is no longer the appearing but rather the appearing of the being and ultimately the being itself , insofar as it appears.18 the path from a phenomenology of the world to a
phenomenology of life , from a phenomenology of transcendence to one of immanence has now been sketched more clearly .
he holds on to this duplicity against the ontological monism that reduces the field of appearing entirely to the laws of intentional display that exhibits objects in the luminous horizon of the world .
nevertheless , for him it is not a question of construing this duality or , rather , duplicity , as a dualism.20 his aim lies rather in grasping the world on the basis of life , without thinking the latter
qua nature of the subject and of being within the apperceptive horizon of the world
( nor therefore in that of being , but also not in that of alterity or a mysterious flesh of the world , etc . ) , but rather purely from out of its
we find this idea already in the definition henry gives of the self in lessence de la manifestation : that which is thus forever burdened with self , only this can we truly call a self .
herein is accomplished the movement without movement in which it receives , as a substantial and burdensome content , that which it is ; it masters itself , arrives at itself , experiences its own profusion .
the self is the surpassing of the self as identical to self.22this ipseization of the subject as it is called in i am the truth takes place in its living bodily praxis , i.e. , purely in the invisible medium of its affectivity as well as practically lived intelligibility .
this suggests that we speak here of a metaphysics of the individual , since for this reason all traditional parameters of onto(theo)logical individuation such as substantiality , spatio - temporality localization , self - determination , self - motivated agency ( selbstttigkeit ) or spontaneous self - determination are suspended and this in favor of the apodicticity of an unshakeable foundation of our practical knowledge of life
( savoir de la vie ) as a purely individuated self - feeling.23 that which is thus forever burdened with self , only this can we truly call a self .
herein is accomplished the movement without movement in which it receives , as a substantial and burdensome content , that which it is ; it masters itself , arrives at itself , experiences its own profusion .
the self is the surpassing of the self as identical to self.22 against this background , henry s central thesis can be elucidated , namely , that the mode of the self - appearing or the self - revelation of life is not the affection by means of something foreign to consciousness , nor external ( as classical philosophy would have it ) , nor is it the affection by means of itself as something other in the medium of time or the faculty of imagination ( as husserl and heidegger would have it ) , but rather the pure auto - affection of life , which is revealed in the feeling it has of itself . thus , henry considers the affective auto - revelation as the ineluctable condition that precedes all intentional relatedness and being - toward - the - world in general .
the impressional content of the world is no longer that which fills the meaning - bestowing intentional ray , but rather and thus runs henry s thesis to a non - intentional auto - affection of life as a pathos interior to the ego .
according to henry this pathetic , i.e. , radically passive , self - appearing constitutes the invisible reality of life in its self - affection .
what henry designates as auto - affection , moreover , precedes the classical distinction between activity and passivity .
it is more primordial than the passivity involved in the suffering of an external force and also the passivity of feelings , emotions , affects , and dispositions .
thus what is at issue is an originary , i.e. , ontological passivity of affectivity . for henry ,
this originary passivity is at once passive and yet constitutive of the ground of selfhood itself , and this represents one of the decisive thematics of his system .
for this is implied in every feeling as its self - feeling , or originarily passive self - relationality.24 there is no difficulty in claiming that henry s theory of the irrepressible pathos of auto - affection is distinct from affective states like sensation , which exhibit an intentional or exterior relation to the world .
so exterior sensations involve a relation to something different than themselves , ( to outside sensory impressions ) , an affection structured by what is known as hetero - affection . for henry , hetero - affection , while a legitimate field of display ,
does not exhaust what affectivity is . or , to express it another way ,
henry is convinced that the events of the world are only occasional causes of our feelings .
it is our affectivity itself which makes it possible for the world to exert an influence upon our interior life . in other words , it is , in each case , our particular
attunement that exposes us to an affection by the world.25 in other words , not what befalls us from outside determines affectivity but rather affectivity in its self - relation interior to itself first makes possible any experience of what might befall us . that all dispositions , feelings , etc .
here are given to themselves without the possibility of recoil in their being - always - already - given - to - themselves , crucified on themselves , as jad hatem puts it,26 implies that they bear the character of suffering .
that said it is not the case that life only suffers in such feelings and dispositions .
henry understands suffering rather as a path . inasmuch as it surpasses itself to return home to itself , this being - itself ,
its self - givenness , is also a fount of pleasure and joy that never runs dry .
power and impotence are thus intertwined in this interior experience of a gift which can not be refused,27 and so draws life from the auto - donation of life inside the ego as a primordial force or
this process of arriving at itself ( venue en soi ) , this gift of life ( don de la vie ) that can never be refused , henry understands as the gift of absolute life , which , traditionally speaking , we would call god .
the transcendental birth of the living ( le vivant ) in ( absolute ) life therefore in no way describes a factual genesis or individuation , but rather a conditio : it concerns the conditioof sonship , as henry notes in his christological transformation of his major phenomenological intuition in i am the truth : to understand man on the basis of christ , who is himself understood on the basis of god , in turn rests on the crucial intuition of a radical phenomenology of life , which is precisely that of christianity : namely , that life has the same meaning for god , for christ , and for man .
this is so because there is but a single and selfsame essence of life , and , more radically , a single and selfsame life .
this life that self - generates itself in god and that , in its self - generation , generates the transcendental arch - son as the essential ipseity in which this self - generation comes about is the life from which man himself takes his transcendental birth , precisely since he is life and is explicitly defined as such within christianity .
he is the son of this unique and absolute life , and thus the son of god.28this trinitarian conception of the self - engendering of absolute life , and of the generation that takes place within the trinity of a first living being
( i.e. , christ ) , through which generation this absolute life realizes itself , and finally this conception of the generation of each living being ( individual ) in and through this life , reaches a christological apex : the first living being born in perfect reciprocity with absolute life christ represents for henry that primordial or
older ipseity of a first self29 through which alone each living being can obtain the unrefusable gift of life.30 to understand man on the basis of christ , who is himself understood on the basis of god , in turn rests on the crucial intuition of a radical phenomenology of life , which is precisely that of christianity : namely , that life has the same meaning for god , for christ , and for man .
this is so because there is but a single and selfsame essence of life , and , more radically , a single and selfsame life .
this life that self - generates itself in god and that , in its self - generation , generates the transcendental arch - son as the essential ipseity in which this self - generation comes about is the life from which man himself takes his transcendental birth , precisely since he is life and is explicitly defined as such within christianity .
he is the son of this unique and absolute life , and thus the son of god.28 henry s turn to christology as a way to explicate life s living relation to humanity should not be dismissed as a theological domestication of phenomenology .
viewed phenomenologically , we see that he forges a noteworthy modification of the concept of self - affection .
henry seems to be responding to the critical charge made against his theory of auto - affection , namely , that pure immanence possesses no structure or history and therefore it excludes every possible relation to otherness , as well as every source of hetero - affection.31 on the contrary , henry introduces the distinction between a strong , absolute , or naturalizing self - affection , on the one hand , and a weak , relative , or naturalized self - affection , on the other hand.the singular self that i am experiences itself only within the movement by which life is cast into itself and enjoys itself in the eternal process of its absolute self - affecting . the singular self self
- affects itself ; it is the identity between the affecting and the affected , but it has not itself laid down this identity .
the self self - affects itself only inasmuch as absolute life is self - affected in this self .
it is life , in its self - giving , which gives the self to itself .
it is life , in its self - revelation , that reveals the self to itself.32although we can not discuss the christological transformation of henry s thought in more detail here , the following point seems to have a particularly salient significance for our context : according to henry , the experience that life has of itself takes place in connection with the aforementioned pathic duality of suffering and enjoyment , i.e. , the antinomic structure of life.33 inasmuch as this antinomy of the fundamental affective tonalities34 of suffering and joy is co - constitutive of life s self - revelation and thus refers back to a primitive unity , it constitutes selfhood , or , as henry also puts it , ipseity . in other words , since human life is given to itself in its pure passivity , it is not self - positing or self - subsisting and thus not the source of its own condition as living rather henry insists that i find myself
thrown into life , self - affected , as he articulates it in i am the truth.35 ipseity is thus neither absolute self - affection , nor does it result from the impersonal execution of anonymous forces or drives.36 ipseity so understood by henry means having a self to the extent that life in its self - affection surpasses itself into affective formations and thus transforms itself by way of that affectivity , i.e. , insofar as in the originary affectivity of feeling - itself it also experiences the essence of its subjectivity .
insofar as ipseity carries the burden of feeling - itself and , in its condition as son , experiences it as the burden of its own given being , it comes into possession of itself and of each of the modalities of its life37and , hence , becomes the subject that takes up a relation to the world .
the singular self that i am experiences itself only within the movement by which life is cast into itself and enjoys itself in the eternal process of its absolute self - affecting .
the singular self self - affects itself ; it is the identity between the affecting and the affected , but it has not itself laid down this identity .
the self self - affects itself only inasmuch as absolute life is self - affected in this self .
it is life , in its self - giving , which gives the self to itself .
it is life , in its self - revelation , that reveals the self to itself.32
to shed light on the innovative significance of his practical philosophy , we must develop a deeper understanding of how henry thinks the aforementioned relation to the world .
it concerns and this is crucial to note a world , which would not be the pre - given aim that is exterior to the self - movement of life . for henry ,
world is not to be understood in the traditional sense of the totality of all things , nor is it to be taken phenomenologically as the horizon of horizons , nor is it even the medium of care . with the term flesh ( chair )
henry designates in sharp contrast to late merleau - ponty who takes it to represent the common milieu of subject and object an immanent arch - praxis , that is , the transcendental i can of the subject . in the phenomenality of this
i can world - content and the execution of our potentialities are inextricably bound together at the basic level of our transcendental affectivity prior to the display of the world : if the world is everywhere unrelenting , if the fabric of the sensible is perpetuated without fail or lacuna and it does not come unraveled at any point , if every fiber and every grain of which it is composed are interminably evocable , it is because every one of the capacities ( pouvoirs ) that lead me to them is the power of a flesh nothing is able to divide ( spare de soi ) , that is always present to itself in a memory without diversion , without thought , without a past , without memory in its immemorial memory .
the unity of the world is thus an immanent unity , it lies in the parousia of my flesh.38
the concrete experience of the world thus demands , henry concludes , as its ultimate possibility , a consciousness without world , an acosmic flesh.39 by this he understands , following maine de biran , the immanent corporeality of our i can .
transcendental i can is to be thought as a living ability given to us , a capacity that first and foremost makes possible the unlimited repetition of our concrete capacities.40 the task of unfolding the auto - affective structure of life thus is assigned to the flesh as the material concretion of the self - givenness of our innermost selfhood , i.e. , ipseity .
the flesh accomplishes , as it were , its translation into affective formations and therefore embodies the fundamental habitus of transcendental life,41 which make up the life - world as a world of life in its innermost essence.42 if the world is everywhere unrelenting , if the fabric of the sensible is perpetuated without fail or lacuna and it does not come unraveled at any point , if every fiber and every grain of which it is composed are interminably evocable , it is because every one of the capacities ( pouvoirs ) that lead me to them is the power of a flesh nothing is able to divide ( spare de soi ) , that is always present to itself in a memory without diversion , without thought , without a past , without memory in its immemorial memory .
the unity of the world is thus an immanent unity , it lies in the parousia of my flesh.38 with this we have a further indication of how transcendence ( i.e. , the world ) arising from immanence ( i.e. , life ) is to be understood then as something other than a non really included43 transcendence ( transzendenz irreellenbeschlossenseins)namely , as affective formation , condensation , or even as the immemorial memory of our flesh . yet might these descriptions of life s self - movement be represented more precisely ?
how are we to think henry s claim that the world s reality has nothing to do with its truth , with its way of showing , with the outside of a horizon , with any objectivity?how are we to think that the reality that constitutes the world s content is life?44 viewed against this background , henry s theory of the duplicity of appearing ostensibly leads to a seemingly insurmountable problem : how can the notion of an acosmic flesh in its
radical independence45 as the sole reality of life actually found that which is outside of it , the world ?
it is precisely this that we must now reflect on more explicitly if we wish to show that his approach can be made useful for problems that arise in the philosophy of society and culture as well as the questions posed by political philosophy .
the main objection to henry s reinscription of the world within life proceeds in the following way : the counter - reduction aims to found the visible display of the world in the invisible self - revelation of absolute life , yet does nt this disqualification of the world set into operation a
with this all too radical inquiry into the originary do we not become trapped in a mysticism of immanence , that remains enclosed in its own night , forever incapable of being expressed and coming into the world ? to summarize bernhard waldenfels exemplary formulation of this critique , does nt the negative characterization of self - affection as non - intentional , non - representational , and non - sighted or non - ecstatic bear a constant reference to the world - relation ( weltbezug ) that it suspends?47 ,
48 henry would reply firmly in the negative to this question . on the contrary
, he would posit that only a non - intentional phenomenology is capable of grounding the reality of the world , as a living reality that has nothing to do with its objective truth i.e .
this is certainly not meant to imply that our life brings forth the concrete content of the world , but rather simply that the lived intelligibility of such content is ontologically possible only as living content . in other words ,
its intelligibility rests on nothing other than the variation , transformation , and augmentation of those concrete individual potentialities that are at work in advance of its worldly appearing . according to henry ,
husserl was completely aware of this , however he failed to follow through on this insight : husserl s phenomenology has not disregarded the non - intentional . on the contrary
, it has designated the non - intentional quahyl as a fundamental stratum of consciousness . [ ] nevertheless this decisive function of the primary impression that would make of hyletic phenomenology the fundamental discipline of phenomenology becomes immediately overturned .
the hyl is nothing more than matter for a form that alone has the power of illuminating it and of making of it a phenomenon , and this form that sheds light on it is intentionality .
thus the original essence of phenomenality as revelation of the impression qua impression , that is , its impressionality , or rather its affectivity , is concealed .
[ ] with this concealment of the secret source and original essence of phenomenality at the hands of ecstatic phenomenality , it is nothing less than the concrete life of men that is carried away from the domain of philosophical thought , and away from that thinking which is opened up by phenomenology itself to exteriority and ultimately to a ruinous objectivism that is covered over by the objectivism of galilean science which was defined by its exclusion of subjectivity , that is , of life.49henry s criticism of husserl notwithstanding , this quote shows the extent to which his work is to be understood as a continuation of the husserlian crisis - diagnosis.50 indeed it should be noted that objectivism here should not be taken to mean only a reduction to the finality of the objective sciences inaugurated by galileo ( and the loss of their significance for life to which it necessarily leads ) , which husserl reproaches for being a weary form of reason.51 objectivism designates here rather the underlying self - conception of the human being as subject in light of ontological monism , given that such a self - conception still underlies the procedure of these sciences and is authorized anew by classical phenomenology .
the human being , insofar as he or she is reduced to the subject of self - knowledge or to self - regarding care ( sorge ) , is subjected to a systematic elimination of his transcendental humanitas .
it is therefore this transcendental humanitas that henry names the field of invisible appearing that makes possible the ego s praxis in the luminosity of the world .
he rather insists that there is a duplicity of appearing , i.e. , both visible world and the invisible transcendental ipseity are qualified by its living auto - affection .
it is only when the field of appearing is reduced to the world alone that phenomenology gives way to a barbaric character , eradicating the invisible life in which each ipseity takes hold of its life .
thus , in contrast to other analyses of barbarism , henry does not speak in la barbarie of auschwitz , the gulag or other cases of genocide .
the barbarism that he has in mind concerns rather a self - destructive reduction of human knowledge , which , according to him , made these twentieth - century catastrophes possible in the first place : scientism . by scientism henry understands the view that there exists no mode of knowing other than galilean science , that is to say , modern physics.52 following the presupposition shared even by the humanities of a universal abstraction of all subjective qualities , we consequently find that our knowledge of the human being becomes circumscribed entirely within the display of the world , its exterior objectivity , its truth . in this context , henry s critique of culture is not merely concerned with the forgetfulness of the life - world nor the one - dimensionality of technology as framing ( gestell ) and its resulting ethical problems , which dominate philosophical discourse today .
these problems are thoroughly thematized in his recurring and decidedly conservative critique of culture.53 at its core , henry s analysis aims at nothing other than the possibility of a destruction of life by means of itself , a possibility that underlies all these manifestations of crisis and becomes more or less deliberately exploited by such crises .
, it has designated the non - intentional quahyl as a fundamental stratum of consciousness . [ ] nevertheless this decisive function of the primary impression that would make of hyletic phenomenology the fundamental discipline of phenomenology becomes immediately overturned .
the hyl is nothing more than matter for a form that alone has the power of illuminating it and of making of it a phenomenon , and this form that sheds light on it is intentionality .
thus the original essence of phenomenality as revelation of the impression qua impression , that is , its impressionality , or rather its affectivity , is concealed .
[ ] with this concealment of the secret source and original essence of phenomenality at the hands of ecstatic phenomenality , it is nothing less than the concrete life of men that is carried away from the domain of philosophical thought , and away from that thinking which is opened up by phenomenology itself to exteriority and ultimately to a ruinous objectivism that is covered over by the objectivism of galilean science which was defined by its exclusion of subjectivity , that is , of life.49 henry s thesis implies that the germ of all barbarism lies in a self - destruction of life .
the possibility that life may wish to destroy itself is no way obvious as already a cursory look at the philosophical tradition that extends from spinoza through hegel and up to nietzsche and heidegger shows . according to henry , however , this possibility is indeed constitutive for life and is based on its duplicity . in order to understand this possibility it is necessary to examine the dynamic in which life in its affects is driven against itself , i.e. , the irreducible arch - passive self - givenness of life . as to henry ,
this passivity of life vis - - vis itself , which , as we have said , is the germ of its augmentation and therefore the condition of any kind of culture , can lead life to turn against itself . under what conditions , however , is this possible ? under the condition that it becomes no longer capable in itself to be augmented , under the condition that it is not adequate to the task of implementing the ground of its being , for such implementation belongs to its essence : the assertion that life experiences itself ( sprouve soi - mme ) implies that it is passive vis - - vis itself and consequently vis - - vis a foundation that sustains it [ ] thence , there is room for a meditation or an effort that seeks to feel this foundation of being within us more lively.54
religion , mysticism , art , ethics , and rational knowledge but also the refined fulfillment of our most basic needs are exemplary cases in the realm of culture of the self - augmenting articulation of the excessive nature of life , i.e. , its irreducibly antinomic nature .
symbolic institutions55 is in no way merely the pre - scientific validity of subjective experience in contrast to the objectivism of the scientific worldview . at play
rather is the innermost essence of this experience itself qua cultural life - praxis that expresses the excessive nature of life without displacing life in what is outside life.56 henry 's critique of traditional philosophical accounts of culture thus focuses on their tendency to regard culture as a means to expend or objectify life 's energy in worldly products .
hence , henry 's task is not , as in husserl , to unveil the history of institutions57 ( stiftungsgeschichte ) that concretely articulates the symbolic matrices of our various socio - cultural life - worlds.58 for henry it is rather a matter of inquiring into the originary passivity of life as that which in its very pathetic self - movement oscillates between the self - delighting capacity for living and the self - agonizing wish to live no longer , but which will never be able to escape from this movement . on these premises
it can be established that what scientism or the scientific knowing attempts to flee when it excludes the concrete field of invisible display internal to the ego , or what we attempt to flee when we pretend to live out our subjective potentialities by appealing to ideal entities , is the self - experiencing ( preuve de soi ) that necessarily involves the self - agonizing aspect of life.59 the assertion that life experiences itself ( sprouve soi - mme ) implies that it is passive vis - - vis itself and consequently vis - - vis a foundation that sustains it [ ] thence , there is room for a meditation or an effort that seeks to feel this foundation of being within us more lively.54 this flight from the invisible sphere of life into the exterior world of history and temporality however effectively takes on monstrous forms when life strives to sever itself from itself in order to bring this flight to its injurious yet unreachable end : the leap outside of the self is a flight into exteriority in which it is a matter of fleeing oneself and thus of ridding oneself of what one is , that is , of the burden ( poids ) of this malaise and suffering . however , this flight remains caught in its own pathos . thus there remains only one way out : in order to destroy purely and simply this malaise and this suffering of which we can not be rid , which indeed have their possibility in our self - experience ( le sprouve soi - mme ) and thus in life , life itself , its proper essence , must be terminated .
this self - destruction is bound to be just as unsuccessful in its aim ( fins ) as is self - flight if it is true that the act of self - destruction is only possible on the condition that it actualizes and affirms the essence that it wishes to annihilate.60
life preserves itself even in its intention to destroy itself .
idle energy,61 an energy that no longer traverses the suffering proper to it for the sake of augmenting itself . out of this unbearable situation of life , which in its attempt to destroy itself can not leave itself behind , results a fury of self-flight.the flight of the self is the title under which almost everything that passes before our eyes is arranged .
not science in itself , which as knowledge of the nature that it defines in its procedures is completely positive but [ ] the belief that this galilean science of nature constitutes the sole possible way of knowing , the sole truth , so that there is no other reality , as true reality , i.e. , real reality , than the object of this science , such that man himself is not real except under this title and such that all knowledge that concerns him can only be a mode or form of this particular science .
an ideology scientism and positivism has here become substituted for science.62 in no way does self - flight take place merely at the theoretical level . in the wake of this self - flight , which sets into motion scientism s tendency to systematically put life out of play ,
we see a proliferation of barbaric practices , in which life is expelled from itself.63 henry identifies several such practices , from the depreciation of life in social engineering , scientific practice and the tele - technological imaging and simulation of life to the destruction of the university . what is common to these practices is that they create an
inhuman world64that is , a world that is perpetually externalwhose truth disposes of the actuality of the life that makes it possible . in such practices ,
life is subjected to a fundamental ontological shock , for it becomes the object of a perpetual simulation of life .
for this reason , however , a form of life is established in which communication becomes its content,65 and consequently a culture from which the culture of life is excluded , and , to put it in nietzsche s words , is sacrificed in view of the
great hunt66 for the truth of the world : the world in fact is a milieu of pure exteriority .
everything that finds within it the condition of its being is never offered as anything but external - being , a section of exteriority , a surface , a shoreline given over to our regard and upon which our regard glides indefinitely without being able to penetrate the inside of that which is concealed from view behind each new aspect , faade , and screen . for this being which is nothing other than exteriority
has no interior , its law is becoming , the incessant outcropping of new faces and new planes and knowledge tracks this succession of lures , each of which presents itself to knowledge only to immediately cover over a being it does not possess and to redirect knowledge to another lure that does the same .
no interior : nothing that is living , that can speak in its proper name , in the name of what itexperiences , in the name of what it is .
they can speak only in the name of things , only in the name of death : in advance of the world and its ecstatic disclosure what exhibits itself and ex - poses itself is only the always - at - hand , the always - outside the object.67
this ontological lie about life situates life entirely within the horizon of the world and its objectivity thereby unveiling not life but , in fact , death .
the debacle of humanism in all its forms68 is , according to henry , thus nothing other than the necessary consequence of this barbarism that is internal to the modern spirit , which still calls for a defense of a genuine humanity precisely where it has already degraded humanity to a lifeless copy , that is , to an automaton.69 the leap outside of the self is a flight into exteriority in which it is a matter of fleeing oneself and thus of ridding oneself of what one is , that is , of the burden ( poids ) of this malaise and suffering .
thus there remains only one way out : in order to destroy purely and simply this malaise and this suffering of which we can not be rid , which indeed have their possibility in our self - experience ( le sprouve soi - mme ) and thus in life , life itself , its proper essence , must be terminated .
this self - destruction is bound to be just as unsuccessful in its aim ( fins ) as is self - flight if it is true that the act of self - destruction is only possible on the condition that it actualizes and affirms the essence that it wishes to annihilate.60 the flight of the self is the title under which almost everything that passes before our eyes is arranged .
not science in itself , which as knowledge of the nature that it defines in its procedures is completely positive but [ ] the belief that this galilean science of nature constitutes the sole possible way of knowing , the sole truth , so that there is no other reality , as true reality , i.e. , real reality , than the object of this science , such that man himself is not real except under this title and such that all knowledge that concerns him can only be a mode or form of this particular science .
an ideology scientism and positivism has here become substituted for science.62 the world in fact is a milieu of pure exteriority .
everything that finds within it the condition of its being is never offered as anything but external - being , a section of exteriority , a surface , a shoreline given over to our regard and upon which our regard glides indefinitely without being able to penetrate the inside of that which is concealed from view behind each new aspect , faade , and screen . for this being which is nothing other than exteriority has no interior , its law is becoming , the incessant outcropping of new faces and new planes and knowledge tracks this succession of lures , each of which presents itself to knowledge only to immediately cover over a being it does not possess and to redirect knowledge to another lure that does the same . no interior : nothing that is living , that can speak in its proper name , in the name of what itexperiences , in the name of what it is .
they can speak only in the name of things , only in the name of death : in advance of the world and its ecstatic disclosure what exhibits itself and ex - poses itself is only the always - at - hand , the always - outside the object.67
the barbarism of which henry bespeaks is not only a crisis of culture in the sense of a self - destruction of those practical laws in accordance with which the self - augmentation of subjective potentialities is actualized . on the contrary ,
nowhere is this exhibited more clearly than in the catastrophes of twentieth - century european politics .
henry examines this form of barbarism in his book , du communisme au capitalisme : thorie dune catastrophe .
i highlight here only its broad argument : fascism and totalitarianism succeed in leading the living to negate themselves in their own ineluctable lives in order to induce them to destroy others in violence : let s say simply here that never can life as such be the origin of crime , that is , of an act turned against itself , unless it engages in the monstrous process of self - negation , which will be the distinctive trait of nihilism and fascism.70
life s ontological self - flight , as henry has paradigmed it in la barbarie , is thus treated in light of its political manifestation , or what henry calls the hypostasis of the political .
this consists in the application of the political as a universal , on the basis of which each individual can both be certain of his own particularity and yet be damned to experience himself in his nullity vis - - vis the universal . in other words ,
the hypostasis of the political is quite literally the exaltation of the political to the status of a real being .
thus viewed , the hypostasis of the political severs the connection to the immanent realization of life on the part of individuals along with their pathic intersubjectivity , and this severing subjects living beings and their invisible relations to each other to the judgment of the visible gaze of the state ( which is epitomized in hegel s notion of the light of the state ( staatslicht):the individual is nothing other than the occupant of a place delimited by the ensemble of processes that constitute the substance of society and being taken into account as such , as the affair of civitas , is what defines the political .
the political is everything , the individual is nothing.71
the debasement of the individual defines , for henry , the core of fascism , its attack on life as such , which is carried out not only directly but structurally.fascism always implies the debasement of the individual and , at the bottom of this will to debasement , there lies the will to deny it .
this negation of the individual is what allows fascism to appear from the outset as a force of death but who is the individual we are speaking of here ? under what aspect or in what part of his being must this be directed , attained and precisely negated so that we can speak of fascism ? in what makes him a living being [ ] , it is where the individual is an individual , where he is this singular individual , in his life , that fascism strikes .
[ ] and it is in this that fascism is veritably a force of death.72fascism attacks life by seeking to throw life outside itself into the sphere of contingent objectivity whereas in reality life s essence appears to itself , is given to itself and is manifest to itself by experiencing itself on the basis of its primordial arch - passivity .
fascism as henry conceives it is an attack on the phenomenological essence of absolute life that constitutes every living .
we are thus confronted with the groundlessness of the hypostasis of the political that is rendered visible in the institution of fascism , its phantasmatic incarnation in the body of a people ( or even in a class - consciousness ,
etc.).if a people has its reality in the individual , the negation of the individual is in truth a self - negation .
the time of the political is the time of despair , the moment when life , or the individual , no longer believing in themselves and wishing to flee themselves , throw themselves outside of themselves and plunge into everything that could motivate such a flight and notably political existence , an existence devoted to the public cause ( chose ) , to history , to society and its problems , and to everything that permits the individual to no longer live his own life and to forget himself.73yet , according to henry , it is not only in the hypostasis of the political indicative of real existing socialism or fascism that the groundlessness of the political can appear .
doubtless there are totalitarian ideologies that can be understood to have instrumentalized man s fear of the groundlessness of communal being - together , insofar as they project this fear onto the ontological inconsistency of the very individual that has been constructed by them.74 however , democracy , too , can fall prey to the hypostasis of the political . and
so the collapse of totalitarian political systems should not detract , henry so argues , from the ever - present reality that democracy can enlist state - sanctioned violence in its own interest to protect its boundaries against individuals .
let s say simply here that never can life as such be the origin of crime , that is , of an act turned against itself , unless it engages in the monstrous process of self - negation , which will be the distinctive trait of nihilism and fascism.70 the individual is nothing other than the occupant of a place delimited by the ensemble of processes that constitute the substance of society and being taken into account as such , as the affair of civitas , is what defines the political .
the political is everything , the individual is nothing.71 fascism always implies the debasement of the individual and , at the bottom of this will to debasement , there lies the will to deny it .
this negation of the individual is what allows fascism to appear from the outset as a force of death but who is the individual we are speaking of here ?
under what aspect or in what part of his being must this be directed , attained and precisely negated so that we can speak of fascism ? in what makes him a living being [ ] , it is where the individual is an individual , where he is this singular individual , in his life , that fascism strikes .
[ ] and it is in this that fascism is veritably a force of death.72 if a people has its reality in the individual , the negation of the individual is in truth a self - negation .
the time of the political is the time of despair , the moment when life , or the individual , no longer believing in themselves and wishing to flee themselves , throw themselves outside of themselves and plunge into everything that could motivate such a flight and notably political existence , an existence devoted to the public cause ( chose ) , to history , to society and its problems , and to everything that permits the individual to no longer live his own life and to forget himself.73 according to henry , the breakdown of democracy here is based not on contingent but indeed undoubtedly alarming events .
this is also true in the case of the ( seemingly unavoidable ) self - legitimating caprice of the self - elected elite or the incompetence of the political representatives,75 which may ultimately be excluded as a sufficient reason for a definitive failure in the context of procedural democracies . for henry , the decisive question concerns rather the fact that the democratic itself lacks grounding .
what he interrogates is the foundation of democracy itself , namely , the freedom and equality of individuals , as they are determined by human rights . as he explains , human rights do not represent a formal principle . on the contrary ,
more precisely , this signifies that they should not be understood as procedurally entitled attributes , since on these premises they can , in principle , also be legitimately negated.76 from this reflection it follows that democracy ultimately can not discover its principle within itself ( with respect to human rights levinas speaks of an extra - territorial claim ) : for how can what lies before every decision result from this ( sc . extra - territorial claim ) , how can it be founded on it?77 yet according to henry this objection does not arise by chance , but rather follows , as he notes , from the concept of political representation.in order to fulfill the democratic principle , political representation was invented , but political representation [ as the substitution of individuals by delegates ; ms ] is the negation of the democratic principle.78this line of inquiry opens up a field of enormous scope with respect to the political , for it implies the fundamental question of whether or not the basic values of democracy can be grounded , and if so , how , politically , ethically , or religiously . for henry , the grounding of democracy can only be thought along ethical lines .
the ethical , however , in relation to the essence of life understood even as the unrefusable gift of a supreme power , i.e. , as the transcendental birth of our i can , which is born out of the hyper - power of absolute life ( hyper - pouvoir de la vie)is synonymous with the religious .
religious and so - called primitive societies would have known , says henry , about this supreme power : they lived with the fundamental life - knowledge that man is not his own ground ; they lived this knowledge culturally , in art for instance , which was thus always religious .
the elimination of the last religious societies in the west , the christian society , which thinks the absolute as life , was , according to henry , the natural result following upon the new science that galileo set into operation , which places life out of play in favor of the material universe.79 this galilean reduction takes place especially where , as we saw , the democratic principle forms a fateful alliance with it.80 because for a social organization that seeks to discover the principle of its organization in itself the ostensibly transcendent origin of the ethical political must necessarily appear as extrinsic to the political domain itself , as if imposed from the outside , i.e. , as an inadmissible exteriority ( extriorit irrecevable ) .
the democratic principle here defined props up a political system , as history makes evident , contradictory to the sacredness of life as it is expressed in henry s concept of the transcendental field of absolute life that gives birth to each of us as living ipseities .
this transcendental field interior to the ego finds no place in a political domain funded by galilean science , and indeed life can never do so .
the reason it can not do so is because the categories of freedom and equality so cherished by democracy are what henry calls empty concepts .
what henry conveys by labeling them as empty is the fact that they fail to render intelligible the irrefusable gift of life by which each living self is co - original within a primordial co - pathetic collectivity that precedes any possible symbolic institution of the social bond .
it is no surprise , then , that henry claims that the consequences for our late modern societies are disastrous given that they possess an ethos preoccupied with modern science , individual freedom and exterior display .
henry formulates the consequences in what follows , even if polemically : in fact , from the moment scientific knowledge is taken as the only true knowledge and the galilean field of the material universe that it apprehends is taken as the sole reality , then what does not appear in such a field
absolute life , which experiences itself outside the world , the ipseity of this life that is its experience of self , any transcendental self drawing its essence from this ipseity [ ] .
the death of god, a dramatic leitmotif of modern thought attributed to some audacious philosophical breakthrough and parroted by our contemporaries , is just the declaration of intent of the modern mind and its flat positivism .
but because this death of god destroys the interior possibility of man , since no man is possible who is not first a living self and a
me , it strikes at the very heart of man himself.81
from this insight into the true nature of the death of god as the distinguishing feature of the modern crisis of the subject ensues one of henry s further cardinal insights
namely , the insight into the loss of an originary ethos , an ethos in accordance with life s self - giving82:this is why , as soon as this condition fails to appear , the imprescriptible love of others also disappears .
the other is now just another person , a person as people are [ ] .
forgetful of their veritable condition and the other s veritable condition , they behave toward themselves and others as mere people .
then the whole edifying morality that wishes to found itself on the mere person , on the rights of man , discovers its emptiness , its prescriptions are flouted , and the world is given over to horror and sordid exploitation , to massacres and genocides .
religious morality has given rise not to a new morality , a secular morality , albeit a morality without any definite foundation , but to the downfall of any morality and to the terrifying and yet daily spectacle of that downfall.83 in order to fulfill the democratic principle , political representation was invented , but political representation [ as the substitution of individuals by delegates ; ms ] is the negation of the democratic principle.78 in fact , from the moment scientific knowledge is taken as the only true knowledge and the galilean field of the material universe that it apprehends is taken as the sole reality , then what does not appear in such a field absolute life , which experiences itself outside the world , the ipseity of this life that is its experience of self , any transcendental self drawing its essence from this ipseity [ ] .
the death of god, a dramatic leitmotif of modern thought attributed to some audacious philosophical breakthrough and parroted by our contemporaries , is just the declaration of intent of the modern mind and its flat positivism .
but because this death of god destroys the interior possibility of man , since no man is possible who is not first a living self and a
me , it strikes at the very heart of man himself.81 this is why , as soon as this condition fails to appear , the imprescriptible love of others also disappears .
the other is now just another person , a person as people are [ ] .
forgetful of their veritable condition and the other s veritable condition , they behave toward themselves and others as mere people .
then the whole edifying morality that wishes to found itself on the mere person , on the rights of man , discovers its emptiness , its prescriptions are flouted , and the world is given over to horror and sordid exploitation , to massacres and genocides .
religious morality has given rise not to a new morality , a secular morality , albeit a morality without any definite foundation , but to the downfall of any morality and to the terrifying and yet daily spectacle of that downfall.83
what i have tried to underline in the preceding reflections is the unfathomed fact that the immanent teleology of lifeits tendency as a force to incessantly surpass itself at the moment when it runs dry and remains idle , or is reduced to
natural needs , ultimately turns against itself and others , qua projections of the self - reproach of the alienated self . for such
empty subjectivity remains , as henry notes , an avid subjectivity.84 even when life is reduced ideologically or scientifically it does not cease living . as empty , however , it is not only in an avid way that subjectivity escapes into ever new representations of life in particular medially and procedurally generated representations as well as into certain representations of itself as an other .
in fact , subjectivity becomes ensnared in this self - flight and , at once , falls ever deeper into the fear of being nothing a fear , with which subjectivity copes only by means of projecting itself precisely into these representations of an absent life
those transcendence - surrogates or yet unexplored possibilities , of which nietzsche spoke.85 the true transcendence ,
the immanence of life in every living being,86 falls prey to a forgetting that , according to henry , is in fact transcendental.87 such a self - forgetting on the part of life , however , does not therefore merely represent a negation of life but in fact is an expression of life itself . when henry speaks of a second birth
that is supposed to set us free,88 we are nevertheless left to wonder if he has not hereby precisely broken up the
, the essential duplicity of appearing and thereby effaced the self s place in the world , giving the impression that the interior self is to be privileged at the expense of the self s creative activity in the exterior world . in transcendence , it is hence necessary to vindicate the life that apprehends itself in immanence , as nothing more but also nothing less than a
biotope.90 hence we must understand the self - forgetting of life as its transcendental condition of possibility .
the last words of christ , as they are transmitted through the gospel of matthew , also testify to this idea : my god , my god , why have you forsaken me ? in them a kenotic truth of the world is expressed , for which we as living beings have to assume responsibility , as jan patocka argues in a commentary on christ s words : there is nothing mystical here .
why have you forsaken me? the answer lies in the question . what would have happened if you had nt forsaken me ?
forsaken precisely so that there will be nothing there , nothing he can cling to .
no thing but this does not mean that this nothing does not in fact contain the whole , das all , in the words of the poet.91 there is nothing mystical here .
why have you forsaken me? the answer lies in the question . what would have happened if you had nt forsaken me ?
forsaken precisely so that there will be nothing there , nothing he can cling to .
no thing but this does not mean that this nothing does not in fact contain the whole , das all , in the words of the poet.91 | this essay explores the practical significance of michel henry s material phenomenology . commencing with an exposition of his most basic philosophical intuition , i.e. , his insight that transcendental affectivity is the primordial mode of revelation of our selfhood , the essay then brings to light how this intuition also establishes our relation to both the world and others .
animated by a radical form of the phenomenological reduction , henry s material phenomenology brackets the exterior world in a bid to reach the concrete interior transcendental experience at the base of all exteriority .
the essay argues that this counter reduction , designed as a practical orientation to the world , suspends all traditional parameters of onto(theo)logical individuation in order to rethink subjectivity in terms of its transcendental corporeality , i.e. , in terms of the invisible display of affective flesh .
the development of this metaphysics of the individual anchors his practical philosophy as he developed it under shifting accents throughout his oeuvre .
in particular , the essay brings into focus henry s reflections on modernity , the industry of mass culture and their barbaric movements .
the essay briefly puts these cultural and political areas of henry s of thinking into contact with his late
theological turn ,
i.e. , his christological account of life and the ( inter)subjective self - realization to which it gives rise . |
over the last decades numerous publications have highlighted the decline of serum testosterone levels in aging men .
this decline has been termed late - onset hypogonadism ( loh ) . rather than chronological age
per se , obesity , but also impaired general health , are the more common causes of low testosterone in aging men .
it has become abundantly clear that the decline of serum testosterone with aging is not an academic question but has important implications for elderly men .
severe loh is associated with substantially higher risks of all - cause and cardiovascular mortality , to which both the level of testosterone and the presence of sexual symptoms contribute independently .
a recent literature review on the risk factors , comorbidities , and consequences of loh demonstrated that advanced age , obesity , a diagnosis of metabolic syndrome , and poor general health status were predictors of loh .
diabetes mellitus was correlated with hypogonadism in most studies but was not established as a risk factor .
although diseases such as coronary heart disease , hypertension , stroke , and peripheral arterial disease did not predict hypogonadism , they did correlate with the incidence of low testosterone .
low levels of testosterone may have important long - term negative health consequences . in the european male ageing study ,
more than 50% of subjects with loh reported the presence of one or more common morbidities .
overall , hypertension ( 29% ) , obesity ( 24% ) , and heart diseases ( 16% ) were the most prevalent conditions .
particularly , sexual symptoms might indicate loh . in one study , an inverse relationship between an increasing number of sexual symptoms and a decreasing testosterone level was observed .
the authors concluded that loh can be defined by the presence of at least three sexual symptoms associated with a total testosterone level of less than 11
nmol / l ( 3.2 ng / ml ) and a free testosterone level of less than 220
testosterone treatment has a number of beneficial effects , particularly on weight , fat distribution , and metabolic factors .
testosterone might be a powerful tool in weight reduction and its associated benefits . in the present study , we analyzed whether the age of subjects receiving testosterone for the treatment of loh is a determinant of the beneficial effects of testosterone administration .
in this cumulative registry study , 561 hypogonadal mainly elderly men from two urology offices were followed through observational registry studies .
a total of 450 men were 65 years of age ( meanstandard deviation [ sd ] , 56.106.29 years ) and 111 men were > 65 years ( meansd , 68.452.91 years ) .
all subjects had sought urological consultation for various urological conditions . a number of subjects , for instance , men with osteoporosis , had been referred by other specialists who suspected the men might have testosterone deficiency .
we performed a pooled analysis of men 65 years of age ( group y ) and men > 65 years ( group o ) of age from both registries . a threshold for testosterone deficiency of 12.1 nmol / l was used , as confirmed by an international group of authors .
all men also had clinical symptoms of hypogonadism as assessed with the aging males ' symptoms ( ams ) scale .
all men received treatment with parenteral testosterone undecanoate 1,000 mg ( nebido , bayer pharma , berlin , germany ) , administered at baseline and 6 weeks and every 12 weeks thereafter for at least 72 months .
exclusion criteria for testosterone administration were a previous diagnosis of primary or secondary hypogonadism ; previous treatment with androgens ; prostate cancer or any suspicion thereof , such as prostate - specific antigen ( psa ) levels>4 ng / ml ; international prostate symptom score ( ipss)>19 points ; breast cancer ; or severe untreated sleep apnea . measurements of anthropometric parameters were performed at baseline ( height , weight , waist circumference ) and at each visit , and blood samples were drawn at each or every other visit before the next injection of testosterone . therefore , testosterone levels were trough levels at the end of an injection interval .
waist circumference provides a unique indicator of body fat distribution , which can identify patients who are at increased risk of obesity - related cardiometabolic disease , above and beyond the measurement of body mass index ( bmi ) .
the significance of waist circumference to assess the accumulation of abdominal fat has been well documented .
the ams scale and the international index of erectile function ( iief ) were used as self - administered quality of life and erectile function instruments .
the ams questionnaire is a 17-item scale assessing health - related quality of life and symptoms in aging men ( e.g. , somato - vegetative , psychological , and sexual symptoms ) on a 5-point likert scale ( 1=not applicable to 5=very severe ) .
the iief erectile function domain ( iief - ef ; maximum score , 30 ; according to cappelleri et al . ) was assessed at baseline and every 3 months .
liver enzymes were measured because reductions in liver enzymes may suggest reductions in liver fat content , as observed by hoyos et al . .
postvoid residual bladder volume and prostate volume were measured by using a sonoace sa 8000 se with 3 ultrasound probes . for abdominal measurement of residual bladder urine volume , a probe with 3 - 7 mhz was used , and for prostate volume , a transrectal probe of 5 - 12 mhz was used .
ethical guidelines as formulated by the german " rzte kammer " ( the german medical association ) for observational studies in patients receiving standard treatment were followed .
all subjects consented to be included in the research of their treatment protocol , which was in accordance with the declaration of helsinki ( http://www.wma.net ) .
all procedures were carried out with the adequate understanding and written consent of the subjects .
the group 65 years of age was labeled as group y ( young : n=450 : mean age , 56.106.29 years ; range , 32 - 65 years ) , and the group > 65 years of age as group o ( old : n=111 ; mean age , 68.452.91 years ; range , 66 - 84 years ) .
all together , we are reporting 3,320 patient - years : 2,587 for group y and 733 for group o. the results of 6 years of testosterone administration are presented in table 1 .
trough levels of total testosterone ( nmol / l ) rose from 8.961.95 to 16.182.73 nmol / l after 1 year and stabilized at 17 - 18 nmol / l in group y. in group o , total testosterone rose from 8.462.26 to 16.644.23
after 1 year and remained stable at 17 - 18 nmol / l thereafter ( fig .
1 ) . mean weight decreased from 102.5215.56 to 90.159.69 kg in group y and from 102.8315.64 to 95.359.03 kg in group o. model - adjusted mean change from baseline was -14.780.35 and -15.140.71 kg , respectively .
percentage change from baseline was -13.56%7.56% in group y and -13.28%7.14% in group o. waist circumference decreased from 106.549.03 to 98.267.1 cm in group y and from 108.9510.75 to 100.729.45 cm in group o. the mean change from baseline was 9.340.2 cm in group y and 10.450.47 cm in group o. bmi ( kg / m ) decreased from 32.585.08 to 29.023.01 in group y and from 32.844.86 to 30.352.61 in group o. the mean change from baseline was -4.720.11 and -4.810.22 , respectively ( p<0.0001 for all ) .
total cholesterol decreased from 268.9245.95 to 193.5616.58 mg / dl in group y and from 268.4452.69 to 191.6921.8 mg / dl in group o. low - density lipoprotein decreased from 159.8736.7 to 119.81 34.87 mg / dl in group y and from 162.4831.63 to 120.8633.56 mg / dl in group o. triglycerides decreased from 262.3573.16 to 192.134.4 mg / dl in group y and from 266.984.37 to 192.27 32.16 mg / dl in group o. high - density lipoprotein ( hdl ) increased from 48.9117.33 to 59.5517.66 mg / dl in group y and from 51.64 16.56 to 61.9916.87 mg / dl in group o. the ratio of total cholesterol to hdl improved from 6.152.42 to 3.541.04 in group y and from 5.67 2.09 to 3.320.91 in group o ( p<0.0001 for all ) . in group
y , iief - ef improved from 14.947.34 at baseline to 19.235.94 after 1 year , 21.585.72 at 2 years , 22.575.53 at 3 years , 22.985 at 4 years , 23.394.96 at 5 years , and 24.214.39 at 6 years .
changes were statistically significant versus baseline at each year ( p<0.0001 ) and at 2 years vs. 1 year ( p<0.0001 ) , 3 years vs. 2 years ( p<0.0001 ) , and 4 years vs. 3 years ( p=0.0071 ) and were sustained thereafter .
o , iief - ef improved from 11.877.58 at baseline to 16.796.35 after 1 year , 18.716.75 at 2 years , 19.466.62 at 3 years , 19.957.19 at 4 years , 20.167.11 at 5 years , and 22.126.41 at 6 years .
changes were statistically significant versus baseline at each year ( p<0.0001 ) and at 2 years vs. 1 year ( p<0.0001 ) and were sustained thereafter .
prostate volume increased in group y from 26.779.4 to 31.5810.9 ml ( p<0.0001 ) and in group o from 33.858.66 to 39.957.6 ml ( p<0.0001 ) ( fig .
psa increased by 0.360.02 ng / ml in group y , from 1.330.91 to 1.650.83 ng / ml ( p<0.0001 ) , and by 0.380.05 ng / ml in group o , from 1.440.98 to 1.880.84 ng / ml ( p<0.0001 ) ( fig .
ipss improved in group y from 7.745 to 3.893.12 ( p<0.0001 ) and in group o from 10.74.07 to 4.632.63 ( p<0.0001 ) .
these changes were statistically significant versus the previous year for all 6 years in group y and the first 5 years in group o and were then maintained throughout the observation period ( fig .
postvoiding residue declined in group y from 34.8723.12 to 16.726.74 ml ( p<0.0001 ) and in group o from 41.4623.48 to 18.845.75 ml ( p<0.0001 ) ( fig .
ams improved in group y from 53.539.47 to 21.686.88 ( p<0.0001 ) and in group o from 54.999.02 to 23.457.91 .
these changes were statistically significant versus the previous year for the first 3 years in both groups and were then maintained throughout the observation period .
c - reactive protein as a marker of inflammation dropped in group y from 4.046.34 to 0.751.06 mg / dl ( p<0.0001 ) and in group o from 2.653.75 to 0.710.51 mg / dl .
the changes in both groups over a period of 6 years are summarized in table 1 .
prostate cancer occurred in 11 men : 7 men in group y ( 1.6% ) and 4 men in group o ( 3.6% ) .
the incidence per 10,000 patient years was 27.1 in the younger and 54.6 in the older group .
age need not be a contraindication against treatment with testosterone . as the guidelines of the international society for the study of the aging male specify , age is not a contraindication to initiate testosterone treatment .
individual assessment of comorbidities ( as possible causes of symptoms ) and potential risks versus benefits of testosterone treatment are particularly important in elderly men .
there is a paucity of information on the benefits of testosterone treatment for elderly men with proven loh . in this report
we provide data on a large cohort of men receiving testosterone for up to 6 years .
the benefits for men older than 65 years of age were compared with those of younger men , and the improvements in body weight , metabolic factors , psychological functioning , and sexual functioning were of the same magnitude in both age groups .
although weight loss was progressive over the 6-year period , effects of testosterone on lipids and on psychological and sexual functioning reached a plateau after approximately 3 years and these effects were sustained .
effects of testosterone on hematopoiesis , on the prostate , and on bladder function were not more severe in older men than in younger men .
we did observe a mild increase in prostate volume and serum psa over time , which is a normal finding in aging men .
maybe somewhat surprising , postvoiding residue and the ipss did not deteriorate with aging but showed a degree of improvement .
some patients received treatment with -blockers ( tamsulosin ) or 5-reductase inhibitors , but these drugs were already being taken before testosterone administration started .
there are now studies showing that testosterone treatment of elderly hypogonadal men improves these parameters .
this may be a direct effect on tissues or may be an indirect effect : the severity of the metabolic syndrome is associated with the severity of lower urinary tract symptoms .
the symptoms of the metabolic syndrome improve upon testosterone treatment and testosterone may thus have a favorable effect on lower urinary tract symptoms .
therefore , it seems reasonable to conclude that the risks of testosterone administration to elderly men are not disproportionately higher in elderly men than in younger men .
our data collection was not large enough to draw solid conclusions as to the risks of prostate cancer in an elderly population of men receiving testosterone treatment , which is , however , an important issue .
despite evidence to the contrary , physicians still harbor a wrongful association between testosterone and the development of prostate pathology ( prostate cancer and benign prostate hyperplasia ) . in the combined group of younger and older patients , 11 cases of
not surprisingly , the incidence of prostate cancer was higher in older men ; however , it was lower than expected in both groups .
these observations suggest that the incidence of prostate cancer in patients receiving testosterone therapy , both in the younger and in the older group , was not greater than in the general population not receiving testosterone treatment . of the 11 cases of prostate cancer ,
7 were 65 years of age when testosterone therapy was initiated , and 4 were older than 65 ( 66 , 67 , 67 , and 73 years of age ) .
so far as these data permit conclusions , it was not the oldest men who developed prostate cancer .
the historical fear that raising testosterone levels will result in more prostate cancer has been dispelled , particularly by the work of abraham morgentaler .
higher serum testosterone levels fail to show an increased risk of prostate cancer , and supraphysiological testosterone does not increase prostate volume or psa in healthy men .
this apparent paradox is explained by the " saturation model , " which posits a finite capacity of androgen to stimulate the growth of prostate cancer .
recent studies indicate no increased risk of prostate cancer among men with serum testosterone in the therapeutic range .
because age is a strong prognosticator of prostate malignancy , follow - up of elderly men receiving testosterone remains mandatory .
two studies showing a relationship between testosterone administration and cardiovascular disease in elderly men have stirred up concerns .
however , these studies have been heavily criticized for methodological flaws and have invited a much needed critical analysis of pro and con arguments .
the relationship between testosterone and cardiovascular disease has recently been extensively reviewed . in the present observational study ,
a very important aspect is that the benefits of testosterone therapy are fully achieved only by long - term treatment . to achieve maximal benefits ,
some recent reports have raised questions about patient compliance to testosterone therapy . in our registry study of 561 patients ,
however , there were only 4 dropouts ( 0.7% ) : 2 patients moved to different locations and 2 patients were lost to follow - up for unknown reasons .
observations were carried out over 6 years in a large population . however , there are some limitations .
the study was not blinded and was not designed to monitor specific benefits and side effects of testosterone administration to elderly men ( > 65 years ) .
a limitation of our study was that we did not record any changes in use of concomitant medications , for instance , phosphodiesterase type 5 ( pde5 ) inhibitors . at baseline ,
nevertheless , some conclusions are warranted : we found that restoring testosterone levels to normal had similar beneficial effects in men older than 65 years of age as in younger men .
in addition , the risks of administration of testosterone to this age group were not higher than in younger men .
. there is , however , a certain hesitation on the part of physicians to prescribe testosterone to men of advanced age .
the results of the present study show that benefits of testosterone treatment are equal in men older than 65 years and in men younger than 65 years .
the men studied were too few in number to make a reliable assessment of the risks in elderly men , but there are now several publications indicating that the risks are acceptable . | purposeto investigate the potential benefits of testosterone administration to elderly men ( > 65 years ) with late - onset hypogonadism ( loh ) in comparison with younger men and to assess the safety of testosterone administration to elderly men.materials and methodsa total of 561 hypogonadal men from two registry studies were divided into age groups of 65 years ( group y , n=450 ; range , 32 - 65 years ) and > 65 years ( group o , n=111 ; range , 66 - 84 years ) . following an initial 6-week interval ,
all men were treated with 3-month injections of parenteral testosterone undecanoate for up to 6 years.resultsover the 6 years , there was a progressive decrease of body weight and waist circumference .
beneficial effects on lipids and other metabolic factors and on psychological and sexual functioning progressed over the first 24 to 42 months and were sustained . rather than a deterioration
, there was an improvement of urinary parameters .
prostate volume and prostate - specific antigen increased moderately .
hematocrit levels increased but remained within safe margins.conclusionsthe benefits of restoring serum testosterone in men with loh were not significantly different between men older than 65 years of age and younger men .
there were no indications that side effects were more severe in elderly men .
the effects on prostate and urinary function and hematocrit were within safe margins .
age itself need not be a contraindication to testosterone treatment of elderly men with loh . |
haemophilus spp . generally colonize the upper respiratory tract and can cause infections such as bronchitis , sinusitis , epiglottitis , pneumonia and meningitis .
h. influenzae has been reported as a rare cause of genitourinary tract infection such as urinary tract infection , , , , , pyelonephritis , , prostatitis , epididymitis , salpingitis and endometritis .
we report here an immunocompetent japanese man with bacteremic pyelonephritis caused by nontypable h. influenzae associated with a left ureteral calculus .
a 44-year - old japanese man with a history of left ureteral renal calculus presented to our hospital with a one - day history of left flank pain , fever and malaise without any respiratory symptoms .
he had left flank pain caused by a left ureteral calculus over the past two decades . otherwise , his past history and family history were unremarkable . on physical examination ,
his blood pressure was 153/91 mm hg , pulse rate 112 beats / min , respiratory rate 20 breaths / min , oxygen saturation on ambient air 98% and body temperature 38.9 c .
laboratory tests showed an elevated white blood cell ( wbc ) count 12.2 10/l and creatinine level of 1.25 mg / dl .
microscopic examination of the urine sediment showed an elevated wbc and red blood cell ( rbc ) count of 50 and 99 per high power field , respectively .
two sets of blood cultures were obtained using bactec plus aerobic / f and anaerobic / f culture bottles ( becton , dickinson and company , sparks , md ) .
urine culture was performed using sheep blood agar and drygalski improved medium ( eiken chemical co , ltd , tokyo , japan ) .
abdominal ultrasound and computerized tomography ( ct ) scan of the abdomen and pelvis without contrast showed a left ureteral calculus ( 17 10 19 mm ) , left hydronephrosis and a normal - sized prostate gland .
after admission , treatment with intravenous ceftriaxone 2 g every 24 h was initiated and the ureteral stent was inserted into his left ureter .
after 32 h of incubation , gram - negative bacilli were isolated from an aerobic blood bottle .
both blood and urine cultures using sheep blood agar and drygalski improved medium showed no growth .
chocolate ii agar ( nissui pharmaceutical co , ltd , tokyo , japan ) for blood and urine based on candle jar method at 37 c were used for culture .
the bacteria required both x factor ( hemin ) and v factor ( nicotinamide adenine dinucleotide ) for its growth .
, the united states ) automated identification and susceptibility testing showed 99.99% probability of -lactamase - negative h. influenzae biotype 3 .
it was also identified as nontypable h. influenzae by influenza bacillus immune serum kit ( denka seiken , co. , ltd , niigata , japan ) .
this organism was susceptible to ampicillin ( mic = 0.25 g / ml ) , amoxicillin clavulanic acid ( mic 1 g / ml ) , ampicillin sulbactam ( mic 0.5 g / ml ) , ceftriaxone ( mic 0.12 g / ml ) , meropenem ( mic 0.12 g / ml ) , clarithromycin ( mic = 4 g / ml ) , levofloxacin ( mic 0.12 g / ml ) and rifampicin ( mic 0.5 g / ml ) and resistant to sulfamethoxazole - trimethoprime ( mic = 4 g / ml ) .
nasopharyngeal culture that was obtained after the initiation of antimicrobials was negative for haemophilus spp .
follow up blood and urine cultures were negative . on hospital day 4 , he became afebrile .
he was discharged home on hospital day 9 and treated with oral amoxicillin 1000 mg three times a day for 5 more days .
fifteen days after discharge , he underwent extracorporeal shock wave lithotrity for the left ureteral calculus .
the non - typable strains generally are not invasive and only the type b strain is preventable by vaccine .
h. influenzae has been reported as a rare cause of genitourinary tract infection and fewer case reports of genitourinary tract infections along with bacteremia due to h. influenzae .
the reported prevalence of h. influenzae in urine samples was 0.04% in women and 0.22% in men .
true prevalence of this organism in the genitourinary tract , may be underestimated , since culture media for this organism are not routinely used for urine specimens . when chocolate agar was used for routine urine cultures
showed that biotype 4 predominated in genitourinary isolates , while biotype 1 in respiratory isolates .
wallace et al . also reported that nontypable h. influenzae of biotype 4 may have been most common for genitourinary tract infection of women .
the results of these studies suggest that h. influenzae biotype 4 might have some mechanism related to the affinity to the genitourinary systems and/or direct invasion to the epithelium .
the patients were stratified by gender due to differences in clinical features . in women , 90% ( 18/20 cases ) of the patients were associated with pregnancy and , notably , 75% ( 15/20 ) had bacteremia , .
non - bacteremic urinary tract infection was only a case reported by morgan et al . .
in men , 25.6% ( 10 out of 39 cases ) of past reported patients had anatomical or functional abnormality of genitourinary tract such as renal calculus , benign prostate hypertrophy , ureteral occlusion or post - operation .
there were only 3 cases of bacteremia among the infections in males ( 7.6% ) .
therefore the true proportion of the patients with underlying diseases was possibly underestimated . regarding the serotypes of h. influenzae , a total of 66.7% ( 26 out of 39 ) was type b and 28.2% ( 11 out of 39 ) was nontypable ( 2 cases was unknown ) . in 29 out of 39 patients , biologic type of h. influenzae
thirteen were type 2 , 9 were type 3 , type 1 or 4 for 3 patients respectively and type 8 was only from one patient .
our patient had left renal calculus and serologic / biologic type was nontypable / type 3 . regardless
gender and serologic type , h. influenzae were mostly biotype 14 ( 39 out of 42 ) .
it is possible that this ability might facilitate colonization or infection in the genitourinary tract . indeed ,
urease production in the urine will produce an alkaline ph which can predispose to stone formation .
we report an immunocompetent patient with a ureteral calculus associated with bacteremic urinary tract infection caused by nontypable h. influenzae ( biotype 3 ) .
h. influenzae is difficult to grow on urine culture and its isolation may be underestimated .
collaboration between clinicians and microbiology laboratory personnel is essential for correct identification of the organism and appropriate therapy for genitourinary tract infections due to this organism .
| haemophilus species are known to colonize the upper respiratory tract and can cause infections .
however haemophilus influenzae has been rarely described as a cause of genitourinary tract infection .
we report a 44-year - old nonimmunocompromised japanese man with bacteremic pyelonephritis caused by a nontypable h. influenzae associated with a left ureteral calculus .
the organism was isolated from both blood and urine cultures .
treatment consisted of 14 days of intravenous ceftriaxone and oral amoxicillin one after than other and insertion of a left ureteral stent . after discharge
, he underwent extracorporeal shock wave lithotrity for the left ureteral calculus .
he had no recrudescence of the symptoms .
h. influenzae should be considered as a genitourinary pathogen among patients with certain risk factors such as anatomical or functional abnormality of genitourinary tract .
collaboration between clinicians and microbiology laboratory personnel is essential for correct identification of the organism and appropriate therapy for genitourinary tract infections due to this organism . |
iniencephaly is a rare , fatal neural tube defect ( ntd ) characterized by occipital bone defects at foramen magnum , fixed retroflexion of head , spinal dysmorphism , and lordosis of cervicothoracic vertebrae .
howkin and lawrie in 1939 classified iniencephaly into two types based on the presence or absence of encephalocele into iniencephaly clausus ( without encephalocele ) and iniencephaly apertus ( with encephalocele ) .
it is usually associated with central nervous system , gastrointestinal , and cardiovascular anomalies . here
we present the radiological and autopsy findings of two cases of iniencephaly clausus ( 29.3 and 24 weeks ) and one case of iniencephaly apertus ( 23 weeks ) with brief review of literature .
a 21-year - old primigavida with 29.3 weeks of gestation was admitted in our institute for termination of pregnancy , as her routine anomalous ultrasonogram ( usg ) done at a primary center revealed defective development of spine , atrial septal defect ( asd ) , aplasia of the right kidney , and encephalocele .
she was not a known diabetic or hypertensive and had a history of nonconsanguineous marriage .
a repeat usg done at our institute confirmed the above - mentioned anomalies . in view of multiple nonviable anomalies , with the informed consent of the parents ,
it was a fetus of ambiguous genitalia , weighed 900 g. the head was enlarged , severely retroflexed , and there was no neck .
head was seen directly resting on the chest and mandibular skin in continuous with chest skin surface .
the ears were low set [ figure 1a ] and the vertebral column was short and irregular .
there was an omphalocele with partly covered membrane containing liver , bladder , and intestine as contents .
posteriorly the skin of the scalp was thick and extended up to lumbar region [ figure 1c ] radiograph of the abortus revealed hyperextension of the neck with occiput touching the cervicodorsal vertebra with soft tissue continuation on the dorsal aspect of the neck . cervical and thoracic vertebra showed defective block vertebra with thoracolumbar lordosis suggestive of iniencephaly [ figure 1d ] .
autopsy of the fetus revealed two lobes in the right lung with hypoplasia of both the lungs [ figure 1e ] .
on opening the cranial cavity , there was defect in the development of occipital bone , which was covered by duramater inside and thin membrane outside .
umbilical cord showed single umbilical artery . considering these features a pathological diagnosis of iniencephaly clausus
( a ) gross photograph of iniencephaly showing low - set ears , absence of neck , and club foot .
( b ) gross photograph of lateral view of iniencephaly showing omphalocele ( arrow ) .
( c ) posterior view of iniencephaly showing thick scalp skin extending up to lumbar region .
( d ) x - ray lateral view reveals short vertebral column , fused cervical vertebrae , malformed thoracic and lumbar vertebrae , and overcrowding of the ribs .
( f ) gross photograph of opened left atrium showing atrial septal defect ( asd ) .
( i ) gross photograph after scalp skin reflexion showing defective development of occipital bone ( arrow ) .
( k ) closer view of the sagittal section showing shallow posterior fossa ( black arrow ) , defective occipital bone development , fused cervical vertebrae , and malformed thoracic and lumbar vertebrae with lordosis . ( case 1 ) a 27-year - old female gravida 2 , para 1 living 1 with 23 weeks of gestation presented to obstetrics department with a history of abdominal pain since a day .
usg revealed features of intrauterine growth retardation ( iugr ) with multiple congenital anomalies and malformed spine and skull .
with informed consent , the pregnancy was terminated and abortus was subjected for pathological examination . at autopsy , it was a female fetus and weighed 510 g. on external examination , the head was retroflexed with no neck .
small occipital encephalocele , low - set ears , and lordotic short spinal column [ figure 2a c ] .
computed tomography ( ct ) scan revealed defective development of endochondral portion of occipital bone , rachischisis of cervical spine , and club foot .
head was hyperextended and foramen magnum was enlarged with fusion of occiput to upper cervical vertebrae .
magnetic resonance imaging ( mri ) in addition to ct scan findings revealed an encephalocele from the occipital defect .
autopsy confirmed all the above findings , and final pathological diagnosis of iniencephaly apertus was constructed .
gross photograph of lateral ( a ) , anterior ( b ) , posterior ( c ) view of iniencephaly showing hyperextended head with encephalocele ( * ) ( d ) computed tomography ( ct ) scan showing fusion of occipital bone with cervicothoracic vertebrae , spina bifida , and malformed ribs .
( case 2 ) gross photograph of iniencephaly showing hyperextended head , low - set ears .
( e&f ) ct scan ( g ) and magnetic resonance imaging ( mri ) ( h ) showing defective development of occipital bone with fusion to the cervicothoracic vertebrae .
( case 3 ) a 24-year - old primigravida with 24 weeks of gestation arrived at casuality with complains of pain in the abdomen and bleeding pervaginum since 2 days .
the pregnancy was terminated and abortus was sent for pathological examination . at autopsy , it was a male fetus and weighed 580 g. on external examination , the head was severely retroflexed and no neck .
ears were low set and short lordotic spinal column [ figure 2e and f ] .
enlarged foramen magnum , short irregular vertebral column , and mild club foot were noted [ figure 2 g ] .
lateral ventricles were dilated in the posterior horn ( colpocephaly ) and configurations of the infratentorial structures were distorted [ figure 2h and i ] .
all other visceral organs were normal . with the above features , a pathological diagnosis of iniencephaly clauses was made .
a 21-year - old primigavida with 29.3 weeks of gestation was admitted in our institute for termination of pregnancy , as her routine anomalous ultrasonogram ( usg ) done at a primary center revealed defective development of spine , atrial septal defect ( asd ) , aplasia of the right kidney , and encephalocele .
she was not a known diabetic or hypertensive and had a history of nonconsanguineous marriage .
a repeat usg done at our institute confirmed the above - mentioned anomalies . in view of multiple nonviable anomalies , with the informed consent of the parents ,
it was a fetus of ambiguous genitalia , weighed 900 g. the head was enlarged , severely retroflexed , and there was no neck .
head was seen directly resting on the chest and mandibular skin in continuous with chest skin surface .
the ears were low set [ figure 1a ] and the vertebral column was short and irregular .
there was an omphalocele with partly covered membrane containing liver , bladder , and intestine as contents .
posteriorly the skin of the scalp was thick and extended up to lumbar region [ figure 1c ] radiograph of the abortus revealed hyperextension of the neck with occiput touching the cervicodorsal vertebra with soft tissue continuation on the dorsal aspect of the neck . cervical and thoracic vertebra showed defective block vertebra with thoracolumbar lordosis suggestive of iniencephaly [ figure 1d ] .
autopsy of the fetus revealed two lobes in the right lung with hypoplasia of both the lungs [ figure 1e ] .
on opening the cranial cavity , there was defect in the development of occipital bone , which was covered by duramater inside and thin membrane outside .
umbilical cord showed single umbilical artery . considering these features a pathological diagnosis of iniencephaly clausus
( a ) gross photograph of iniencephaly showing low - set ears , absence of neck , and club foot .
( b ) gross photograph of lateral view of iniencephaly showing omphalocele ( arrow ) .
( c ) posterior view of iniencephaly showing thick scalp skin extending up to lumbar region .
( d ) x - ray lateral view reveals short vertebral column , fused cervical vertebrae , malformed thoracic and lumbar vertebrae , and overcrowding of the ribs .
( f ) gross photograph of opened left atrium showing atrial septal defect ( asd ) .
( i ) gross photograph after scalp skin reflexion showing defective development of occipital bone ( arrow ) .
( k ) closer view of the sagittal section showing shallow posterior fossa ( black arrow ) , defective occipital bone development , fused cervical vertebrae , and malformed thoracic and lumbar vertebrae with lordosis . ( case 1 )
a 27-year - old female gravida 2 , para 1 living 1 with 23 weeks of gestation presented to obstetrics department with a history of abdominal pain since a day .
usg revealed features of intrauterine growth retardation ( iugr ) with multiple congenital anomalies and malformed spine and skull .
with informed consent , the pregnancy was terminated and abortus was subjected for pathological examination . at autopsy , it was a female fetus and weighed 510 g. on external examination , the head was retroflexed with no neck .
small occipital encephalocele , low - set ears , and lordotic short spinal column [ figure 2a c ] .
computed tomography ( ct ) scan revealed defective development of endochondral portion of occipital bone , rachischisis of cervical spine , and club foot .
head was hyperextended and foramen magnum was enlarged with fusion of occiput to upper cervical vertebrae .
magnetic resonance imaging ( mri ) in addition to ct scan findings revealed an encephalocele from the occipital defect .
autopsy confirmed all the above findings , and final pathological diagnosis of iniencephaly apertus was constructed .
gross photograph of lateral ( a ) , anterior ( b ) , posterior ( c ) view of iniencephaly showing hyperextended head with encephalocele ( * ) ( d ) computed tomography ( ct ) scan showing fusion of occipital bone with cervicothoracic vertebrae , spina bifida , and malformed ribs .
( case 2 ) gross photograph of iniencephaly showing hyperextended head , low - set ears .
( e&f ) ct scan ( g ) and magnetic resonance imaging ( mri ) ( h ) showing defective development of occipital bone with fusion to the cervicothoracic vertebrae .
a 24-year - old primigravida with 24 weeks of gestation arrived at casuality with complains of pain in the abdomen and bleeding pervaginum since 2 days .
usg revealed anomalous fetus with absent cardiac activity . with the informed consent , the pregnancy
was terminated and abortus was sent for pathological examination . at autopsy , it was a male fetus and weighed 580 g. on external examination , the head was severely retroflexed and no neck .
ears were low set and short lordotic spinal column [ figure 2e and f ] .
enlarged foramen magnum , short irregular vertebral column , and mild club foot were noted [ figure 2 g ] .
lateral ventricles were dilated in the posterior horn ( colpocephaly ) and configurations of the infratentorial structures were distorted [ figure 2h and i ] .
all other visceral organs were normal . with the above features , a pathological diagnosis of iniencephaly clauses was made .
the word inion is derived from greek meaning occiput . the first description of iniencephaly is attributed to saint - hilare in 1836 . incidence of this rare anomaly is 0.1 - 10 in 10,000 pregnancies .
in this condition , posterior most part of the head ( occiput ) is fused with the back leading to severe retroflexion of the head . till date
environmental factors such as poor socioeconomic conditions , lack of folic acid supplementation , obesity , drugs ( sulfonamides , tetracycline , antihistaminic ) , genetic factors such as monosomy x , trisomy 13 , trisomy 18 , and chromosomal abnormalities are implicated in the etiology of this condition .
balci et al . in their study observed mutation in methyl tetrahydrofolate reductase gene ( 677ct ) in parents of iniencephaly indicating possible etiology .
the defects in iniencephaly occur in the early pregnancy prior to the closure of cephalic neural fold at 24 days of gestational age .
the paravertebral mesoderm differentiates into two parts wherein ventral mass forms the vertebral bodies , pedicles , and cranial homologues .
dorsal mass forms the neural arches and bones of the cranial vault . in iniencephaly ,
initially it was believed that neural tube closure begins in the cervical region and extends rosterally and caudally , but now evidences suggest that neural tube closure is a multisite initiation process and five closure sites are proposed ( site 1 : mid cervical , 2 : between prosencephalon and mesencephalon , site 3 : stomodeum , site 4 : caudal end of rhombencephalon , and site 5 : caudal end of neural tube ) .
the observed central nervous system anomalies include anencephaly , encephalocele , cyclopia , hydrocephalus , and holoprosencephaly .
other systemic anomalies reported in the literature are cardiac septal defects , cleft lip and palate , pulmonary hypoplasia , right lung with two lobes , omphalocele , congenital diaphragmatic hernia ( cdh ) , intestinal atersia , imperforate anus , horseshoe kidney , bronchogenic cysts , genital malformations , and skeletal abnormalities such as club foot and overgrowth of upper limbs compared with lower limbs . in the present case series , pulmonary hypoplasia , omphalocele , disproportionate limbs , accessory spleen , asd , spina bifida , colpocephaly , and club foot were noted .
accessory spleen association with iniencephaly observed in the present case is previously not reported in the literature .
pulmonary hypoplasia , cdh , and omphalocele occur due to increased pressure in the thoracic and abdominal cavity due to exaggerated spinal retroflexion .
halder et al . , in their large series on iniencephaly of 19 cases , observed 3 cases of cardiac septal defects , 5 cases of pulmonary hypoplasia , 1 case of horseshoe kidney , 2 cases each of ompalocele , gastrointestinal malformation and dandy walker malformations , 3 cases of adrenal hypoplasia , and 1 case of thymic hypoplasia
. congenital retroflexion of spine is seen in conditions such as iniencephaly , anencephaly , klippel - feil syndrome ( kfs ) and nuchal tumors .
there will be partial or complete absence of cranial vault with retroflexed head , which is not covered by skin , and cervical vertebrae are normal .
in contrast , in iniencephaly , the head retroflexed and covered by skin and cervical vertebrae is abnormal .
kfs is a surgically treatable condition , which is due to the failure of segmentation of cervical vertebrae leading to fusion , but retroflexion of head is not seen , which is a feature of iniencephaly .
a detailed examination of the spine and cranial bones is possible with ct and mri , but fetal autopsy is gold standard to document the associated anomalies .
mri is safe in pregnancy as there is no radiation exposure and gives better detailed visualization of organs .
till date there are only six fetuses recorded in the literature with long - term survival .
gartman et al . suggested that the fibrosed trapezius muscle is the cause for the retroflexion but edincler et al .
iniencephaly is a rare fatal ntd , which occurs due to the defect in the neural tube closure at sites 1 and 4 . because the condition is incompatible with life ,
careful and early usg diagnosis is required so that termination can be offered for willing parents . | iniencephaly is a rare form of neural tube defect with an incidence of 0.1 - 10 in 10,000 pregnancies .
it is characterized by the presence of occipital bone defects at foramen magnum , fixed retroflexion of head , spinal dysmorphism , and lordosis of cervicothoracic vertebrae .
it is usually associated with central nervous system , gastrointestinal , and cardiovascular anomalies .
we present radiological and autopsy findings in a series of 3 cases of iniencephaly ( gestational ages 29.3 , 23 , and 24 weeks ) first fetus in addition showed omphalocele , pulmonary hypoplasia , two lobes in right lung , accessory spleen , atrial septal defect , bilateral clubfoot , ambiguous genitalia , and single umbilical artery .
second fetus was a classical case of iniencephaly apertus with spina bifida .
third fetus had colpocephaly and bifid spine . |
this case describes the development of acute kidney injury following oral ingestion of 3,4-methylenedioxymethamphetamine ( ecstasy ) with histological demonstration of venous thrombosis .
a 22-year - old man presented with acute bilateral loin pain and was referred by his general practitioner for a urological consultation with suspected renal stones .
he was on no regular medication , and at the beginning of his admission , he denied taking any recreational drugs . however , later on , he admitted taking ecstasy 1 day prior to his admission .
when examined , he was apyrexial but hypertensive with a blood pressure of 145/95 mmhg .
initial biochemical investigations showed evidence of acute kidney injury with a creatinine of 189 mol / l .
other biochemistry results were na 138 mmol / l , k 4.2 mmol / l and urea 7.7 mmol / l .
anti - neutrophil cytoplasmic antibodies ( anca ) and anti - nuclear antibodies ( ana ) were negative ; complement and immunoglobulins were normal .
his urine dipstick was positive for both blood and protein . the protein creatinine ratio was 92 mg / mmol .
renal ultrasound showed normal - sized kidneys and no renal stones . given the unexplained acute kidney injury with significant proteinuria and haematuria
, he underwent ultrasound - guided renal biopsy . over the next 3 days of admission
, his renal function continued to improve , and his proteinuria started to disappear ( table 1 ) .
progression of renal function with proteinuria during admission renal biopsy histology : a small vein at the corticomedullary junction was occluded by thrombus with evidence of early organization ( figure 1 ) .
a small vein at the corticomedullary junction was occluded by thrombus with evidence of early organization .
a renal doppler ultrasound did not show a thrombus in the main renal vein bilaterally .
he was discharged with the diagnosis of acute kidney injury due to renal venous thrombosis induced by ecstasy .
its use has been associated with different organ failure such as acute kidney injury and fulminant hepatic failure .
firstly , vasoconstriction caused by mdma is described in animal models with effects such as increase in mean blood pressure , cutaneous vasoconstriction and coronary artery vasoconstriction . moreover , necrotizing vasculopathy was described in a case report of a patient who used mdma 10 days prior to presentation with acute kidney injury resulting in chronic renal failure .
necrotizing vasculopathy was also reported in two transplanted renal grafts taken from the same donor who regularly used ecstasy ; both grafts failed within the first week due to necrotizing graft vasculopathy .
finally , there have been case reports of extra - renal venous thrombosis associated with the use of ecstasy such as a case of cerebral venous thrombosis in a 22-year - old woman who ingested one tablet of ecstasy prior to presentation with headache and photophobia .
although mdma can cause disseminated intravascular coagulation , venous thrombosis could also be a result of hyperthermia and dehydration induced by mdma .
we describe a case of acute kidney injury most likely caused by renal venous thrombosis induced by ecstasy ingestion . the venous thrombosis involved small intra - renal veins , evident on renal biopsy , but did not include the main renal veins .
this is supported by the degree of the acute kidney injury and the resolution of both the injury and the proteinuria . | the use of 3,4-methylenedioxymethamphetamine , also known as mdma or ecstasy , has been associated with vascular and end - organ damage .
this case report describes , with histological evidence , the development renal venous thrombosis presenting with acute kidney injury following oral ingestion of 3,4-methylenedioxymethamphetamine ( ecstasy ) . |
currently , basalbolus insulin therapy is used to provide the insulin secretory patterns most physiologically similar to normal .
basalbolus therapy includes multiple daily injection ( mdi ) and continuous subcutaneous insulin infusion ( csii ) therapies .
the recent advent of rapidacting insulin analogs and longacting insulin analogs , and the development of csii therapy using rapidacting insulin have clarified the role of basal and bolus insulin .
few studies , however , have focused on factors associated with basal insulin doses . although basal insulin doses for japanese patients with type 1 diabetes mellitus were reported to be lower than those of type 1 diabetes mellitus patients in europe and the us , the reasons for this have not been clarified .
japanese meals are known to have a higher carbohydrate energy ratio of meal ( cer ) and a lower fat energy ratio of meal ( fer ) than western meals . according to the statistics of the united nations , based on the amounts supplied ,
the fer in japan is 28% while those in western countries exceed 35% or even 40% .
we speculated that differences in meal contents might be responsible for the differing basal insulin doses . to investigate determinants of basal insulin doses for basalbolus therapy in japanese patients with type 1 diabetes mellitus
, we examined associations of potential factors , including age , sdsbmi ( standard deviation score of body mass index ) , hba1c levels and meal contents based on meal records , with basal insulin doses .
fortyone outpatients with type 1 diabetes mellitus between the ages of 3 and 26 ( 20 males and 21 females , means age 15.9 ) were enrolled .
patients with a duration of diabetes of < 2 years , hba1c levels of 9.0% or more , diabetic complications , or who were pregnant and/or had other diseases were excluded .
the following variables were examined : gender , age , height , weight , sdsbmi , hba1c level , duration of diabetes , insulin administration method ( mdi or csii ) , daily bolus insulin doses ( bod ) , daily basal insulin doses ( bad ) , total daily insulin doses ( tdd ) , bad / tdd ( bt ratio ) , the value obtained by dividing bad by bw ( bad / bw ) and meal contents . in order to calculate the sdsbmi for the 12 patients aged 19 or over ( for which there is no recorded data in japan ) , each age used the data of 18.5 years old . the value for hba1c ( % )
is estimated as a national glycohemoglobin standardization program ( ngsp ) equivalent value ( % ) calculated by the formula hba1c ( % ) = hba1c ( jds : japan diabetes society ) ( % ) + 0.4% , considering the relational expression of hba1c ( jds ) ( % ) measured by the previous japanese standard substance and measurement methods and hba1c ( ngsp ) .
insulin doses for mdi therapy were obtained from the patients themselves or from medical records .
insulin doses for csii therapy were obtained by reading the records on the pump used via the comstation ( mmt7301u ) ( medtronic diabetes , northridge , ca , usa ) .
the doses of daily bolus insulin and daily basal insulin were checked every day for 3 days when the patients took pictures of their meals as described in detail below and the average values of three data were used for analysis .
all subjects were treated with either mdi or csii employing basalbolus therapy . as to rapidacting insulin analogs , either lispro or aspart was used .
patients given csii were received a bolus of normal type for bolus insulin dosing , during the study period .
patients were instructed to adjust the basal insulin dose to keep blood glucose levels stable while they were not eating or not receiving the additional insulin .
instructions of carbohydrate counting were provided at outpatient visits by a physician or by a dietitian working in our hospital .
the photographs were taken with a digital camera or a mobile phone equipped with a camera .
the meals included all breakfasts , lunches , dinners , and snacks , as well as supplemental food consumed at the time of hypoglycemia attacks . when the photographs were taken , something ( e.g. , a coin ) was placed beside the food to provide a size marker for length assessment .
the dietitians estimated all ingredients and their weights from the photographs and calculated the ingested amounts ( in grams ) of protein , fat , and carbohydrate .
they entered data for each ingredient using the computer software program excel eiyokun , version 4.0 ( kenpaku co. ltd . ,
excel eiyokun is a program designed to calculate amounts of protein , fat , and carbohydrate by entering the name and weight of a meal ingredient .
the mean ingested amount of each nutrient ( daily protein , fat , and carbohydrate intakes ) as determined by the three dietitians was used for the analysis .
the protein energy ratio of meal ( per ) was calculated by the following equation : daily protein intake ( g ) 4/daily total energy ( kcal ) .
the fat energy ratio of meal ( fer ) was calculated by the following equation : daily fat intake ( g ) 9/daily total energy ( kcal ) .
the carbohydrate energy ratio of meal ( cer ) was calculated by the following equation : daily carbohydrate intake ( g ) 4/daily total energy ( kcal ) .
the ethics committee of osaka city university institutional review board , osaka , japan , approved this protocol .
intergroup comparisons were performed for age , sdsbmi , hba1c levels , duration of diabetes , bt ratio , bad / bw and meal contents , according to gender and therapy ( mdi or csii ) , using the ttest or the mann
subsequently , to evaluate simple correlations with basal insulin doses , associations of the bt ratio or bad / bw with age , sdsbmi , hba1c levels , duration of diabetes and each meal content variable were examined using pearson s productmoment correlations .
next , multiple regression analysis was performed for all subjects and for each age group to evaluate factors influencing bad without interactions among the variables examined . the dependent variable selected was bad / bw .
the independent variables selected were : gender ( female = 0 , male = 1 ) , age , sdsbmi , hba1c levels , therapy ( mdi = 0 , csii = 1 ) , and fat energy ratio . for the analysis of age effect , patients were divided into four age groups .
patients aged 9 or younger were age group 1 , patients between the age of 10 and 13 were age group 2 , patients between the age of 14 and 18 were age group 3 , and patients aged 19 or above were age group 4 .
then patients were divided by sex ( male and female group ) and hba1c levels ( good < 7% and poorly controlled >
7% group ) . differences with p < 0.05 were considered to be statistically significant . for multiple regression analysis , the stepwise method was used with the following criteria : probability of entering f : 0.050 and probability of removing f : 0.100 .
fortyone outpatients with type 1 diabetes mellitus between the ages of 3 and 26 ( 20 males and 21 females , means age 15.9 ) were enrolled .
patients with a duration of diabetes of < 2 years , hba1c levels of 9.0% or more , diabetic complications , or who were pregnant and/or had other diseases were excluded .
the following variables were examined : gender , age , height , weight , sdsbmi , hba1c level , duration of diabetes , insulin administration method ( mdi or csii ) , daily bolus insulin doses ( bod ) , daily basal insulin doses ( bad ) , total daily insulin doses ( tdd ) , bad / tdd ( bt ratio ) , the value obtained by dividing bad by bw ( bad / bw ) and meal contents . in order to calculate the sdsbmi for the 12 patients aged 19 or over ( for which there is no recorded data in japan ) , each age used the data of 18.5 years old . the value for hba1c ( % )
is estimated as a national glycohemoglobin standardization program ( ngsp ) equivalent value ( % ) calculated by the formula hba1c ( % ) = hba1c ( jds : japan diabetes society ) ( % ) + 0.4% , considering the relational expression of hba1c ( jds ) ( % ) measured by the previous japanese standard substance and measurement methods and hba1c ( ngsp ) .
insulin doses for mdi therapy were obtained from the patients themselves or from medical records .
insulin doses for csii therapy were obtained by reading the records on the pump used via the comstation ( mmt7301u ) ( medtronic diabetes , northridge , ca , usa ) .
the doses of daily bolus insulin and daily basal insulin were checked every day for 3 days when the patients took pictures of their meals as described in detail below and the average values of three data were used for analysis .
all subjects were treated with either mdi or csii employing basalbolus therapy . as to rapidacting insulin analogs , either lispro or aspart was used .
patients given csii were received a bolus of normal type for bolus insulin dosing , during the study period .
patients were instructed to adjust the basal insulin dose to keep blood glucose levels stable while they were not eating or not receiving the additional insulin .
instructions of carbohydrate counting were provided at outpatient visits by a physician or by a dietitian working in our hospital .
the photographs were taken with a digital camera or a mobile phone equipped with a camera .
the meals included all breakfasts , lunches , dinners , and snacks , as well as supplemental food consumed at the time of hypoglycemia attacks . when the photographs were taken , something ( e.g. , a coin ) was placed beside the food to provide a size marker for length assessment .
the dietitians estimated all ingredients and their weights from the photographs and calculated the ingested amounts ( in grams ) of protein , fat , and carbohydrate .
they entered data for each ingredient using the computer software program excel eiyokun , version 4.0 ( kenpaku co. ltd . ,
excel eiyokun is a program designed to calculate amounts of protein , fat , and carbohydrate by entering the name and weight of a meal ingredient .
the mean ingested amount of each nutrient ( daily protein , fat , and carbohydrate intakes ) as determined by the three dietitians was used for the analysis .
the protein energy ratio of meal ( per ) was calculated by the following equation : daily protein intake ( g ) 4/daily total energy ( kcal ) .
the fat energy ratio of meal ( fer ) was calculated by the following equation : daily fat intake ( g ) 9/daily total energy ( kcal ) .
the carbohydrate energy ratio of meal ( cer ) was calculated by the following equation : daily carbohydrate intake ( g ) 4/daily total energy ( kcal ) .
the ethics committee of osaka city university institutional review board , osaka , japan , approved this protocol .
intergroup comparisons were performed for age , sdsbmi , hba1c levels , duration of diabetes , bt ratio , bad / bw and meal contents , according to gender and therapy ( mdi or csii ) , using the ttest or the mann
subsequently , to evaluate simple correlations with basal insulin doses , associations of the bt ratio or bad / bw with age , sdsbmi , hba1c levels , duration of diabetes and each meal content variable were examined using pearson s productmoment correlations .
next , multiple regression analysis was performed for all subjects and for each age group to evaluate factors influencing bad without interactions among the variables examined . the dependent variable selected was bad / bw .
the independent variables selected were : gender ( female = 0 , male = 1 ) , age , sdsbmi , hba1c levels , therapy ( mdi = 0 , csii = 1 ) , and fat energy ratio . for the analysis of age effect , patients were divided into four age groups .
patients aged 9 or younger were age group 1 , patients between the age of 10 and 13 were age group 2 , patients between the age of 14 and 18 were age group 3 , and patients aged 19 or above were age group 4
. then patients were divided by sex ( male and female group ) and hba1c levels ( good < 7% and poorly controlled >
7% group ) . differences with p < 0.05 were considered to be statistically significant . for multiple regression analysis , the stepwise method was used with the following criteria : probability of entering f : 0.050 and probability of removing f : 0.100 .
the averages of key characteristics were as follows : age , 15.9 5.4 years ; sdsbmi , 0.86 0.83 ; duration of diabetes , 9.8 5.7 years ; hba1c levels 7.2 0.7% ; bt ratio , 0.35 0.09 ( mdi : 0.33 , csii : 0.37 ) ; and bad / bw , 0.35 0.12 u / kg ( mdi : 0.36 csii : 0.34 u / kg ) .
the average of differences for 3 days in fer was 6 3% .
tdd , total daily insulin doses ; bt ratio , daily basal insulin doses / total daily insulin doses ; bad / bw , daily basal insulin doses / body weight ; tdd / bw , total daily insulin doses / body weight ; per , protein energy ratio of meal ; fer , fat energy ratio of meal ; cer , carbohydrate energy ratio of meal ; energy , total energy intake .
based on the assessments made by the three experienced dietitians , the mean per was 0.16 , the mean fer was 0.30 , and the mean cer was 0.52 in 41 patients .
the total daily grams of protein , fat , and carbohydrate were 107 , 76 , and 359 g , respectively .
protein energy , fat energy , and carbohydrate energy ratios were 0.16 , 0.26 , and 0.55 , respectively . in intergroup comparisons ,
regardless of gender or insulin administration method ( mdi or csii ) , there were no intergroup statistical differences in sdsbmi , bad / bw , bt ratio and fer . however , age and duration of diabetes of the mdi group were significantly higher than that of the csii group , and hba1c levels of the csii group were significantly higher than that of the mdi group .
simple correlations of the bt ratio or bad / bw with age , sdsbmi , hba1c level and each meal content variable are given in table 2 .
in all subjects , there was a positive correlation between age and tdd ( r = 0.452 , p = 0.003 ) or sdsbmi and tdd ( r = 0.320 , p = 0.041 ) .
additionally , there was a positive correlation between bad / bw and fer ( r = 0.493 , p = 0.001 ) and a negative correlation between bad / bw and cer ( r = 0.541 , p < 0.001 ) .
the bod / bw tended to correlate negatively with hba1c level ( r = 0.281 , p = 0.075 ) . among patients under 19 years old ( age groups 1 , 2 and 3 ) ,
there was a positive correlation between bad / bw and sdsbmi ( r = 0.472 , p = 0.010 ) or bad / bw and fer ( r = 0.452 , p = 0.014 ) .
correlations between the dose of insulin and age , sdsbmi , hba1c levels , duration of diabetes or each meal content variable using pearson s productmoment correlations .
tdd , total daily insulin doses ; bt ratio , daily basal insulin doses / total daily insulin doses ; bod / bw , daily bolus insulin doses / body weight ; bad / bw , daily basal insulin doses / body weight ; tdd / bw , total daily insulin doses / bw ; per , protein energy ratio of meal ; fer , fat energy ratio of meal ; cer , carbohydrate energy ratio of meal .
multiple regression analyses are shown in table 3 . in all subjects , when bad / bw was selected as the dependent variable , fer was obtained as an entered variable ( p = 0.001 ) .
in age group 1 or age group 2 , no value was obtained as an entered variable . in age group 3 ,
fer was obtained as an entered variable ( p < 0.001 , standardized coefficient = 0.844 , adjusted r = 0.686 ) . in age group 4 , fer was also obtained as an entered variable ( p = 0.048 , standardized coefficient = 0.580 , adjusted r = 0.270 ) . among patients aged 14 or above ( age groups 3 and 4 ) ,
fer was obtained similarly ( p < 0.001 , standardized coefficient = 0.683 , adjusted r = 0.443 ) .
the correlation between fer and bad / bw in age group 3 and 4 ( n = 25 ) is shown in figure 2 . in the male group or female group , no value was obtained as an entered variable . in the well
controlled group , fer was obtained as an entered variable ( p = 0.003 , standardized coefficient = 0.631 , adjusted r = 0.365 ) while none was obtained in the poorly controlled group .
independent variables were : gender , age , sdsbmi , hba1c levels , therapy ( mdi or csii ) , and fat energy ratio . in all subjects , age group 3 , age group 4 , age group 3 and 4 or well controlled group , when bad / bw was selected as the dependent variable ,
bad / bw , daily basal insulin doses / body weight ; fer , fat energy ratio of meal ; well controlled group , hba1c < 7.0 ; poorly controlled group , hba1c > 7.0 .
the correlation between fer and bad / bw . among patients aged 14 or above ( age group 3 and 4 ) ,
fer : fat energy ratio of meal , bad / bw : daily basal insulin doses / body weight .
in this study , estimation of meal ingredients from photographs taken by patients was chosen as the method for examining meal contents .
williamson et al . reported that estimates of portion sizes related to food selections , plate waste , and food intake correlated highly with actual food weights by both the digital photography and the direct visual estimation method .
the correlation coefficient between the estimated amounts of whole meals using digital photography and the actual amounts of meals ranged from 0.89 to 0.94 , indicating a high correlation .
reported that a person with considerable knowledge of nutrition , such as a senior nutritionist at a university , could assess nutrient intakes using digital images and that the results of these assessments were accurate . in the present study , three experienced registered dietitians assessed digital images , and their evaluation results were combined to obtain mean values .
therefore , given the previous reports , the method used in the present study was considered to be appropriate and to have yielded valid results .
furthermore , we asked the subjects to take pictures of food during normal daily life .
the nutritional data obtained from the 3 days of food photographs was not so variable day by day . the data of insulin dosage used in this study was also obtained from the same 3 days as the food pictures .
therefore , the relationships between the nutritional data and basal insulin dose seems to be reliable .
reported that in the ppsg ( the pediatric pump study group ) study conducted in 23 facilities in 9 european countries and israel , the mean bt ratio was 0.48 in 377 patients , with a mean age of 12.9 years , receiving csii therapy and that there were differences among facilities .
reported that the mean bt ratio was 0.67 in children with type 1 diabetes mellitus receiving csii therapy in the usa .
studies in europe and the usa have often found the bt ratio to be at least 0.50 .
studies in japan , on the other hand , have demonstrated bt ratios tending to be lower than those in europe and the usa .
the bt ratios were usually below 0.50 in patients receiving basalbolus therapy ( csii or mdi ) , regardless of age , at many japanese facilities . particularly , kuroda reported < 0.30 of bt ratio in japanese type 1 diabetes using csii therapy as inpatients with 5060% cer meals .
the bt ratios in europe and the usa , however , are not always high .
reported the mean bt ratio to be 0.28 in children with type 1 diabetes mellitus receiving csii therapy in warsaw .
therefore , bt ratios are not consistently high in european patients , such that factors other than race and ethnicity are thus considered to be involved .
reported bt ratios to correlate positively with age and diabetes duration and to correlate negatively with blood concentrations of cpeptide in type 1 diabetes mellitus patients under 19 years of age in poland .
in the present study , however , the bt ratio showed no association with age or disease duration .
blood concentrations of cpeptide were not examined in this study , and this relationship thus remains unclear .
disease duration and endogenous insulin were thought not to influence bt ratios significantly because patients with disease durations of < 2 years were excluded from this study .
many studies worldwide have reported bt ratios , but few have examined associations between bad / bw and other factors .
the results of our studies show bad / bw is likely to be associated with sdsbmi in type 1 diabetes mellitus patients of < 19 years of age .
this suggests that obesity increases insulin resistance in pediatric type 1 diabetes mellitus patients , as has been observed in other general populations . on the other hand ,
reported that in 1486 japanese patients with type 1 diabetes mellitus between 16 and 90 years of age , those with higher bmi had higher tdd / bw .
although it must be recognized that bad / bw is different from tdd / bw , their results were similar to ours .
as to insulin resistance and obesity , reinehr et al . reported that tdd / body surface area and tdd / ideal body weight correlated more with sdsbmi than tdd / bw in 4124 german patients with type 1 diabetes mellitus .
they proposed that glucose uptake occurring mainly in the lean body mass was one of the explanations for this finding , and sdsbmi was associated with a decrease in tdd / bw and an increase in tdd / body surface area and tdd / ideal body weight .
the utility of carbohydrate counting for dose adjustment of bolus insulin was recently confirmed , and our department also uses this method for patient education .
carbohydrate counting , which allows determination of the doses of bolus insulin based on the amounts of carbohydrate contained in meals , provides very good glycemic control .
however , the optimal insulin dosing method with carbohydrate counting when a large amount of protein or fat is ingested has not yet been clearly defined , and no method of achieving this goal has been established . in fact , various studies have focused on identifying an insulin dosing method which allows adjustments for protein and fat intake . according to the results of the national health and nutrition survey conducted in japan in 2008 , the mean fer for the entire japanese population was 0.25 and the mean fer by age group ( 16 , 714 , and 1519 years of age ) were 0.280.29 .
the mean per of all three age groups was 0.14 and the mean cer was 0.550.57 .
therefore , the present study subjects had slightly higher per and fer and lower cer readings than the mean values for the japanese population as a whole
. generally , when a high protein , high fat meal is consumed , blood glucose levels are elevated for at least 5 h afterwards . since the blood glucose elevation due to intake of large amounts of protein and/or fat
can be managed by increasing bad , patients , who routinely consume large amounts of protein and fat , are expected to require higher bad .
our study revealed that the bad / bw tended to be higher in those with higher fer especially aged 14 or above .
this may be considered to explain the aforementioned expectation of increased fat intakes making higher basal insulin doses . to our knowledge , so far , no other studies have shown associations between meal contents and bad .
however , this tendency was not seen in the younger age groups . when subjects were divided into four age groups to consider the effect of age , the result of multiple regression analyses was different .
while in age groups 1 and 2 ( the age of 13 or younger ) no variable was associated with bad / bw , in age groups 3 and 4 ( the age of 14 or above ) the fer variable was associated with bad / bw .
first , because the dose of basal insulin increased as patients grew up , the difference in the older group might easily become significant .
all of their parents have received nutritional instruction regarding calories and balanced nutrition at the onset of their child s type 1 diabetes mellitus . according to the guideline of japanese diabetes association , a cer of 5060% , an fer of approximately 30% , and per of approximately 20% were recommended .
therefore , it was thought that in patients consuming balanced meals under the management of their parents , variability in cpf ratio ( carbohydrate : protein : fat energy ratio ) would be minimal .
while among patients aged 14 or over , an association between bad / bw and fer was shown in the multiple regression analysis . this might be because when the patients grow up and assume responsibility for their own meals , they made their bad requirements adequately . in our study of the multiple regression analysis , dividing the subjects into well and poorly controlled groups , an association between bad / bw and fer was shown in only the well controlled group .
this may be because the better controlled patients or their families administer suitable doses of insulin .
as described above , the contents of meals , especially carbohydrate amounts , which greatly influence blood glucose levels , and cpf ratio may be an important factor related to bt ratios of insulin doses in mdi .
meal contents vary between countries worldwide , and the possibility that the differences in reported bt ratios among facilities are attributable to meal contents can not be ruled out .
thus , we calculated cpf ratio based on the report entitled daily food supply per person in a country published by the fao ( food and agriculture organization of the united nations ) in 2005 , in order to obtain summaries of meal contents .
the supplied fat energy / supplied total energy ratios in asian countries were 0.28 ( japan ) , 0.27 ( china ) , and 0.25 ( korea ) .
on the other hand , they were 0.30 in poland , 0.36 in germany , 0.41 in france , and 0.41 in the usa .
this indicates that fer based on amounts supplied in asian countries , including japan , are lower than those in european countries and the usa .
the supplied fat energy / supplied total energy ratio in poland was 0.30 , but , relatively similar to that in japan . as reported by pankowska
, people in poland have a lower bt ratio than those in other european countries .
however , since these results were calculated based on amounts of food supplied , surveying the meals consumed by each patient , as in our present study , may be necessary to obtain results allowing comparisons among countries .
in conclusion , this study demonstrated an association between bad / bw and fer , in particular among the patients aged 14 or above .
this may explain the expectation of increased fat intake causing higher basal insulin doses in type 1 diabetes mellitus patients . | abstractaims / introduction : the daily basal insulin doses / body weight and the daily basal insulin doses / total daily insulin doses of japanese type 1 diabetes mellitus patients are less than those of western type 1 diabetes mellitus patients .
it is known that western meals are richer in fat than japanese meals .
we speculated that fat intake might be associated with basal insulin dose in type 1 diabetes mellitus patients.materials and methods : fortyone outpatients with type 1 diabetes mellitus ( 20 males , 21 females , mean age 15.9 ) were enrolled .
variables investigated included : gender , sdsbmi , hba1c , duration of diabetes , therapy ( mdi or csii ) , insulin doses and meal contents .
meal contents were recorded for 3 days using a digital camera .
correlation and multiple regression analyses were performed for all subjects and each age group.results : the mean daily basal insulin doses / total daily insulin doses was 0.35 . in the multiple regression analysis among all subjects , when daily basal insulin doses / body weight was used as a dependent variable ,
fat energy ratio of the meal was obtained as an entered variable ( p = 0.001 ) .
this tendency was particularly strong among the patients aged 14 or above ( p < 0.001 , standardized coefficient = 0.683).conclusions : in the type 1 diabetes patients who are aged 14 or above , an association between daily basal insulin doses / body weight and fat energy ratio of meal was suggested .
this may explain the aforementioned expectation of increased fat intakes making higher basal insulin doses .
( j diabetes invest , doi : 10.1111/j.20401124.2011.00171.x , 2011 ) |
left ventricular free wall rupture ( lvfwr ) is reported to occur in 2 - 6% of acute myocardial infarction ( mi ) cases .
lvfwr is presumed responsible for as much as 20 - 80% of infarct - related deaths.1 ) there is a history of previous mi in 25% of cases , lvfwr can often be the first presentation of ischemic heart disease.2 ) we describe the case of a 60-year - old man without history of ischemic heart disease who was admitted with chest pain ; lvfwr and bacterial pericarditis were detected by urgent echocardiography and managed successfully .
a 60-year - old man was a smoker and drinker with no history of coronary artery disease , hypertension and hyperlipidemia except diabetes , which was diagnosed several years ago .
the man presented in the emergency room with complaints of chest pain for 2 days . the patient was conscious and physical examination showed heart rate 128 beats / min , blood pressure 95/70 mm hg , temperature 36.5. heart sound was faint without murmur and bruits .
initial electrocardiogram ( ecg ) showed atrial fibrillation and pathological q waves in v 1 - 6 .
blood tests were negative for troponin i and creatine kinase - mb ( ck - mb ) .
inflammatory markers were raised ( white cell count of 23.12109/l , neutrophil percentage 88.4% , c reactive protein 16.84 mg / dl ) , and hemoglobin was mildly reduced ( 11.7
kidney and liver enzymes were severely elevated ( aspartate aminotransferase 2325 iu / l , alanine aminotransferase 1005 iu / l , -guanosine triphosphate 130 iu / l , lactate dehydrogenase 2989
iu / l , blood urea nitrogen 124.2 mg / dl , creatinine 7.29 mg / dl ) .
echocardiography showed reduced left ventricular ( lv ) systolic function and lv dilatation , especially at the lv apex .
there was a small abnormal shunt from the lv apex to the pericardial space ( fig .
1 ) . thus , lvfwr was strongly suspected . in order to establish a diagnosis of lvfwr ,
chest computerized tomography ( ct ) was conducted and showed lv pseudoaneurysm with rupture in the anteroinferior wall with associated hemopericardium ( fig .
the postoperative pathology revealed focal coagulation necrosis and abscess presented with blood clots and fibrin clots .
postoperatively , intravenous antibiotic treatment was continued and the patient was discharged after 2 weeks with a good recovery .
three months later , coronary ct showed total occlusion of the mid left anterior descending coronary artery .
left ventricular free wall rupture is a dramatic complication of mi in which there is a rupture of infarcted lv free wall tissue .
the rupture is commonly insidious with bleeding into the pericardial sac and subsequent cardiac tamponade.3 ) it is third to cardiogenic shock and arrhythmias as the leading cause of death following an mi.4 ) the most common location of lvfwr is in the anterior or lateral walls.5 ) it often appears within the first week following an acute mi , usually on the 4th or 5th day post - mi , although the time to manifestation may range from a few minutes to more than one month after the acute mi.6 - 9 ) clinically , ruptures can be divided into 3 types : acute rupture , which results in death within a few minutes due to massive hemorrhage into the pericardial cavity ; subacute rupture , which is characterized by a smaller tear that may temporarily be sealed by a clot or fibrinous pericardial adhesion and may be compatible with life for several hours or even longer ; and chronic rupture with false aneurysm formation , which occurs when the leakage of blood is slow and when surrounding pressure on the epicardium temporarily controls the hemorrhage.10 ) the recent significant increase in the number of lvfwr diagnosed before death and the number of surgical repairs attempted is largely due to the widespread availability and use of cardiac imaging , particularly echocardiography.11 ) echocardiography provides invaluable diagnostic information and indicates the possible extent and location of a rupture prior to surgery .
the most frequent echocardiographic finding in the case of lvfwr is a localized pericardial effusion overlying the infarcted akinetic area .
other signs include echogenic ' specks ' within the effusion and visible wall defects.12 ) in the current case , we found that pericardial effusion by echocardiography established the diagnosis of lvfwr by chest ct . on the basis of troponin
i , ck - mb and ecg , we thought that the anterior mi had occurred previously and led to lvfwr .
at first , we had suspected coronary atherosclerotic heart disease was the reason of mi .
in addition , we also could not rule out bacterial endocarditis , other sources of infection and embolization .
atrial fibrillation could cause embolization ( such as septic embolism ) into coronary artery , then mi due to just embolism or abscess ( caused by septic embolism ) could occur without atherosclerotic coronary artery disease .
for this reason , ct angiography of the patient showed no evidence of coronary artery disease in other vessels except in the left anterior descending coronary artery occlusion . a pathologic report could not differentiate whether mi was atherosclerotic , or embolic , or secondary change according to abscess .
physicians need to be aware of the risk factors , clinical signs , ecg and so on . this diagnosis can be made more accurately by using echocardiography and chest ct .
most of all , survival depends on early assessment of the patient , prompt investigation and diagnosis , and urgent surgical treatment . | left ventricular free wall rupture ( lvfwr ) is a serious complication of myocardial infarction .
it presents with a very high mortality rate and can be rescued by accurate diagnosis and emergency surgery .
lvfwr can occur with sudden overt clinical symptoms or present insidiously .
this report highlights the case of a man with no prior history of coronary artery disease , who presented with lvfwr and pericardial effusion that evolved to severe bacterial pericarditis . |
ocular involvement is common in autoimmune disorders ; conversely , patients with autoimmune polyglandular syndrome ( aps ) frequently show vitiligo , alopecia , pernicious anemia , chronic hepatitis and uveitis1 , 2 . transgenic mice deficient for the autoimmune regulator gene develop type 1 aps and uveitis3 .
therefore , searching for other autoimmunity diseases is mandatory in the clinical suspect of aps .
the combination of type 1 diabetes , vitiligo , thyroid disorders and adrenal insufficiency suggests the presence of type 2 aps4 ; other combinations of organspecific autoimmune disease are compatible with type 3 or type 4 aps ; however , the autoimmune disease might occur progressively within a wide age span , making the diagnosis difficult .
with all this in mind , we suspected an autoimmune origin of iridocyclitis that developed in a patient with vitiligo , type 1 diabetes , alopecia and thyroid disease .
a 61yearold man with type 1 diabetes and longlasting poor metabolic control ( glycated hemoglobin 100 mmol / mol , 11.3% ) despite a 96iu basal
bolus insulin regimen was referred to our clinic at the department of clinical and experimental medicine university of pisa , pisa , italy .
his clinical history showed vitiligo , a previous hyperthyroidism corrected with methimazole , a cholecystectomy for gall bladder stones and a prior diagnosis of chronic obstructive pulmonary disease . at the age of 43 years , after a sudden retinal detachment in the left eye , diabetes was diagnosed ; insulin therapy was started without further characterization of the disease .
four months before coming to our attention , the patient suffered for 2 months from a remittent fever ( 38.5c ) , not associated with shivering , and weight loss ( 10% ) ; routine blood tests were normal except for mild abnormalities in aspartate aminotransferase , alanine aminotransferase , glutamyl transferase and alkaline phosphatase .
then he developed a progressive reduction of visual acuity in the right eye ; an iridocyclitis was diagnosed , whose etiology was not investigated .
he also reported unstable glycemic control , requiring a significant increase in total daily insulin dose , associated with weight loss , fatigue , diffuse arthralgias persisting throughout the day and declining during the night , and sporadic recurrence of mild fever ( 3737.5c ) .
an eye examination found bilateral mild miosis with a turbid right iris , and depigmented redness around the cornea and conjunctiva .
a neurological examination was normal , apart from a reduction of visual acuity in the right eye .
complete blood count , creactive protein , international normalized ratio , activated partial thromboplastin time , blood urea nitrogen , creatinine , uric acid , total protein , bilirubin , lipid profile , creatine kinase , pancreatic amylase and lipase showed normal values .
antimitochondrial antibodies , antismooth muscle antibodies , antineutrophil cytoplasmic antibodies , extractable nuclear antigens , antiliver kidney microsomal antibodies , scleroderma70 kd extractable immunoreactive fragment antibodies , rheumatoid arthritis test , antitissue transglutaminase antibodies , endomysial antibodies , deamidated gliadin peptide antibodies and hormones ( thyroidstimulating hormone , free triiodothyronine , free thyroxine , adrenocorticotrophic hormone , cortisol , luteinizing hormone , folliclestimulating hormone , free testosterone , growth hormone , prolactin , parathyroid hormone ) were all normal , as well as thyroid autoantibodies ( antithyroglobulin autoantibodies and antithyroid peroxidase autoantibodies : < 1 iu / ml for both ) .
gammaglutamyl transferase ( 115 u / l ; normal value < 60 ) , alkaline phosphatase ( 194 u / l ; < 115 ) and erythrocytes sedimentation rate ( 38 mm / h ) showed altered values .
the presence of glutamic acid decarboxylase and tyrosine phosphataselike protein ia2 antibodies with undetectable cpeptide confirmed type 1 diabetes . despite insulin uptitration ,
glucose values were extremely variable , with mild hypoglycemic episodes and frequent , severe hyperglycemia .
the combination of vitiligo , alopecia , type 1 diabetes , previous hyperthyroidism and the presence of positive adrenal antibodies allowed the suspicion of type 2 aps , but adrenal function was normal . therefore ,
lowgrade fever , fatigue and weight loss , increased cholestasis enzymes , insulin resistance , and mild inflammation focused our clinical workup on the granulomatous causes of iridocyclitis : the sexual habits of the patient were investigated ; treponemal screening was 40.95 s / co ( normal value < 1.0 ) and a treponemal test found : treponema pallidum hemoagglutination assay > 1:40960 , immunoglobulin g and immunoglobulin m positivity for treponema pallidum , and fluorescent treponemal antibody absorption positive 3+/4 + .
although international guidelines for treatment suggest penicilling as the firstchoice drug for the treatment of all stages of syphilis5 , we started with ceftriaxone and doxycycline , continuing at home with benzathine penicillin ( four intramuscular doses of 2.4 mu each at 1week interval ) . immediately after the first dose of ceftriaxone and doxycycline , the patient developed a jarish herxheimer reaction ( as a result of a massive release of treponemal antigens ) ; however , in the light of such a reaction and in order to prevent a possible worsening of the ocular manifestations , i.v .
methylprednisolone had been administered before starting antimicrobial therapy . to investigate central nervous system involvement ,
brain magnetic resonance imaging was carried out ( signs of a previous optic neuritis ) .
lumbar puncture was postponed , as the patient was hivnegative and iridocyclitis was the only neurological manifestation of the disease . during the follow up , the patient reported subjective wellbeing with rapid improvement of visual acuity and eye inflammation ( figure 1 ) .
glycemic control , though still suboptimal , was obtained with a total of 58 iu of insulin .
six months after the diagnosis , treponema pallidum hemoagglutination assay was 1:10240 and venereal disease research laboratory 1:16 .
the ( a ) eyes status and ( b ) vitiligo ( detail of the hands ) 4 months after the diagnosis .
an eye examination found bilateral mild miosis with a turbid right iris , and depigmented redness around the cornea and conjunctiva .
a neurological examination was normal , apart from a reduction of visual acuity in the right eye .
complete blood count , creactive protein , international normalized ratio , activated partial thromboplastin time , blood urea nitrogen , creatinine , uric acid , total protein , bilirubin , lipid profile , creatine kinase , pancreatic amylase and lipase showed normal values .
antimitochondrial antibodies , antismooth muscle antibodies , antineutrophil cytoplasmic antibodies , extractable nuclear antigens , antiliver kidney microsomal antibodies , scleroderma70 kd extractable immunoreactive fragment antibodies , rheumatoid arthritis test , antitissue transglutaminase antibodies , endomysial antibodies , deamidated gliadin peptide antibodies and hormones ( thyroidstimulating hormone , free triiodothyronine , free thyroxine , adrenocorticotrophic hormone , cortisol , luteinizing hormone , folliclestimulating hormone , free testosterone , growth hormone , prolactin , parathyroid hormone ) were all normal , as well as thyroid autoantibodies ( antithyroglobulin autoantibodies and antithyroid peroxidase autoantibodies : < 1 iu / ml for both ) .
gammaglutamyl transferase ( 115 u / l ; normal value < 60 ) , alkaline phosphatase ( 194 u / l ; < 115 ) and erythrocytes sedimentation rate ( 38 mm / h ) showed altered values .
the presence of glutamic acid decarboxylase and tyrosine phosphataselike protein ia2 antibodies with undetectable cpeptide confirmed type 1 diabetes . despite insulin uptitration ,
glucose values were extremely variable , with mild hypoglycemic episodes and frequent , severe hyperglycemia .
the combination of vitiligo , alopecia , type 1 diabetes , previous hyperthyroidism and the presence of positive adrenal antibodies allowed the suspicion of type 2 aps , but adrenal function was normal .
therefore , lowgrade fever , fatigue and weight loss , increased cholestasis enzymes , insulin resistance , and mild inflammation focused our clinical workup on the granulomatous causes of iridocyclitis : the sexual habits of the patient were investigated ; treponemal screening was 40.95 s / co ( normal value < 1.0 ) and a treponemal test found : treponema pallidum hemoagglutination assay > 1:40960 , immunoglobulin g and immunoglobulin m positivity for treponema pallidum , and fluorescent treponemal antibody absorption positive 3+/4 + .
although international guidelines for treatment suggest penicilling as the firstchoice drug for the treatment of all stages of syphilis5 , we started with ceftriaxone and doxycycline , continuing at home with benzathine penicillin ( four intramuscular doses of 2.4 mu each at 1week interval ) .
immediately after the first dose of ceftriaxone and doxycycline , the patient developed a jarish
herxheimer reaction ( as a result of a massive release of treponemal antigens ) ; however , in the light of such a reaction and in order to prevent a possible worsening of the ocular manifestations , i.v . methylprednisolone had been administered before starting antimicrobial therapy . to investigate central nervous system involvement ,
brain magnetic resonance imaging was carried out ( signs of a previous optic neuritis ) .
lumbar puncture was postponed , as the patient was hivnegative and iridocyclitis was the only neurological manifestation of the disease . during the follow up , the patient reported subjective wellbeing with rapid improvement of visual acuity and eye inflammation ( figure 1 ) .
glycemic control , though still suboptimal , was obtained with a total of 58 iu of insulin .
six months after the diagnosis , treponema pallidum hemoagglutination assay was 1:10240 and venereal disease research laboratory 1:16 .
the ( a ) eyes status and ( b ) vitiligo ( detail of the hands ) 4 months after the diagnosis .
despite the presence of a clinical phenotype suggestive of aps , the nature of this iridocyclitis was not related to the autoimmune disorder .
acute anterior uveitis can be idiopathic , sometimes hlab27linked or granulomatous , the latter either associated with systemic infectious processes , such syphilis , lyme disease , tuberculosis or herpetic viral infections , or sarcoidosis . in this perspective , specific serology tests should be arranged in case of intractable uveitis of uncertain origin , especially in hiv patients , where macular palcoid chorioretinitis can occur6 . in italy ,
720 cases of syphilis were recorded in the national health registry in 2007 ; 78% occurred in male subjects .
according to 2011 world health organization data , the estimated number of new cases of curable sexually transmitted diseases in adults , in europe , is 20 million , of which , in 2010 , 17,884 were cases of syphilis7 . in the postwar period ,
a relationship between syphilis and diabetes was described ; recently , an increased prevalence of type 2 diabetes has been reported among patients with neurosyphilis ; these patients were also characterized by a worse metabolic pattern , with higher fasting glucose and glycated hemoglobin levels8 .
ocular manifestation of syphilis can occur at any stage of the disease , mimic a wide range of ocular disorders , and lead to misdiagnoses and delay in the administration of appropriate antimicrobial therapy .
syphilis is a rare cause of uveitis ; it might occur 6 weeks after primary infection , sometimes being the only systemic sign of secondary syphilis , often characterized by optic neuropathy .
the diagnosis of syphilis in the present patient was , in fact , delayed ; a meditated interpretation of the clinical history and a criticallyoriented analysis of laboratory tests would have allowed us to rapidly formulate a correct diagnosis .
in fact , as early as 4 months before coming to our attention , the patient started to report symptoms not clearly related to a rise in the hepatobiliary indices in a long termcholecystectomized individual .
therefore , a screening for hepatotropic viruses , syphilis and hiv should had been carried out even at that time , because of the presence of risk behaviors .
when iridocyclitis developed , it was considered an autoimmune epiphenomenon of a coexisting aps , with this view being supported by the lowgrade inflammation and the worsening glycemic control .
such a hypothesis allowed us to also explain flulike symptoms and fever ; on the same basis , the suspicion of syphilitic etiology could have been made previously .
we attributed the modest rise in liver enzymes to hepatosteatosis , likely worsened by the metabolic and inflammatory derangement .
however , it is good practice , when there is an increase in transaminases or cholestasis indexes in the absence of hepatitis or hepatobiliary disease , to carry out screening tests for syphilis , given that various degrees of hepatobiliary involvement are reported in early and late stages of syphilis9 , 10 .
an attempt to correctly define the patient 's phenotype , even in the presence of an atypical presentation of the disease ( figure 2 ) , is of particular relevance in a perspective of prevention and early treatment aiming at reducing severe complications and improving the prognosis . the natural history of syphilis in the patient .
| abstractwe present a rare cause of iridocyclitis in a patient with vitiligo and type 1 diabetes who showed poor metabolic control , and suffered from remitting fever , weight loss , fatigue , diffuse arthralgias and reduced visual acuity .
mild systemic symptoms coupled with increased cholestasis enzymes , insulin resistance , mild inflammation and a functioning adrenal gland focused our clinical workup on granulomatous causes of iridocyclitis .
specific tests confirmed syphilis , with no involvement of the central nervous system .
ocular syphilis , despite being unusual , can be the only manifestation of the disease .
the workup of any unexplained ocular inflammation should include testing for syphilis so as to not delay the diagnosis . |
plant root - released volatile and non - volatile chemicals transmit information to other plants or edaphon organisms ( badri and vivanco 2009 ; bais et al .
2006 ; rasmann et al . 2005 ; van tol et al . 2001 ; wenke et al . 2010 ) .
so far , technical limitations have rendered analyses of rhizosphere compound blends difficult , particularly under natural conditions or in field studies .
important methodological parameters for analysis of rhizochemicals include the way roots are kept prior to analysis , the sampling technique , and the ( ad)sorbent used .
sophisticated radioactive or stable isotope labeling techniques , applicable even in field studies , have delivered comprehensive knowledge on carbon fluxes ( e.g. , carbohydrate transfer from plant roots to mycorrhizal fungi ) in soil ( derrien et al .
such labeling techniques , however , require complex and expensive experimental setups and often destructive processing of test plants .
other less complex and less costly studies on rhizochemicals apply methods that affect the release of root chemicals since the roots are not kept in a ( close - to ) natural way . for example , ( i ) extraction by solutions in which excavated plant roots are dipped ( gransee and wittenmayer 2000 ; strm et al .
1994 ) , ( ii ) extraction from roots of hydroponic - grown plants ( mostly sterile and hypoxic conditions ) ( cieslinski et al . 1997
2000 ) , or ( iii ) extraction from root systems of plants grown in agar , soda - glass beads or on filter paper ( eldhuset et al .
root - derived compounds further have been analyzed from cut roots ( steeghs et al .
2004 ) , frozen and sliced root mats ( gahoonia and nielsen 1991 ) , or extracts of pulverized roots ( rasmann and turlings 2007 ) .
dynamic ( active ) sampling techniques for rhizochemicals use air- or water flow around the ( often substrate - free ) plant roots and through an adsorbent ( e.g. , ali et al .
such dynamic sampling techniques are suitable and most frequently used to analyze plant volatiles aboveground ( tholl et al .
. however , air- or water flow around the plant roots is problematic due to the risk of drought or flooding .
again , the disturbance to the rhizosphere may be so drastic that the plants can not be used for further experiments .
static ( passive ) chemical sampling methods ( without air- or water flow ) are based on diffusion of compounds into a substrate or sorbent .
widely used passive sampling techniques are solid phase microextraction ( spme ) and stir - bar sorptive extraction ( sbse ) ( baltussen et al .
numerous adsorbents that bind analytes to their adsorptive sites and sorbents into which analytes are diffused and retained are commercially available ( baltussen et al .
woolfenden ( 2010 ) outlines a wide range of factors that need consideration when selecting an ( ad)sorbent ; sampling and desorption efficiency of the ( ad)sorbent as well as artifact risks are parameters that significantly determine the quality of analysis .
well - known ( ad)sorbents range from activated alumina , silica , zeolithes ( aluminosilicates ) to octadecyl - modified silica , activated carbon , and synthetic polymeric materials , e.g. , tenax ta consisting of 2,6-diphenylene - oxide polymer resin , or polydimethylsiloxane ( pdms )
. passive sampling of compounds on sorptive tubes based on polydimethylsiloxane ( pdms , silicone ) has been accomplished successfully in the past and allows sampling of volatiles of widely divergent polarity and volatility ( individually or in complex mixtures ) , and even those with low emission rates ( bartelt 1997 ; nyasembe and torto 2014 ; tholl et al .
polydimethylsiloxane ( pdms ) tubes are robust , easy to handle , and can be cut with scissors or blades into pieces of desired length .
furthermore , the tubes are inexpensive , and due to their size and hollowness , they provide a larger active surface than the active surface of a pdms - covered fiber ( kallenbach et al .
is commonly combined with liquid chromatography , i.e. , hplc ( mohney et al . 2009 ; ohara 2009 ; van pinxteren et al .
2009 , and references therein ) , and has , been applied e.g. , to study the transport of allelopathic substances through mycorrhizal networks ( barto et al .
( 2009 ) used a similar method for collection of rhizosphere chemicals ; they showed that pdms tubes inserted into soil ( solid phase root zone extraction ; spre ) were more efficient in collecting allelopathic rhizosphere compounds than any other tested ( ad)sorbent .
sampling on pdms tubing also may be combined with gas chromatography ( gc ) . as the trapped compounds
are not eluted with solvents , but thermally desorbed prior subjection to gas chromatography , a noise - free baseline can be obtained ( bartelt 1997 ) .
( 2014 ) presented a high - throughput technique for the collection of volatiles from aerial plant parts in field experiments using pdms tubes .
this study aimed ( i ) to develop a set - up that allows inexpensive , simple , and reliable collection of volatiles ( using pdms tubing for sampling ) as well as non - volatile hydrophilic compounds ( using aqueous substrate extraction ) from a rhizosphere that is not exposed to disturbing conditions like air- or water flow through the root zone , and ( ii ) to elucidate conditions at which rhizosphere compounds adsorb optimally on pdms tubes .
a rhizosphere mesocosm was designed from which volatiles were extracted on pdms tubes without root damage , air- or water flow .
we focused on the analysis of rhizosphere volatiles because of their ecological relevance in root - associated food webs ( hiltpold et al . 2010 ; robert et al .
2012 ) . in order to evaluate the potential use of the mesocosm for aqueous extraction of polar , water - soluble compounds
, we also analyzed plant root - derived sugars as one example of this type of compound .
we focused on sugars because of their quantitative dominance in rhizospheres ( azaizeh et al .
1995 ) . for analysis of sugars , minor amounts of substrate encasing the roots were harvested , and extracted sugars were derivatized prior to gc / ms analysis .
rhizosphere compounds belong to diverse chemical classes with divergent affinity to pdms and to the substrate or soil .
we analyzed how well pdms tubes compete with different adsorbing substrates by determining recovery rates of synthetic reference compounds that have been identified previously in natural rhizospheres .
we further tested whether protecting pdms tubes against clogging by soil particles could enhance the recovery rates of volatiles .
the ubiquitous ( stewart - wade et al . 2002 ) and pharmacologically relevant ( williams et al . 1996 ) ruderal plant dandelion ( taraxacum sect .
ruderalia , kirschner , llgaard et tpnek ) was chosen to test the suitability of the newly developed rhizosphere mesocosm for analysis of rhizosphere chemistry .
dandelion seeds ( treppens & co samen gmbh , berlin ) were surface - sterilized as described by krgel et al .
all plants were watered with nutrient solution modified from arnon and hoagland ( 1940 ) , see reinecke et al .
young plants were grown in a climate chamber at 22 c , 70 % r.h .
, l : d 16:8 h. when plants were 5-wk - old , they were transplanted into rhizosphere mesocosms ( see below ) .
1a ) , containing two horizontally stacked perforated polytetrafluoroethylene ( ptfe ) discs ( 124 holes of 2 mm diam ) mounted on a stainless steel thread bar ( fig .
the size of holes in the discs was adjusted to the size of the roots of the plants .
the discs were designed similar to desiccator plates , but the lower disc had a spacer rim of 1.5 cm height .
each rhizosphere mesocosm was divided into three compartments by the discs : ( i ) a sand - filled compartment ( 1.2 l , top ) , ( ii ) a central compartment between the discs ( 250 ml , height = 1.5 cm ) , and ( iii ) a drainage compartment ( 2.1 l , bottom ; fig .
due to the high moisture content of both adjacent compartments , the central compartment contained air of high humidity , and thus , roots reaching this compartment were not exposed to drought .
a shading aluminum foil cover was wrapped around the outside of each mesocosm and prevented algal growth inside the glass vessels .
the inside of each mesocosm was lined with clean polyester fleece , which provided a barrier between roots , substrate , and glass .
thus , roots could not attach to the glass walls , and the entire plant and root ball could gently be removed from the mesocosm and inserted back again when necessary without damaging the roots .
non - destructive removal of the root ball from the mesocosms was essential for sampling of rhizosphere volatiles ( see section
a the size of the mesocosm may be adapted to the number and size of plants : the mesocosm shown in this photo basically consisted of the same components as the mesocosm used in the experiments ( except for the fleece in the substrate compartment ) , but a 1 l schott beaker glass replaced the glass vessel .
b the mesocosm consisted of a substrate compartment ( filled with plant roots and sand ) , a central compartment ( filled with humid air , buffer zone between substrate and drainage compartment ) , and a drainage compartment ( containing leached aqueous nutrient solution that was not taken up by the plants and accumulated here over 5 d ; approx . 5 mm , v = 80 ml solution in total ) .
the plant roots grew through the perforated polytetrafluoroethylene ( ptfe ) discs ( 124 holes , diam 2 mm ) , which were mounted on a stainless steel thread bar ( length : 25 cm , diam 1 cm ) .
due to static ( passive ) volatile collection on polydimethylsiloxane ( pdms ) tubes , no artificial air flow was required .
c the thread bar , which carried the ptfe discs , allowed gentle removal of rhizospheres prior to insertion of pdms tubes for sampling of volatiles .
fleece surrounding the substrate minimized friction between the plant roots , substrate , and the glass vessel when removing the plant with root ball from the substrate compartment for insertion of pdms tubes in the central and/or drainage compartment rhizosphere mesocosm for collection of root - derived volatile and water - soluble compounds .
a the size of the mesocosm may be adapted to the number and size of plants : the mesocosm shown in this photo basically consisted of the same components as the mesocosm used in the experiments ( except for the fleece in the substrate compartment ) , but a 1 l schott beaker glass replaced the glass vessel .
b the mesocosm consisted of a substrate compartment ( filled with plant roots and sand ) , a central compartment ( filled with humid air , buffer zone between substrate and drainage compartment ) , and a drainage compartment ( containing leached aqueous nutrient solution that was not taken up by the plants and accumulated here over 5 d ; approx . 5 mm , v = 80 ml solution in total ) .
the plant roots grew through the perforated polytetrafluoroethylene ( ptfe ) discs ( 124 holes , diam 2 mm ) , which were mounted on a stainless steel thread bar ( length : 25 cm , diam 1 cm ) .
due to static ( passive ) volatile collection on polydimethylsiloxane ( pdms ) tubes , no artificial air flow was required .
c the thread bar , which carried the ptfe discs , allowed gentle removal of rhizospheres prior to insertion of pdms tubes for sampling of volatiles .
fleece surrounding the substrate minimized friction between the plant roots , substrate , and the glass vessel when removing the plant with root ball from the substrate compartment for insertion of pdms tubes in the central and/or drainage compartment five - wk - old plants were transplanted into sand - filled rhizosphere mesocosms .
groups of 10 plants each were combined in order to compensate for differences among individuals .
plants were grown for an additional period of 3.5 wk in the mesocosm prior to insertion of pdms tubing pieces for volatile sampling .
sampling of pdms tubing pieces for chemical analysis of volatiles and collection of substrate samples for chemical analysis of sugars was conducted when the plants were 9-wk - old . in total ,
rhizosphere chemicals of six mesocosms , i.e. , groups of 10 plants each were analyzed .
the conditions at which plants were kept in the mesocosms were 22 c , 70 % r.h .
, l : d 16:8 h , and 791.37 31 molsm par light intensity . plants in the rhizosphere mesocosms were irrigated daily until a defined drainage from the substrate was observed in the drainage compartment ( approx .
5 mm , i.e. , 80 ml in total collected over 5 d ) .
sugars were extracted from the substrate in the upper ( substrate ) compartment and lower drainage compartment .
volatiles were collected on pdms tubes ( inner diam : 1.5 mm , o.d .
: 2.3 mm , length : 3 mm , reichelt chemietechnik , germany ) .
pdms bulk tubes were cut into pieces of 3 mm using a custom made cutting mask with razor blades in guiding rails at 3 mm distance .
tube pieces were cleaned for 8 h in 4:1 ( v / v ) acetonitrile : methanol , and afterwards heated to 230 c for 12 h under nitrogen flow ( filtered on activated charcoal ) in a tube conditioner ( tc 2 , gerstel , mlheim , germany ) prior to analysis .
as it was unknown from which compartment the volatiles could be extracted most efficiently , we collected volatiles from all three compartments ( substrate , central , and drainage ) of the rhizosphere mesocosms ( fig .
three tube pieces in one compartment were regarded as technical replicates , and the average of these technical replicates was considered one biological replicate .
for control , volatiles were collected from six identically treated but plant - free mesocosms . in the upper ,
substrate - filled compartment , pdms tube pieces were placed at 34 cm depth into plant rhizospheres or substrate - filled control compartments of mesocosms .
for insertion of the pdms tubes into the central and drainage compartments , the intact plants with root balls were gently withdrawn from the rhizosphere compartments together with the ptfe discs by pulling the thread bar ( fig .
the plants and root ball remained on the upper ptfe disc , and the screw holding the lower ptfe disc was moved downward to obtain a 5 cm slot for insertion and removal of pdms tubes into the central compartments ( between the two ptfe discs ) .
although plant roots sporadically grew through the holes in the perforated discs , they were not ruptured nor abraded by the ptfe discs or the glass vessel during the insertion or removal procedure . in a preliminary experiment , different extraction durations
( 12 , 24 , 48 , 96 , and 144 h ) were assessed by sampling volatiles from three dandelion mesocosms , each containing 10 plants ( n = 3 ) .
the proportions of total peak areas of plant compounds , i.e. , compounds not present in substrate controls were : 12.9 2.4 % ( 12 h ) , 10.2 2.2 % ( 24 h ) , 32.6 10 % ( 48 h ) , 100 % ( 96 h ) , 83.7 17.7 % ( 144
h ) relative to the highest measured peak area for each compound ( mean s.d . ) . as sampling efficiency was highest for both 96 and 144 h and did not significantly differ between these durations ( p > 0.05 , wilcoxon signed - rank test ) , we chose an intermediate sampling duration of 120 h for further analyses .
after sampling , pdms tubes were removed from rhizosphere mesocosms and stored individually in 1.5 ml screw - cap glass vials at 20 c until analysis .
for collection of a rhizosphere substrate sample for sugar extraction , the intact plants with root balls were gently removed together with the substrate by withdrawing the ptfe discs , thread bar , and overlapping fleece from the mesocosms ( fig . 1 ) .
then , two substrate samples ( two technical replicates , each 15 ml ) were collected from each substrate compartment ( n = 6 mesocosms ) .
in addition to samples from the substrate compartment , we also sampled the leached nutrient solution collected over 5 d in the drainage compartment ( two technical replicates of 15 ml per sample ; fig . 1 ) .
remaining leached nutrient solution was discarded , and mesocosms were not sampled again for sugars .
samples of substrate and leached nutrient solution were kept on ice during collection ( approx .
15 min ) and later stored at 80 c until extraction and analysis .
the ( root - free ) substrate samples were extracted by adding 15 ml distilled water to 15 ml substrate .
the filtrate was obtained by using a suction filter , and it was cooled on ice . after repeating this procedure , approx .
25 ml filtrate per sample were obtained , which were immediately frozen at 80 c , lyophilized to dryness , and dissolved in pyridine to 2 g l .
the samples taken from the drainage compartment ( 15 m ) also were lyophilized to dryness and dissolved in pyridine to 2 g l . to determine substrate specific recovery rates of reference compounds , we used substrate - filled glass vials ( 40 ml screw top , supelco , sigma - aldrich ) into which pdms tubes were inserted ( for volatile sampling ) , or from which the substrates were extracted ( for sampling of sugars ) .
recovery rates were compared for distinct quantities of commercially available rhizosphere compounds that were added to water , sand , vermiculite , a fraction of processed ( heated and sieved ) soil ( tables 1 and 2 ) , and unprocessed soil ( table 3 ) ( see below for details on the substrates and tested compounds ) .
glass vials ( 40 ml screw top , supelco , sigma - aldrich ) were filled with 21 ml water or 25 ml dry substrate ( see below ) supplemented with 21 ml water .
the non - dried field soil was supplemented by 10 ml water to reach a comparable moisture level .
commercially available compounds were added to these substrates in distinct quantities ( see below).table 1determination of recovery rates ( % ) of standard volatiles applied to different substrates in a glass vial and sampled by polydimethylsiloxane ( pdms ) tubes with contact to substrate or by gauze - protected pdms tubescompound
setupsubstrateswatersandvermiculiteprocessed soil
butyl acetate
contact pdms0.3 0.0 ( c )
1.2 0.2 ( b )
2.8 0.4 ( a )
1.3 0.1 ( b * )
protected pdms2.2 0.6 ( b * )
1.9 0.4 ( b )
5.4 1.7 ( a * )
0.7 0.3 ( c )
1-hexanol
contact pdms0.0 0.0 ( c )
0.4 0.1 ( a )
0.2 0.0 ( b )
0.0 0.0 ( c )
protected pdms1.4 0.4 ( a * )
1.9 0.4 ( a * )
1.6 0.5 ( a * )
0.2 0.1 ( a )
-pinene
contact pdms5.0 0.5 ( c )
12.0 1.4 ( b )
15.2 1.2 ( a )
7.2 0.8 ( c )
protected pdms19.2 7.4 ( b * )
9.7 1.1 ( bc )
32.9 5.6 ( a * )
6.7 0.8 ( c )
benzaldehyde
contact pdms0.6 0.1 ( c )
5.1 0.9 ( a * )
2.5 0.4 ( b )
5.1 0.8 ( a * )
protected pdms1.3 0.2 ( a * )
2.4 1.1 ( a )
2.3 0.3 ( a )
2.6 0.4 ( b )
hexyl acetate
contact pdms0.4 0.1 ( b )
0.9 0.1 ( a )
0.9 0.1 ( a )
0.3 0.0 ( b )
protected pdms5.1 1.6 ( b * )
1.6 0.4 ( c * )
9.3 1.7 ( a * )
0.2 0.0 ( d )
linalool
contact pdms2.4 0.3 ( b )
6.1 1.0 ( a )
2.6 0.3 ( b )
6.9 0.8 ( a )
protected pdms6.6 0.3 ( ab * )
7.4 0.3 ( a )
7.9 0.3 ( a * )
5.9 0.5 ( b )
methyl salicylate
contact pdms0.1 0.0 ( c )
0.7 0.1 ( a )
0.1 0.0 ( c )
0.2 0.0 ( b )
protected pdms1.8 0.2 ( b * )
1.9 0.2 ( b * )
2.8 0.4 ( a * )
0.4 0.2 ( c )
cinnamal
contact pdms1.2 0.1 ( c )
3.3 0.6 ( a )
1.6 0.2 ( b )
3.1 0.5 ( ab )
protected pdms2.6 0.4 ( a * )
4.3 0.6 ( a )
1.8 0.3 ( a )
4.6 0.4 ( b )
-elemene
contact pdms45.6 3.6 ( a )
5.9 1.4 ( b )
11.4 1.5 ( b )
8.3 1.6 ( b )
protected pdms43.7
2.9 ( a )
7.8 0.4 ( c )
18.3 1.5 ( b * )
8.5 1.8 ( c )
-farnesene
contact pdms13.9 1.0 ( a )
9.1 0.7 ( b )
15.0 1.5 ( a )
0.7 0.2 ( c )
protected pdms12.8 1.3 ( a )
9.6 0.9 ( a )
14.6 0.8 ( a )
2.1 0.6 ( b * )
-farnesene
contact pdms22.3 3.1 ( b )
21.5 2.0 ( b )
36.0 3.4 ( a )
0.9 0.4 ( c )
protected pdms19.2 5.9 ( b )
18.7 2.3 ( b )
35.3 8.7 ( a )
2.1 0.6 ( c * )
farnesyl acetate
contact pdms0.1 0.0 ( c )
2.5 0.3 ( a )
0.4 0.1 ( b )
2.6 0.3 ( a )
protected pdms3.5 1.4 ( a * )
3.5 1.2 ( a )
0.4 0.1 ( b )
1.8 0.6 ( ab )
compounds ordered by ki ( kovats retention index ) ; 100 ng of each volatile were applied per vial ; 100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
sieved and heated soil
compounds that have been studied with respect to interactions between dandelion roots and a rhizophagous insect ( eilers et al . 2012 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 ) * asterisks indicate significant differences between contact and protected pdms within one compound and substrate category at p 0.05 ( wilcoxon signed - rank test ) ; different letters indicate significant differences between substrates at p 0.05 ( kruskal - wallis h - test and post - hoc mann
whitney u - tests with bonferroni correction)table 2recovery rates ( % ) of standard sugars applied to different substrates in a glass vial and sampled by aqueous extractioncompound
substrateswatersandvermiculiteprocessed soil fraction
arabinose ( c5 )
42.2 11.9 ( a )
15.3 5.7 ( b )
3.8 1.2 ( c )
1.5 0.3 ( c )
xylose ( c5 )
86.6 7.1 ( a )
12.4 1.3 ( b )
4.2 0.7 ( c )
1.6 0.2 ( d )
mannose ( c6 )
58.7 8.1 ( a )
17.4 2.4 ( b )
3.6 0.8 ( c )
3.2 0.4 ( c )
fructose ( c6 )
46.4 7.2 ( a )
10.5 0.5 ( b )
2.6 0.5 ( c )
1.2 0.5 ( d )
glucose ( c6 )
70.7 10.4 ( a )
16.6 2.9 ( b )
4.4 0.4 ( c )
2.2 0.1 ( d )
sucrose ( c12 )
25.3 8 ( a )
13.5 1.8 ( b )
2.6 0.5 ( c )
1.6 0.3 ( c )
maltose ( c12 )
85.8 7.6 ( a )
6.3 0.1 ( b )
1.1 0.3 ( c )
1.0 0.1 ( c )
ordered by ki ( kovats retention index ) ; 10 g of each sugar was applied per vial .
100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
sieved and heated soil
compounds that were also detected in rhizospheres of other plants than dandelion ( dennis et al .
2010 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 )
different letters indicate significant differences between substrates at p 0.05 ( kruskal - wallis h - test and post - hoc mann
whitney u - tests with bonferroni correction)table 3recovery rates ( % ) and total amounts of volatiles applied to unprocessed soil and sampled by polydimethylsiloxane ( pdms ) tubes with contact to soilcompound
amounts applied to unprocessed field soil100 ng
500 ng1 g5 gvolatiles%ngngngng butyl acetate
0.0 0.00.0 0.00.1 0.00.0 0.00.0 0.0 1-hexanol
0.0 0.00.0 0.00.1 0.00.1
0.11.1 0.5 -pinene
0.2 0.00.2 0.114.9 1.232.5 2.661.2 4.1 benzaldehyde
0.0 0.10.0 0.00.1 0.02.6 0.27.6 0.8 hexyl acetate
0.0 0.20.1 0.00.1 0.00.0
0.00.0 0.0 linalool
0.0 0.10.0 0.07.8 0.610.1 0.85.5 0.6 methyl salicylate
0.2 0.10.1 0.01.7 0.42.2 0.244.0 3.6 cinnamal
0.3 0.20.1 0.02.2 0.34.1 0.412.1 0.9 -elemene
0.0 0.00.0
0.01.2 0.31.3 0.42.6 0.3 -farnesene
0.0 0.00.0 0.01.6 0.21.4 0.12.5 0.4 -farnesene
0.0 0.00.0 0.00.5 0.10.5 0.11.0
0.4 farnesyl acetate
0.3 0.20.1 0.06.1 0.57.4 0.09.0 1.4
sorted by ki
100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
compounds that have been studied with respect to interactions between t. sect .
2012 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 ) determination of recovery rates ( % ) of standard volatiles applied to different substrates in a glass vial and sampled by polydimethylsiloxane ( pdms ) tubes with contact to substrate or by gauze - protected pdms tubes
compounds ordered by ki ( kovats retention index ) ; 100 ng of each volatile were applied per vial ; 100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
sieved and heated soil
compounds that have been studied with respect to interactions between dandelion roots and a rhizophagous insect ( eilers et al . 2012 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 ) * asterisks indicate significant differences between contact and protected pdms within one compound and substrate category at p 0.05 ( wilcoxon signed - rank test ) ; different letters indicate significant differences between substrates at p 0.05 ( kruskal - wallis h - test and post - hoc mann
whitney u - tests with bonferroni correction ) recovery rates ( % ) of standard sugars applied to different substrates in a glass vial and sampled by aqueous extraction
ordered by ki ( kovats retention index ) ; 10 g of each sugar was applied per vial .
100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
sieved and heated soil
compounds that were also detected in rhizospheres of other plants than dandelion ( dennis et al .
2010 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 )
different letters indicate significant differences between substrates at p 0.05 ( kruskal - wallis h - test and post - hoc mann
whitney u - tests with bonferroni correction ) recovery rates ( % ) and total amounts of volatiles applied to unprocessed soil and sampled by polydimethylsiloxane ( pdms ) tubes with contact to soil
100 % = peak area of 100 ng of each compound directly subjected to gc / ms analysis ( mean sd ,
n = 4 )
compounds that have been studied with respect to interactions between t. sect .
2012 )
compounds that were also detected in dandelion rhizospheres ( compare table 4 ) the incubation time , i.e. , the time lag between spiking of the test compounds into the substrate - filled vials and obtainment of samples for determination of recovery rates by gc - ms analysis was 1 h ( volatiles ) or 15 min ( sugars ) .
volatiles were collected on pdms tubes as described above for volatile collection in the dandelion mesocosm ; three conditioned pdms tube pieces ( 3 mm long ) were inserted into each vial , either directly into the water or substrate , or protected by a polyetheretherketone ( peek ) gauze sheath ( 100 m mesh openings , 32 % open area , sefar peektex , sefar ag , heiden , switzerland ) .
the gauze was closed with steel staple clips , and a steel spring provided for a constant volume of approx
2gauze sheath for polydimethylsiloxane ( pdms ) tubes in volatile recovery experiments with standard components . in order to evaluate the effect of contact between the polydimethylsiloxane ( pdms ) tubes and the tested substrates ( and water ) on the recovery of applied standard volatiles ,
pdms tubes were either placed directly into the matrix or enveloped in 100 m peek ( polyether ether ketone ) gauze , as illustrated in this figure .
a stainless steel spring was placed inside the gauze sheath to maintain a constant volume ( 0.69 cm ) .
both ends of the gauze sheath were sealed with steel staple clips gauze sheath for polydimethylsiloxane ( pdms ) tubes in volatile recovery experiments with standard components . in order to evaluate the effect of contact between the polydimethylsiloxane ( pdms ) tubes and the tested substrates ( and water ) on the recovery of applied standard volatiles ,
pdms tubes were either placed directly into the matrix or enveloped in 100 m peek ( polyether ether ketone ) gauze , as illustrated in this figure .
a stainless steel spring was placed inside the gauze sheath to maintain a constant volume ( 0.69 cm ) .
both ends of the gauze sheath were sealed with steel staple clips we compared the recovery rates from water and the following substrates : sand : ph ( cacl2 ) = 5.5 , particle size = 0.40.8 mm , specific surface area ( ssa ) : approx . 0.010.005 mg , cation exchange capacity ( cec ) : 0 meq100 g ( all particle size , ssa and cec values according to ahl et al .
2000).vermiculite : ph ( cacl2 ) = 6.92 , cluster size = 23 mm ,
80 meq100 g.processed ( sieved and heated ) fraction of field soil : sandy clay loam , collected from a dandelion - rich meadow in hessenthal , bavaria , germany ( 4993n , 926o ) in october 2010 and april 2011 .
the organic fraction was partly removed by sieving ( 1 mm ) and heating to 200 c for 3 h. after processing , the ph ( cacl2 ) was 5.9 , indicating a humus content of 15 % ( kerschberger et al .
supposing that the composition of this sandy clay loam matches the known composition of this type of soil ( 2530 % clay , 525 % silt , 5560 % sand , and 15 % humus ) , the cec is expected to range between 5561 meq100 g.natural , untreated soil : sandy clay loam , collected from a dandelion - rich meadow in berlin , germany ( 52.45n , 13.31o ) in march 2014 and used for experiments without further processing .
the ph ( cacl2 ) was 6.5 , indicating a humus content of 1215 % ( kerschberger et al .
supposing that the composition of this untreated soil matches the known composition of this type of soil ( 3035 % clay , 525 % silt , 4550 % sand , and 1215 % humus ) , the cec is expected to range between 5466 meq100 g. sand : ph ( cacl2 ) = 5.5 , particle size = 0.40.8 mm , specific surface area ( ssa ) : approx . 0.010.005 mg , cation exchange capacity ( cec ) : 0 meq100 g ( all particle size , ssa and cec values according to ahl et al . 2004 ; ph ( cacl2 ) was determined according to kerschberger et al .
vermiculite : ph ( cacl2 ) = 6.92 , cluster size = 23 mm , ssa : 6080 m g , cec : approx .
80 meq100 g. processed ( sieved and heated ) fraction of field soil : sandy clay loam , collected from a dandelion - rich meadow in hessenthal , bavaria , germany ( 4993n , 926o ) in october 2010 and april 2011 .
the organic fraction was partly removed by sieving ( 1 mm ) and heating to 200 c for 3 h. after processing , the ph ( cacl2 ) was 5.9 , indicating a humus content of 15 % ( kerschberger et al .
2000 ) . supposing that the composition of this sandy clay loam matches the known composition of this type of soil ( 2530 % clay , 525 % silt , 5560 % sand , and 15 % humus ) , the cec is expected to range between 5561 meq100 g. natural , untreated soil : sandy clay loam , collected from a dandelion - rich meadow in berlin , germany ( 52.45n , 13.31o ) in march 2014 and used for experiments without further processing .
the ph ( cacl2 ) was 6.5 , indicating a humus content of 1215 % ( kerschberger et al .
supposing that the composition of this untreated soil matches the known composition of this type of soil ( 3035 % clay , 525 % silt , 4550 % sand , and 1215 % humus ) , the cec is expected to range between 5466 meq100 g. the volatiles tested here were chosen because ( i ) they were detected in dandelion rhizospheres , or ( ii ) they are presumably relevant in an interaction between a rhizophagous insect ( melolontha melolontha larvae ) and dandelion roots ( eilers et al .
the sugars were chosen because they were identified in the rhizosphere of dandelion ( glucose , fructose , sucrose ) or have been described in root exudates of other plants ( dennis et al .
the following synthetic volatiles were used : -elemene ( aapin chemicals limited , abingdon , oxfordshire , uk ) , benzaldehyde , butyl and hexyl acetate , cinnamaldehyde ( cinnamal ) , - and -farnesene , farnesyl acetate , 1-hexanol , linalool , methyl salicylate ( all sigma - aldrich , steinheim , germany ) , -pinene ( fluka , steinheim , germany ) .
a mix of these compounds was dissolved in dichloromethane ( 100 ng l of each compound ) .
a volume of 1 l of the mixture was added to each vial filled with substrate as described above ( n = 4 vials for each type of substrate or water ) ( table 1 ) . in natural , untreated soil , we additionally tested the recovery of higher amounts : 5 l of the mixture ( 500 ng of each compound ) , 10 l ( 1 g of each compound ) , and 50 l ( 5 g of each compound ) ( table 3 ) . spiked substrate and water samples were vortexed for 2 min to distribute the compounds evenly and expose them to the substrate particles .
afterwards , pdms tubes were inserted and kept for 60 min before they were subjected to thermal desorption and gc / ms analysis .
the following sugars were included in recovery experiments ( table 2 ) : sucrose , maltose , mannose ( all sigma aldrich , steinheim , germany ) , glucose , fructose , arabinose , and xylose ( all roth , karlsruhe , germany ) .
we added 20 ml water and 1 ml sugar solution to each vial ( n = 4 vials for each type of substrate or water ) and vortexed the mix intensively for 2 min to distribute the compounds in the matrix .
thereafter , the samples were kept for 15 min before the entire sample was extracted , lyophilized , derivatized , and analyzed by gc / ms .
analytical separation of volatiles and derivatized sugars was carried out using an agilent 7890a gas chromatograph ( gc ) ( agilent technologies ; waldbronn , germany ) connected to an agilent 5975 c mass spectrometer ( ms ) .
helium was used as carrier gas ( constant flow 1 ml min ) . for analysis of volatiles ,
the instrument was equipped with a thermal desorption unit ( tdu , gerstel ) , coupled to a programmable temperature vaporization ( ptv ) injector unit ( gerstel , kas 4 ) .
the volatiles were separated on an hp-5 ms column ( 30 m 0.25 mm i.d . with 0.25 m - film coating , agilent technologies ) .
after insertion of pdms sampling tubes ( one tube per analysis ) , the tdu temperature increased from 30 c to 210 c at a rate of 30 c min and was kept for 10 min ( 1 min initial delay time , total desorption period = 18 min ) .
the gc run started by heating the injection system at a rate of 12 c s to 220 c , kept for 5 min .
the gc oven was kept at 40 c for 5 min , heated at 5 c min to 260 c , and kept for 7 min ( total run time 56 min ) .
kovats retention time indices ( ki ) were calculated for each compound based on comparison of retention times to n - alkane standard compounds ( c8-c20 , 100 ng l in hexane , supelco , sigma - aldrich ) .
the identification of volatiles was accomplished by comparison of ki and mass spectra to authentic reference compounds when available : -elemene ( aapin chemicals limited ) , 2-ethyl-1-hexanol , methyl salicylate , 2-phenoxyethanol , -farnesene , farnesyl acetate ( all sigma - aldrich ) ; -selinene and -selinene from celery ( apium graveolens ; hardt et al .
1995 ) as well as pethybrene and -isocomene from butterbur ( petasites hybridus ; saritas et al .
2002 ) , kindly provided by stephan h. von reu , mpi ce jena , germany .
the remaining compounds were identified tentatively by comparison of mass spectra and ki values with those available in the national institute of standards and technology ( nist ) library or in the massfinder terpenoid library ( hochmuth , hamburg , germany ) .
see table 4 for information on compound identification.table 4estimation of absolute quantities of volatiles and sugars detected in rhizosphere mesocosms containing taraxacum sect .
ruderalia plants and sampled by polydimethylsiloxane ( pdms ) tubes with direct contact to substrate ( volatiles ) or by aqueous extraction ( sugars)#ref.compound
ki ( hp-5 ms)amount ( ng ) ( mean sd)volatiles 1
2-ethyl-1-hexanol ( likely a contamination )
1032156.2 129.6 2methyl salicylate119520.6 47.5 32-phenoxyethanol122111.2 24.7 4panaginsene1335not quantified 5pethybrene137752 41.2 6african-2-ene1385not quantified 7-isocomene1388187.9 218.5 8-elemene13987.1 5.9 9
m / z : 119(100 ) , 189(76 ) , 161(70 ) , 204(32 ) , 91(24 ) ; m : 2041447not quantified 10-farnesene14581.9 3.1 11
m / z : 109(100 ) , 110(56 ) , 204(43 ) , 79(41 ) , 93(28 ) ; m : 2041463not quantified 12-selinene1485not quantified 13-selinene ( eudesma-4(14),11-diene)149348.1 80.2 14-selinene149762.2 95.4 15farnesyl acetate18430.6 1.85sugars 16fructose40.7 48.6 17glucose197 143.3 18sucrose279.9 239.8
extraction of compounds from the central compartment ( volatiles ) and substrate compartment ( sugars ) of a rhizosphere mesocosm with dandelion plants ( n = 6 biological replicates , corresponding to 6 mesocosms containing groups of 10 plants each ) .
all 15 volatiles have been found in the central compartment , whereas the substrate and drainage compartments contained only 12 and 8 of the compounds , respectively : identification via nist library ( mass spec .
match > 95 % and ki 5) : identification via massfinder : compared to reference compound ( mass spec .
match > 95 % and ki 5 ) estimation of absolute quantities of volatiles and sugars detected in rhizosphere mesocosms containing taraxacum sect .
ruderalia plants and sampled by polydimethylsiloxane ( pdms ) tubes with direct contact to substrate ( volatiles ) or by aqueous extraction ( sugars )
extraction of compounds from the central compartment ( volatiles ) and substrate compartment ( sugars ) of a rhizosphere mesocosm with dandelion plants ( n = 6 biological replicates , corresponding to 6 mesocosms containing groups of 10 plants each ) .
all 15 volatiles have been found in the central compartment , whereas the substrate and drainage compartments contained only 12 and 8 of the compounds , respectively : identification via nist library ( mass spec .
match > 95 % and ki 5 ) : identification via massfinder : compared to reference compound ( mass spec .
match > 95 % and ki 5 ) for analysis of derivatized sugars , an hp-1 ms capillary column ( 30 m 0.25 mm i.d . with 0.25 m - film coating , agilent technologies )
the gc - injection port for liquid samples was kept at 240 c and operated in splitless mode .
the initial oven temperature of 60 c was kept for 3 min and then increased at 4 c min to a final temperature of 300 c kept for 1 min ( total run time 64 min ) .
sugars were identified by comparison of mass spectra and retention times to those of authentic standards ( all roth , karlsruhe , germany ) .
lyophilized samples ( extracted from rhizosphere substrate ) , dissolved in 50 l pyridine were derivatized with 50 l n , o - bis(tri - methylsilyl)trifluoroacetamide ( bstfa ) supplemented with 1 % tri - methylsilyl chloride ( sigma - aldrich , steinheim , germany ) .
the samples were kept for 90 min at 37 c on a thermo shaker .
furthermore , a volume of 900 l pyridine was added to each sample prior to analysis ( total volume = 1 ml , injection volume = 1 l ) .
prior comparison of relative quantities and estimation of absolute quantities of detected compounds in mesocosms ( within compartments and between compartments ) , a standard response factor was calculated for each sample based on the peak area of an internal standard ( r.f .
: measured peak area of a standard in sample divided by the peak area of the same amount of standard directly subjected to gc / ms ) . for volatiles , 1-bromodecane ( sigma - aldrich , germany ; 100 ng in 1 l in dichloromethane , added directly onto the tube piece ) was added to an extraction sample prior to analysis ; for sugars , phenyl--d - glucoside ( fisher scientific , germany ; 100 ng in 1 l pyridine ) was added as internal standard prior to sample derivatization .
the peak area of each compound was normalized ( multiplied ) by the standard response factor for each sample .
for estimation of absolute quantities of detected compounds , calibration curves of reference compounds ( see section
analytical procedures ) were obtained : for volatiles at concentrations of 100 , 50 , 20 , and 5 ng per l , solved in dichloromethane and for sugars at concentrations of 0.2 , 0.8 , 1 , 2 , 5 , and 10 g per l , solved in pyridine .
one l of the dilution was applied to a pdms tube and for sugars , one l of the dilution was derivatized and analyzed in the same way as the sugar samples obtained from substrates .
absolute quantities were estimated by comparison of peak areas in samples ( corrected by the standard response factor , i.e. , internal standard phenyl--d - glucoside ) to peak areas from calibration curves obtained from reference compounds .
peak areas of each volatile compound recovered from different substrates and water were compared to peak areas of reference compounds directly subjected to thermal desorption and gc / ms analysis ( i.e. , in table 1 , peak area of 100 ng directly thermal - desorbed and analyzed compound was defined as 100 % ) .
similarly , in experiments using higher concentrations of volatiles , peak areas of compounds recovered from soil were compared to peak areas of reference compounds directly subjected to gc / ms ( table 3 ) . in recovery experiments with sugars ,
samples were extracted , lyophilized , derivatized , and analyzed in an analogous manner to the samples of sugars obtained from substrates .
a final volume of 1 ml was obtained after derivatization , of which 1 l was subjected to gc / ms . we used the statistics software
peak areas detected on contact and protected pdms within one compound and substrate category ( table 1 ) were compared by wilcoxon signed - rank tests ( p 0.05 ) .
substrate - specific recovery rates ( tables 1 and 2 ) were determined by kruskal - wallis h - test and post - hoc testing with mann whitney tests including bonferroni correction ( p 0.05 ) .
we developed a new tripartite mesocosm for sampling volatiles on pdms tubes combined with aqueous extraction of sugars .
the upper mesocosm compartment contained substrate and the plant rhizosphere , the central compartment was a perforated ptfe disc that contained moist air , and the lower drainage compartment contained air and leached aqueous nutrient solution .
sand was used as a substrate in mesocosms , due to its inertness and low specific surface area ( ssa ) . for several reasons , dandelion ( taraxacum sect .
ruderalia ) was chosen as a model plant to test the function of the mesocosm : it is a globally distributed weed growing in soils of different properties ( stewart - wade et al . 2002 ) .
the root chemistry is multi - faceted ; total extracts of roots are rich in secondary compounds of pharmacological interest ( williams et al .
1996 ) . when sampling from the newly - developed rhizosphere mesocosms with undamaged t. sect .
ruderalia plants , we detected 15 volatiles and 3 sugars in total ( table 4 ) .
ten volatiles were identified as sesquiterpenes , and the sugars were identified as fructose , glucose , and sucrose . in the central compartment of the rhizosphere mesocosms , the highest number of volatiles was detected ( overall 15 as compared to 12 and 8 in the substrate and drainage compartment , respectively ) .
all volatiles present in the substrate and the drainage compartments were detected in the central compartment as well .
the comparison of volatiles detected in the rhizosphere mesocosms to those sampled from plant - free control mesocosms allowed determining of background chemicals . in plant - containing mesocosms , the lowest level of substrate- or nutrient - solution derived compounds , and at the same time highest number and amount of plant - derived compounds , was found in the central compartment : 76.5 8.7 % ( mean sd ) of the total peak area was annotated to substrate- or nutrient - solution background compounds as compared to 94.9 10.4 % and 80.4 8.9 % in the substrate and drainage compartment , respectively ( example in fig .
3detection of volatile rhizosphere chemicals collected in different compartments of the novel rhizosphere mesocosm ( a ) with substrate and taraxacum sect .
ruderalia plants and ( b ) without plants , but with substrate (= control ) ; example tic ( total ion chromatograms ) of volatiles .
the percentage of dandelion compound peak areas of all detected compounds ( unlabeled peaks were also found in plant - free controls ) was highest in the central compartment ( mean sd : 23.5 8.7 % ) compared to substrate and drainage compartments ( mean sd : 5.1 10.4 % and 19.6 8.9 % ) detection of volatile rhizosphere chemicals collected in different compartments of the novel rhizosphere mesocosm ( a ) with substrate and taraxacum sect .
ruderalia plants and ( b ) without plants , but with substrate (= control ) ; example tic ( total ion chromatograms ) of volatiles .
the percentage of dandelion compound peak areas of all detected compounds ( unlabeled peaks were also found in plant - free controls ) was highest in the central compartment ( mean sd : 23.5 8.7 % ) compared to substrate and drainage compartments ( mean sd : 5.1 10.4 % and 19.6 8.9 % ) sugars were found exclusively in the substrate compartment but were not present in the drainage compartment .
quantities of sugars varied in average by less than 20 % between the two technical replicates ( i.e. , substrate samples of one mesocosm ) and quantities of volatiles varied by less than 10 % between the three technical replicates ( i.e. , pieces of pdms tubing in one mesocosm compartment ) .
the variance among biological replicates was , however , relatively high ( table 4 ) . we determined recovery rates of commercially available volatiles from pdms and sugars by aqueous extraction from plant - free substrates with different sorptive capacity ( tables 1 and 2 ) , and from natural , untreated soil ( table 3 ) .
we used pdms tubes that were left unprotected and pdms tubes that were protected by a mesh , and thus , had no direct contact with the substrate . in general , adsorption capacities of substrates and recovery rates of volatiles did not follow a uniform trend . when using unprotected pdms tubes with direct contact to the substrate , the recovery of the volatiles methyl salicylate and 1-hexanol was better from sand compared to other substrates or even water ( 0.7 % of methyl salicylate and 0.4 % of 1-hexanol were recovered from sand , as compared to 0.1 % or 0 % from water , respectively ) .
other volatiles , such as - and -farnesene , -pinene and butyl acetate were recovered best from the clay mineral vermiculite ( 36 , 15 , 15.2 , and 2.8 % , respectively ) . except for -farnesene ,
none of the compounds was extracted best from the processed ( sieved and heated ) fraction of field soil ( table 1 ) or from untreated soil ( table 3 ) ; however , extractability from the processed field soil fraction and sand was similar for six of the 12 tested volatiles on pdms with contact to substrate . while 1-hexanol was neither recovered from the processed nor the untreated field soil when 100 ng were applied ( tables 1 and 3 ) , the recovery of methyl salicylate was similar from the processed and untreated field soils ( 0.2 % for both , respectively ) .
the recovered amounts of 1-hexanol and of the esters butyl acetate and hexyl acetate from field soil ( table 3 ) were marginal , even when high amounts were applied ( 1.1 , 0 , and 0 of the 5 g applied were recovered , respectively ) .
for some of the volatiles , the recovery rates from the processed fraction of field soil were much lower than from sand and vermiculite , i.e. , for - and -farnesene , -pinene , hexyl acetate , and 1-hexanol ( 0.9 , 0.7 , 7.2 , 0.3 , and 0 % , respectively , table 1 ) .
the highest recoveries from natural , unprocessed field soil treated with 5 g per compound were achieved for the ester methyl salicylate ( 44 ng ) ; the aldehydes benzaldehyde and cinnamal ( 7.6 and 12.1 ng , respectively ) , and the monoterpenes -pinene and linalool ( 61.2 and 5.5 ng , respectively ) ( table 3 ) . when protecting the pdms from contact to the substrate by a mesh cover , recovery rates were significantly improved for 9 , 3 , 7 , and 2 out of the 12 volatiles applied to water , sand , vermiculite , and the processed soil fraction , respectively .
for instance , the recovery rates of 1-hexanol and methyl salicylate were significantly improved for water , sand and vermiculite , and slightly improved for the processed soil fraction ( table 1 ) . protecting the pdms tubes improved the recovery of -pinene and butyl acetate from vermiculite ( 32.9 and 5.4 % as compared to 15.2 and 2.8 % , respectively ) and - and -farnesene from the field soil fraction ( from 0.9 and 0.7 to 2.1 % in both cases ) .
however , the recovery of several compounds remained the same with and without the mesh cover , i.e. , for - and - farnesene in vermiculite or all sesquiterpenoids in water .
some of the substances were even recovered at a reduced rate from protected compared to pdms tubes with substrate contact ( e.g. , recovery of benzaldehyde and linalool from the processed soil fraction decreased from 5.1 and 6.9 % to 2.6 and 3.3 % , respectively ) .
in contrast to the complex pattern of volatile recovery rates in the substrates tested , the recovery rates of water - extracted sugars followed a clear uniform trend : best recovery was in all cases achieved from water , followed by sand ; lowest recovery rates were obtained from vermiculite and the sieved and heated soil fraction ( table 2 ) .
total ion current chromatograms of derivatized sugars extracted from different substrates are shown in fig .
4determination of recovery rates of standard sugars applied to different substrates in a glass vial ( 10 g of each sugar per vial ) .
example ( total ion chromatograms ) tic of recovered compounds from sand , vermiculite , and a processed ( sieved and heated ) fraction of field soil compared to a standard mix in water ( n = 4 ) .
trimethylsilyl derivatives of arabinose , xylose and glucose were each represented by two peaks , a phenomenon that is commonly observed in gas chromatograms of silylated monosaccharides ( medeiros and simoneit 2007 ) determination of recovery rates of standard sugars applied to different substrates in a glass vial ( 10 g of each sugar per vial ) .
example ( total ion chromatograms ) tic of recovered compounds from sand , vermiculite , and a processed ( sieved and heated ) fraction of field soil compared to a standard mix in water ( n = 4 ) .
trimethylsilyl derivatives of arabinose , xylose and glucose were each represented by two peaks , a phenomenon that is commonly observed in gas chromatograms of silylated monosaccharides ( medeiros and simoneit 2007 )
this study demonstrates a convenient and non - destructive sampling technique for root - exuded metabolites .
the combination of three rhizosphere compartments separated by permeable but inert material allows simultaneous determination of root - derived hydrophobic volatiles as well as non - volatile , water - soluble compounds , such as sugars .
the recovery experiments indicate that the sampling method also is suitable for monoterpenes , ketones , and ( aromatic ) aldehydes .
hence , the set - up allows characterizing complex rhizosphere chemical profiles by use of silicone ( pdms ) tubes for sampling of rhizochemicals .
the robustness of the results ( 10 % variance among technical replicates within one sampling unit ) matched the one reported by kallenbach et al .
2014 ( 1025 % ) who sampled aboveground plant volatiles by using pdms tubes .
volatile sampling from roots was most efficient when pdms tubes were placed in the central mesocosm compartment in immediate vicinity of the rhizosphere , but without direct substrate - contact .
air- or water flows were not required , thus avoiding flooding , drying of the root zone , or root damage .
plant roots were not obviously damaged , however , the degree of disturbance due to their removal from mesocosms has not directly been tested . to avoid removal of plants
, it may be appealing to combine the application of pdms with the use of a so - called rhizobox , as presented by wenzel et al .
( 2001 ) . within the rhizobox , membrane systems separate growing roots and root hairs from surrounding substrates .
unfortunately , the establishment of substrate - specific rhizosphere bacterial communities ( marschner et al .
2004 ) and substrate - specific exudation of soluble and volatile compounds from roots ( rovira 1969 ) are suppressed in such rhizobox systems . to identify plant root - derived compounds that are potential belowground infochemicals , the here - presented experimental design offers the advantage that the plant roots remain in , or in close contact to the substrate .
it , therefore , represents a step forward in approaching natural conditions when analyzing rhizosphere infochemicals .
although background components could not be classified due to their presence in plant - free but substrate and nutrient containing mesocosms , it can not be excluded that
target components , listed in table 4 , are not exclusively plant - derived but also microbe - derived .
if studies aim to unravel directly plant - derived compounds rather than microbially produced ones , the experiments could be conducted under sterile conditions thereby avoiding microbial degradation of compounds .
for instance , application of ag containing agents during collection of root exudates avoids microbial degradation after sampling ( e.g. , gransee and wittenmayer 2000 ) .
interestingly , we found that undamaged dandelion plants emit a unique volatile blend , even without herbivore damage ( table 4 ) .
in addition to volatiles , two monosaccharides ( fructose , glucose ) and one disaccharide ( sucrose ) were detected in dandelion rhizosphere samples .
their detection in rhizospheres under non - sterile conditions indicates their availability to soil biota including herbivorous insects , but the amounts were low , i.e. , in the nanogram range .
however , it has to be considered that only a small fraction of the total substrate volume ( 15 out of 1200 ml ) per plant mesocosm was sampled for sugar analysis .
insects may detect extremely small amounts of volatile infochemicals in the nanogram range and below ( e.g. , review by leitch et al .
still , strong adsorption of root - released compounds to the substrate matrix ( see below ) may hinder diffusion and the establishment of gradients along which soil living animals may orient over a distance , or the attractiveness of some compounds may be masked by others ( reinecke et al .
thus , it remains to be determined whether the detected amounts of volatiles and sugars are sufficient to guide orientation of soil insects .
the mesocosm setup with plants grown in ( non - sterilized ) vermiculite also allowed detecting sugars in dandelion rhizospheres .
the release of sugars from roots has so far been shown mainly for plants grown in sterile hydroponics ( reviewed by badri and vivanco 2009 ; dakora and phillips 2002 ; dennis et al .
2010 ) . sugars often account for the largest proportion of root - emitted compounds ( azaizeh et al .
1995 ) but are rapidly degraded with a half - life of about 0.5 to 2 h in non - sterile rhizospheres ( jones and darrah 1996 ; ryan et al .
nevertheless , dandelion - feeding insect larvae have been shown to follow artificial gradients of sucrose in an arena assay ( eilers 2012 ) .
the release of sugars into the rhizosphere is a passive process , following a concentration gradient from plant tissue to soil solution ( jones and darrah 1993 ) .
however , continuous release of sugars from roots might be used for close - range orientation of edaphon species to roots .
the recovery of various volatiles and sugars , some of which were present also in dandelion mesocosm rhizospheres ( table 4 ) , was tested in water and various substrates applied to glass vials .
the potential of the substrate matrix to clog the pdms surface during volatile sampling also was assessed .
compounds belonging to the same chemical classes were surprisingly recovered to different extents from the same substrate , particularly when placing pdms directly into the substrate ( table 1 ) .
clear patterns of relationships between recovery rates , chemical structures , and substrate type became evident only for sugars , but not for volatiles .
as a general trend , high volatility and low water - solubility ( i.e. , -pinene , -elemene , - and - farnesene ) resulted in higher recovery from all substrates compared to compounds with high water - solubility , i.e. , high polarity ( e.g. , butyl acetate and 1-hexanol ) .
recovery rates of volatiles that were applied to processed ( sieved , heated ) soil ( table 1 ) and to untreated field soil ( table 3 ) were low when compared to other substrates , and the amount of compounds recovered was close to the detection limit .
first , the low amounts of pdms material and short sampling duration of only 1 h in these recovery analyses might have caused the low recovery rates .
the use of a higher amount of pdms material ( here : tube length of 3 mm ) , or extended sampling duration ( here : 1 h , but 120 h in mesocosm experiments ) may increase the recovery of sampled volatiles .
the comparison of recovery rates from processed to untreated natural soil ( see above ) indicates that bacterial degradation could indeed be a major factor determining the availability of volatiles to both chemical analysis and other soil biota .
third , clogging of the pdms surface by substrate particles may reduce recovery rates of rhizochemicals . in the recovery experiments ,
pdms tubes were either placed directly into the substrate , or substrate contact was avoided by protective gauze .
for most compounds and substrates , recovery rates tended to be similar or higher when pdms was protected by gauze .
similarly , the results from experiments in mesocosms indicate that some air space around the pdms tubes may be required for the sorption of root volatiles in sufficient quantities for analysis , as best results were obtained when placing the pdms tubes close to , but separated from the substrate , in the central compartment .
the used mesh width of 100 m in the recovery experiments may still allow smaller clay particles to interact or cover the pdms surface , or clog the mesh itself .
hence , the gauze properties ( material , mesh size , open area ) further could be optimized and adapted to specific substrates .
it contained organic matter , which was largely removed by sieving from the processed soil samples and had not been heated , i.e. , microorganisms were present .
as stated , bacterial degradation may be one reason for close to zero recovery from this substrate .
on the other hand , numerous publications substantiate that the organic soil fraction adsorbs organic compounds of environmental concern such as herbicides and volatile hydrocarbons ( e.g. chiou et al . 1979 ; shea 1989 ; balseiro - romero and monterroso 2013 ) .
extremely low recovery rates from untreated soil in experiments are in agreement with these reports and point at soil organic matter as an important sink for root - derived volatiles .
if indeed adsorption to soil organic matter retains root - derived volatiles , this suggests another hypothesis . living organic structures such as mycorrhizal networks act as transport routes for allelochemicals ( barto et al .
dead organic matter could in contrast act as a storage facility establishing lasting infochemical gradients that might inform foraging soil - inhabiting herbivores via taste or close range olfaction on plant roots in their vicinity .
in addition to an optimization and adaptation of the gauze properties and size of holes in the ptfe discs with regard to the used substrate , other studies investigating smaller or larger plants could adapt the mesocosm to the plant size .
furthermore , organisms interacting with the roots ( e.g. , rhizobacteria , mycorrhizal fungi , nematodes ) could be added to the mesocosm to address questions on organism - dependent rhizosphere chemicals .
water soluble components other than sugars ( i.e. , amino acids , organic acids , flavonoids , glucosinolates ) may be less prone to microbial degradation than sugars , and future studies need to unravel whether they occur not only in the substrate compartment , but also ( primarily or in similar proportions ) in the drainage compartment of a mesocosm .
in addition to solvent extraction as performed here , non - volatile compounds may be purified and concentrated by ion - exchange chromatography ( e.g. , using xad resin : tang and young 1982 ) .
we further expect that a simultaneous investigation of both volatile and water - soluble components also is feasible when plant rhizospheres are grown in sterile hydroponics ; in this case , extraction and analysis protocols similar to those established here could be applied .
hence , the presented microcosm provides a tool that is easily adjustable to various experimental requirements and , thus , might be useful to address a wide range of future questions on plant rhizosphere chemistry . | most studies on rhizosphere chemicals are carried out in substrate - free set - ups or in artificial substrates using sampling methods that require an air flow and may thus cause disturbance to the rhizosphere .
our study aimed to develop a simplified and inexpensive system that allows analysis of rhizosphere chemicals at experimentally less disturbed conditions .
we designed a mesocosm in which volatile rhizosphere chemicals were sampled passively ( by diffusion ) without air- and water flow on polydimethylsiloxane-(pdms ) tubes .
dandelion ( taraxacum sect .
ruderalia ) was used as model plant ; roots were left undamaged .
fifteen volatiles were retrieved from the sorptive material by thermal desorption for analysis by gas chromatography / mass spectrometry ( gc / ms ) .
furthermore , three sugars were collected from the rhizosphere substrate by aqueous extraction and derivatized prior to gc / ms analysis . in order to study how the quantity of detected rhizosphere compounds depends on the type of soil or substrate , we determined the matrix - dependent recovery of synthetic rhizosphere chemicals . furthermore , we compared sorption of volatiles on pdms tubes with and without direct contact to the substrate .
the results show that the newly designed mesocosm is suitable for low - invasive extraction of volatile and non - volatile compounds from rhizospheres .
we further highlight how strongly the type of substrate and contact of pdms tubes to the substrate affect the detectability of compounds from rhizospheres . |
a living kidney donor can improve the quality of life and offer a survival advantage to the recipient .
since 1998 , as per organ procurement and transplantation network data , there have been approximately 309,319 kidney transplantations and of these approximately 108,150 are from living kidney donors .
the shortage of living kidney donors is one of the central issues that prolongs transplantation .
this shortage could be due to the stringent criteria that must be applied to protect the health of the donor not only in the perioperative period but long term as well .
the evaluation of a donor presents unique issues that are addressed in a far different manner compared to patients undergoing other types of surgeries where the risk / benefit assessment is of a completely different nature .
a balance between doing no harm to the donor while doing good for the recipient must be achieved .
this risk / benefit analysis is not always straightforward and may not be readily apparent to the nontransplant evaluator .
the issues created by many donors can be complex and thought provoking and require thorough and detailed evaluations .
not only is a comprehensive review of their medical health necessary , but also a complete assessment of their social and psychosocial well being must be performed as well .
in addition , ethical and legal issues must be taken into consideration . aside from the general cardiovascular risk assessment which is needed for all donors as well as addressing immunological issues including blood typing and crossmatching ,
this article will focus on the multifaceted issues that arise as a donor is being evaluated , paying particular attention to the most common dilemmas and challenges that we face when evaluating them .
these issues can be divided into two general categories : assessments to protect the health and safety of the donor and donor assessments to protect the health and safety of the recipient .
both can be further subdivided into medical , renal , lifestyle , and psychosocial issues .
while diabetic patients and prediabetic patients regularly undergo surgery , the approach in the potential kidney donor is entirely different .
one concern is that the presence of a single kidney in a diabetic patient may result in an accelerated decline in kidney function if diabetic nephropathy develops .
most centers initiate screening with hemoglobin a1c ( a1c ) , and/or fasting blood glucose ( fbg ) . if a person has any risk factors for developing diabetes mellitus or any abnormalities in these initial tests , further evaluation with a two - hour oral glucose tolerance test ( 2h - ogtt ) is done .
a fasting blood glucose is performed after at least eight hours of no caloric intake .
a 2h - ogtt is done two hours after a 75 g oral glucose load is ingested . per american diabetes association guidelines ,
diagnosis of diabetes mellitus requires an a1c 6.5% , fasting glucose 126 mg / dl ( 7.0 mmol / l ) , 2h - ogtt 200 mg / dl ( 11.1 mmol / l ) , or a person with classic symptoms of hyperglycemia or a random plasma glucose 200 mg / dl ( 11.1 mmol / l ) . due to the long - term microvascular and macrovascular morbidity associated with diabetes mellitus and the potential to accelerate the course of diabetic nephropathy , these donors should not be allowed to donate .
the next group of donors includes those who do not meet the criteria for diabetes .
prediabetes group , is defined as having an a1c of 5.76.4% , fasting glucose of 100 mg / dl [ 5.6
mmol / l ] , or a 2h - ogtt value of 140 mg / dl [ 7.8
in addition to their increased risk of developing diabetes by 5% to 10% per year , being in this group is a risk factor for cardiovascular disease .
furthermore , this group has high associations with obesity , hyperlipidemia , metabolic syndrome , or hypertension . according to a 2007 survey of kidney transplant centers across the united states ,
49% of programs exclude donors based on elevated fasting blood glucose , however different cutoffs were used to define this . donors in this group
should be counseled appropriately on weight loss , diet modifications , and exercise and should be discouraged from donating . donors with a history of obesity , gestational diabetes , or a strong family history of diabetes mellitus should undergo testing with fasting glucose , 2-hr ogtt , and a1c . checking anti - islet cell antibodies in potential donors with a family history of diabetes can be done but its long - term cost effectiveness is unknown .
this needs to be carefully addressed in potential kidney donors as loss of renal mass in a hypertensive patient may increase the risk of parenchymal renal disease developing , or may accelerate the decline of kidney function in those patients in whom other types of kidney disease develop .
the joint national committee seventh report introduced a classification for hypertension which included the term prehypertension .
they defined prehypertension as blood pressure ranging from 120139 mmhg systolic and/or 8089 mmhg diastolic .
the classification in hypertensives is divided into stage 1 hypertension ( systolic blood pressure ( sbp ) 140159 mmhg and/or diastolic blood pressure ( dbp ) 9099 mmhg ) and stage 2 hypertension ( sbp greater than or equal to 160 mmhg and/or dbp greater than or equal to 100 mmhg ) . with this classification system ,
individuals with prehypertension could be easily identified and counseled due to their increase risk of developing hypertension and cardiovascular disease . for donors , at least two blood pressure readings on two separate occasions should be performed . a donor who has any elevated blood pressure reading
is generally sent for 24 hr ambulatory blood pressure monitoring to rule out white coat hypertension or to confirm the abnormal finding .
if that donor is on one medication , then he can continue as a donor as long as he does not have evidence of target end organ damage . according to the 2007 survey
, 41% of centers consider donors with well - controlled hypertension on one medication and only 8% will consider donors on two medications .
for the donors on one antihypertensive agent , documenting the absence of microabuminuria , left ventricular hypertrophy , or other cardiac disease , dyslipidemia , obesity , or ophthalmologic changes characteristic of hypertension is imperative .
in addition , this donor must demonstrate that his blood pressure had been well controlled for at least six months prior to evaluation and that he will have proper followup postdonation for his blood pressure .
racial variations in the development of hypertension and chronic kidney disease in african americans and hispanics have been described postdonation . because hypertension may have poorer outcomes in african americans and hispanics
, ethnicity may play a role in excluding a larger proportion of this population of hypertensive potential donors or even these potential donors with an increased risk of developing hypertension .
these donors are counseled in detail about lifestyle modification and their increased risk of developing hypertension and cardiovascular disease . of these who opt to donate , postnephrectomy followup by their primary care provider should be suggested .
individuals with a family history of hypertension are again counseled , as there is a genetic predisposition to developing hypertension .
both paternal and maternal hypertension are strongly associated with the development of hypertension during adult life .
advanced age ( defined as age above 65 ) may be associated with decreased recipient graft and patient survival .
biopsies from donors over the median age of 57 years showed age - related nephrosclerosis which may explain these inferior outcomes . nevertheless , especially due to the shortage of live donors , older donors should not be excluded .
conversations should be undertaken with both recipient and donors in regard to potential long - term disadvantages .
the majority of centers exclude donors younger than 18 years [ 5 , 11 ] .
young donors should be evaluated on an individual basis , and depending on their level of comprehension of kidney donation , their maturity level , their ability to provide voluntary and valid consent , and their general well being , they should be allowed to donate .
minors and guardians should be informed of the gaps in our knowledge regarding the long - term outcomes in minors with a solitary kidney .
in addition , given recent data on pregnancy and donation , all female donors of childbearing age who are contemplating pregnancy should be counseled on the potential risks of donating .
women postdonation may have an increased risk for gestational diabetes , gestational hypertension , preeclampsia , prematurity , and fetal loss .
another study confirmed the increased risk of preeclampsia and hypertensive disorders of pregnancy . although there is no clear evidence as to the timing of pregnancy postdonation , it is suggested that women should delay pregnancy until at least two months to a year postdonation to allow and assess for the degree of renal compensation and to assess baseline blood pressures , gfr , and microalbuminuria [ 16 , 17 ] .
a high level of surveillance and monitoring is suggested for all pregnant women postdonation . while removal of a kidney in a person with two normal functioning kidneys should not result in progressive kidney failure , a large reduction in nephron mass in a person who already has a decreased baseline glomerular filtration rate ( gfr ) can have adverse consequences . the most common approach to estimating gfr is with a 24-hour urine for creatinine clearance .
inadequate collection , low protein diet , and other factors may lead to low creatinine clearances in those with actually normal kidney function .
if the 24-hour creatinine clearance is borderline , further evaluation with radionuclide methods can be done .
the general cutoff for most centers is gfr of 80 ml / min/1.73 m [ 5 , 11 ] .
the majority of programs assess proteinuria with a 24-hour urine collection [ 5 , 11 ] .
other tests that have been used but may not be able to replace the 24-hour protein collection include the spot urine protein to creatinine ratio , spot urine albumin to creatinine ratio , urine microalbumin , or 24-hour urine for albumin excretion [ 5 , 11 ] . while most programs use > 300 mg / day in a 24-hour urine collection as the cutoff , a minority use > 150 mg / day as a cutoff .
all programs should perform a urinalysis looking for microscopic hematuria . obtaining any past urinalysis as well as repeating another one would be helpful to determine if the hematuria is transient or persistent .
hematuria is considered significant by one - third transplant centers if there are more than 3 red blood cells / high powered field ( rbc / hpf ) .
renal ultrasound which is the normal part of the donor workup should be able to reveal most structural causes of hematuria at the level of the kidney .
family history of renal disease including polycystic kidney disease , thin basement membrane disease , or alport 's syndrome or the presence of concomitant proteinuria can suggest an underlying glomerular cause of hematuria in the donor . in such cases , renal biopsy should likely be performed .
dysuria , urinary frequency , and the presence of pyuria can suggest a urinary tract infection . a donor with a family history of renal cell carcinoma or with a strong smoking history should be evaluated for malignancy with a urologic evaluation , urine cytology , and cystoscopy . in men ,
prostate issues should be ruled out . in women , making sure the urine specimen was not taken at the time of their menstruation is important .
. almost half of transplant centers will accept donors with > 10rbc / hpf if renal and urologic evaluations are negative .
correlations between a time zero biopsy and donor characteristics have been made leading to the following correlations : interstitial fibrosis and age , tubular atrophy and diastolic blood pressure and proteinuria , arteriolar hyalinosis and serum creatinine and estimated glomerular filtration rate , mesangial increase , and body mass index .
long - term allograft outcomes in donors with these histologic characteristics are still uncertain . more recently , in a study of deceased donors , a donor risk score using ten risk factors was calculated and used to prognosticate the chronic allograft damage index ( cadi ) using a histologic grading system .
this study showed a correlation between donor risk scores , greater degree of histologic lesions , and worse graft outcomes . whether kidney biopsy should be done in all potential living donors
requires further studies , taking into account risk of the biopsy itself , and sampling error .
definite indications may be persistent unexplained microscopic hematuria or proteinuria , or any other uncertainty of a possibility of underlying glomerular disease in the potential donor .
nephrolithiasis with development of obstruction of a single kidney can have catastrophic consequences yet a history of nephrolithiasis need not preclude donation as long as certain conditions are met .
the number of stones the potential donor has formed in the past needs to be determined .
for example , if a donor has a history of nephrolithiasis but the disease has been quiescent for years and there are no abnormalities in metabolic or radiologic testing , then donating can be acceptable .
this donor , however , should be informed that his lifetime risk for developing another stone can be 14% at 1 year , 35% at 5 years , and 52% at 10 years .
if , however , a donor has had recurrent episodes of nephrolithiasis , he or she should be discouraged from donating .
the other end of the spectrum includes those who do not give a history of nephrolithiasis but are found to be asymptomatic stone formers .
stones may be noted incidentally while doing screening renal ultrasounds as part of the donor evaluation .
these donors can likely be accepted but must be cautioned about their increased risk of developing another kidney stone in their solitary kidney during their lifetime .
some of these relate to the perioperative period where the risk of complications during this time is increased .
however , there are special long - term considerations as well and donation of a kidney in someone who is obese may enhance their risk of eventually developing kidney disease .
it should also be noted that the increasing prevalence of obesity may prove to be a substantial limiting factor to rates of kidney donation .
body mass index ( bmi ) is then calculated which is defined as weight in kilograms divided by height in meters squared .
those with bmi between 25 kg / m and 29.9 kg / m are considered overweight and those with bmi greater than 30 kg / m are considered obese .
kg / m ) , class ii ( bmi 35.039.9 kg / m ) , and class iii ( bmi greater than 40 kg / m ) .
higher baseline bmi has been shown to be an independent risk factor for end - stage renal disease .
the majority of transplant centers exclude potential donors with a bmi over 35 kg / m .
those with bmi over a 30 kg / m but less than 35 kg / m should be counseled on lifestyle modifications especially weight loss . in addition , they should be made aware of the long - term associations of obesity with the development of hypertension , diabetes mellitus , metabolic syndrome , and kidney disease , including end - stage renal disease .
smoking is an area of concern for a number of reasons but is currently not a contraindication to donate .
while the postoperative risks of poor wound healing and pulmonary infections in smokers are known , the long - term effects of smoking on the kidneys are uncertain . in a recent study
, smoking has been shown to be associated with an increased risk of developing proteinuria possibly through glomerular hyperfiltration .
on the contrary , in a study of living donors , smoking status was not a risk factor for long - term decline in glomerular filtration rate . with this in mind , due to the negative postoperative risks and extrarenal long - term effects of smoking
, donors should be counseled on smoking cessation and advised about the negative consequences of smoking .
donors should have behavioral screening in addition to infectious disease testing . sexual history and drug history
issues of concern are promiscuity ; men who have had sex with other men in the previous five years ; anyone exposed to or who has had sexual relations with someone at risk for , or with hiv , hcv , or hbv in the preceding year ; donors with a history of hemophilia ; donors with history of blood transfusions or any human - derived clotting factor concentrates ; intravenous , intramuscular , or subcutaneous drug use in the preceding five years ; men or women who have had sex in exchange for money or drugs in the previous five years ; incarceration .
a substantial disadvantage of these donors is the potential to transmit infectious diseases , namely , hbv , hcv , and hiv . depending on the situation , donors can be excluded if their risk to the recipient is very high .
if they are currently engaging in any high - risk behavior , they should be advised of their obligation to avoid the high - risk behavior .
if they can not avoid it , they should be given the choice to opt out of donating with a medical excuse .
due to the high risk , the recipient advocate and recipient should be made aware of the potential increased risk to the recipient for infectious diseases .
it is controversial at this point whether to repeat serology or perform nat for hbv , hcv , and hiv in high - risk donors two to four weeks prior to transplant .
the purpose of nat would be to detect the disease at the time period between infection and detectability of antibodies .
the downside of nat is that it costly , takes longer to perform and may result in false positives .
ideally , this evaluation should be done by a social worker and/or psychiatrist who has experience in evaluating donors .
the goal of this evaluation is to evaluate the psychological , social , and emotional stability of the potential donor .
in addition , the donor 's ability to give informed consent , their underlying motivation to donate , their relationship to the recipient , and any economic or social hardships that the donor may encounter during or after the process are sought after .
an in - depth evaluation of the donor 's current mental condition should be done .
any donor with a history of any psychiatric disorder , including major depressive disorder , bipolar disorder , generalized anxiety disorder , mood disorder , or any personality disorder should be assessed for any signs or symptoms of instability .
a donor should be asked about any childhood or adolescent problems that suggest mental health issues , suicidality , homocidality , inpatient psychiatric hospitalizations , or outpatient psychiatric evaluations .
a thorough assessment of any medications that they were taking or currently taking should be done , keeping in mind any medications that could potentially be nephrotoxic .
for example , if a donor has been stable on lithium carbonate and can not be taken off of it due to fear of relapse , that donor should likely not donate .
in addition , the evaluator must try to foresee any potential problems that can arise during the postdonation period , and any risk of recurrence .
in fragile donors , evaluation and counseling should extend to the donor during the postdonation period to assure psychological well being . testing focusing on the donor 's level of cooperation , eye contact , speech , language , mood ,
affect , thought process , thought content , memory , orientation , insight , and judgment should all be noted .
donors should not have any evidence of active drug or alcohol abuse . generally , a period of at least six months of abstinence is acceptable for a donor .
again , the high - risk donor can be identified at this time as well with a detailed and thorough sexual history and counseled in an appropriate manner .
the relationship between the donor and the recipient should be established , as well as what is motivating the donor to donate .
any suggestion of coercion or guilt should be further evaluated , making sure that the donor is not threatened in any way and that there is no gain either financial or personal .
in other words , the donor should be making decisions out of his free will .
other donor and family relationships should be addressed , looking for any signs of marital problems , or any hardships that the donor or evaluator can foresee as a result of donation .
if time off from work during the postdonation period could cause the donor to lose his job or lose major income , the donor should reconsider .
any out - of - pocket expenses for travel , lodging , or miscellaneous expenses related to transplant or inability to obtain sick leave should be discussed with the donor [ 12 , 28 ] .
education level and socioeconomic status should be evaluated , making sure that these do not play a role in the donation process .
these can sometimes be a barrier to informed consent or to the donor 's understanding of the entire process .
the donor 's religious beliefs should be evaluated as well , making sure that the donor is not being motivated by certain beliefs .
the donor should demonstrate understanding of the recipient 's illness and should understand the other options for the recipient , including deceased donor transplantation or dialysis
. he should be able to describe to the best of his knowledge why the recipient needs a transplant
. the donor should be able to demonstrate understanding of the nephrectomy including the risks and benefits to the recipient , the risks and benefits to the donor , the postoperative course , the short- and long term - outcomes associated with nephrectomy , the possibility of rejection , and the possibility that the donor may need the sacrificed organ in the future .
all preoperative workup , expenses , and postoperative surgical complications and expected outcomes should be fully disclosed to the donor .
the donor should demonstrate that he is free of coercion or any outside influence and that his decision to donate is voluntary [ 12 , 13 ] .
the donor must also be made aware of his right to reconsider donation at any point during the evaluation , of his right to opt out with a medical excuse , and of the confidentiality of the donation process .
kidney donation entails the risk of transmission of an infectious disease to a recipient who will be subjected to significant immunosuppression .
this testing should include human immunodeficiency virus 1 and 2 ( hiv-1 and hiv-2 ) , human t lymphotropic virus type-1 ( htlv - i ) and type-2 ( htlv - ii ) , hepatitis a , hepatitis b , hepatitis c , and syphilis .
in addition , donors are further tested for cytomegalovirus ( cmv ) , epstein - barr virus ( ebv ) , toxoplasmosis , herpes simplex virus ( hsv ) , and varicella zoster virus ( vzv ) .
screening for hiv should include serum enzyme - linked - immunosorbent assay ( elisa ) for hiv-1 and hiv-2 using u.s .
combination hiv-1/hiv-2 tests are available . screening for hepatitis b should include qualitative detection of hepatitis b surface antigen ( hbsag ) and antibodies to hepatitis b core antigen ( anti - hbc , igg , and igm ) and to hbsag ( anti - hbs ) . screening for hepatitis c can be with antibody testing to hepatitis c virus ( anti - hcv ) . whether nucleic acid testing ( nat ) should be done in addition to or in place of the above is unknown and case - dependent . per centers for disease control and prevention ( cdc ) recommendations
, all recipients should be counseled that negative tests for hbv , hcv , and hiv do not confer absence of disease , as the donor could have donated after infection but before serocoversion .
syphilis can be screened for with venereal disease research laboratory ( vdrl ) or rapid plasma reagin ( rpr ) .
having positive testing for hiv , hcv , hbsag , or anti - hbc , igm positivity would be a contraindication to donate .
hepatitis b core antibody igg positivity in an hbsag - negative donor carries a small risk of transmission and such donors should be excluded .
being anti - hbs positive is not a contraindication as this could signify vaccination or long - term immunity . here
having past infection with toxoplasmosis , cmv , vzv , hsv , or ebv is not a contraindication to donate .
syphilis does not contraindicate a person from donating as the risk of transmission is very low but the donor should be treated prior to donation and the recipient should be treated with penicillin posttransplant .
being htlv - i - positive should ideally be a contraindication given there could be increased risk for myelopathy and adult t cell leukemia in the immunosuppressed recipient .
given minimal chance of htlv - i / ii disease in the recipient approximately 12 months posttransplant , careful consideration can be given to these donors .
long - term outcomes , however , are not available and one should proceed with caution , after obtaining appropriate informed consent . screening for strongyloides , trypanosoma cruzi , west nile virus , tuberculosis with purified protein derivative testing or the interferon gamma release assay should be done on donors from endemic areas if these diseases are suspected .
kidney donation entails a potential risk of transmission of malignancy to the immunosuppressed recipient . with careful evaluation of donors , unintentional transmission of cancer
the evaluation of potential malignancy in the donor should begin with age appropriate health maintenance screening .
preventative task force are as follows : all women who are sexually active and have a cervix or those above 18 years of age should have pap smears . biennial screening mammography for women beginning at age 50 should be performed . screening for colorectal cancer using fecal occult blood testing , sigmoidoscopy , or colonoscopy , in adults ,
age 50 or above , whether symptomatic or not , prostate screening testing should be performed with digital rectal examination and prostate specific antigen ( psa ) . if there is a family history of malignancy , screening should be done at an earlier age after discussion with the donor . according to the disease transmission advisory committee ( dtac ) malignancy subcommittee ,
donors can be categorized into risk categories including no significant risk , minimal , low , intermediate , high , or unknown risk .
absolute contraindications and high - risk donors include those with a history of lung , breast , renal , or other urologic cancers , melanoma , neuroendocrine tumors , or small cell carcinoma any site of origin , monoclonal gammopathy , choriocarcinoma , testicular cancer , gastrointestinal , or hematologic malignancy such as leukemia or lymphoma [ 2 , 33 ] .
minimal risk category patients include those with basal cell carcinoma of the skin , squamous cell carcinoma of the skin , in situ cervical cancer , in situ vocal cord carcinoma , and certain thyroid cancers .
transmission risks are meant to provide a basis from which to suggest levels of concern , as true transmission frequency estimates have not yet been established [ 33 , 34 ] .
if a suspicious mass or nodule is found , biopsy should be performed and prompt frozen section examination should be done .
the final decision for transplantation should respect the donor 's autonomy and decision to donate , the recipient 's right to accept , and the respective transplant teams ' medical decision making to proceed for living donor transplantation . all parties involved must be free of coercion .
the risk and benefit analysis for the donor should be to provide more good than harm . if the opposite is the case , then transplantation should not proceed . | evaluation of the potential kidney donor is a complex activity that differs substantially from other types of preoperative assessments .
the well being of the donor , who derives no medical benefit from this surgery , must be assured in both the short term and long term , and the potential adverse consequences to the recipient must be determined as well .
the criteria that must be met for a person to donate a kidney are rigorous and include medical , social , psychosocial , ethical , and legal issues .
donor evaluation can be divided into assessments to protect the health and safety of the donor and assessments to protect the health and safety of the recipient .
this article provides an approach to evaluating a donor , focusing on the complex issues that an evaluator is faced with . a careful assessment of risks and benefits to both
the donor and recipient can lead to favorable outcomes . |