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clinical series of endovascular or surgical management in sca aneurysms were usually limited in the distal segment of sca aneurysms or incorporated in the literatures of basilar artery ( ba ) or posterior circulation aneurysms.1)4)7)10)13 - 16)18)22)24)25 ) posterior circulation aneurysms including sca aneurysms were regarded challenging to neurosurgeon due to narrow operative field , their intimate relationship to perforating vessels and cranial nerves , high morbidity and morbidity rates.2)18 ) although endovascular treatment of sca aneurysms has been reported to be effective and safe,10)17 ) sca aneurysms have peculiar characteristics different from those of basilar apex aneurysms , including relatively easy surgical accessibility to the lesions using a conventional pterional approach and poor perforating arteries related to the aneurysms .
we describe here our clinical experiences with endovascular and microsurgical treatment of sca , as well as classifying sca aneurysms by angiographic features .
between june 1997 and august 2007 , 33 patients with sca aneurysms ( 9 men , 24 women ; mean age 53.7 years ; range , 29 - 76 years ) underwent microsurgical ( n = 12 ) or endovascular treatment ( n = 21 ) in our institution .
sca was classified into anterior mesencephalic ( s1 ) , lateral mesencephalic ( s2 ) , cerebellomesencephalic ( s3 ) , and cortical ( s4 ) segment.19 ) of the 33 sca aneurysms , 30 ( 90.9% ) were in the junction between the sca and the ba , two in s1 and one in s2 - 3 .
patients with sca aneurysms plus other vascular malformations such as moyamoya disease or arteriovenous malformation were excluded from this analysis .
of the 33 patients , 22 presented with subarachnoid hemorrhage ( sah ) and one suffered from third nerve palsy due to mass effect .
the aneurysms were incidentally discovered in five patients and they were associated with coexisting aneurysms in five . in 14 patients ( 42.4% ) , we observed other aneurysms in addition to the sca aneurysm .
the 22 patients presenting with sah were classified by the hunt - hess scale;11 ) one patient was grade i , nine were grade ii , six were grade iii , and seven were grade iv .
based on conventional angiography obtained at the time of evaluation , the morphology of sca aneurysms were saccular in 32 patients and dissecting in one patient .
classification of these aneurysms by size and neck width showed that 15 small aneurysms were very small ( 5 mm ) with a small neck ( < 4 mm ) ; however , one was very small with a wide neck ( 4 mm ) , three were > 5 mm and < 10 mm in diameter with a small neck , six were small with wide necks , and seven were large ( 10 mm ) with wide necks . due to the loss of conventional radiography results in one patient
patients with sca aneurysms in the junction between sca and ba were categorized according to the main direction of the aneurysm sac and by the angle between the ba and an imaginary line from the center of the aneurysm sac to the mid - point of the neck as having lateral ( angle > 105 degrees ) , 2 ) lateral horizontal ( angle 90 degrees , 105 degrees ) , 3 ) lateral inferior ( angle < 90 degrees ) , and 4 ) posterior sca aneurysms .
the decision as to whether to treat surgically or by other methods was discussed among the physicians , including neurovascular surgeons and neurointerventionists .
factors indicating treatment of unruptured aneurysms included 1 ) multiple aneurysms ( n = 5 ) , 2 ) bleb formation ( n = 3 ) , 3 ) large aneurysm ( n = 2 ) , and cranial nerve palsy ( n = 1 ) . indications for microsurgery included anticipated endovascular difficulty because of aneurysm size or morphology ( n = 11 ) and attempted embolization ( n = 1 )
. indications for endovascular treatment included 1 ) poor neurological status ( m = 5 ) ; 2 ) expected difficulty of surgical access due to aneurysm location or morphological characteristics ( n = 5 ) ; 3 ) morphological characteristics indicating embolization ( n = 8) ; 4 ) attempted microsurgery ( n = 1 ) ; 5 ) the need for an alternative to microsurgery due to previous surgical clipping for other coexisting aneurysms ( n = 1 ) ; and 6 ) embolization trial ( n = 1 ) .
follow - up time of flight magnetic resonance angiography ( mra ) was performed six months after embolization if the lesions had been completely occluded .
subsequent follow - up mra was performed annually in patients with no interval changes compared with the previous evaluation .
conventional angiography was used to confirm coil compaction or regrowth of the aneurysm in patients with progression of incompletely occluded aneurysms .
radiological results without relationship to treatment alternatives included complete obliteration , residual neck and residual aneurysms.20)21 ) complete occlusion was defined as a complete absence of contrast agent filling of the aneurysms following embolization or microsurgery on conventional angiography or cta , residual neck was defined as lesions with residual filling of part of the neck , and residual sac was defined as incomplete occlusion of the aneurysm sac because of partial filling of the coils or incomplete surgical clipping .
patients were clinically followed - up mainly at out - patient clinical visits or by telephone interviews .
in classifying these 29 patients with sca aneurysms in the junction between the ba and the sca , based on our angiographic data we found that 20 patients ( 69.0% ) had lateral - superior aneurysms .
in addition , six patients ( 20.7% ) had lateral - horizontal , two ( 6.9% ) had lateral - inferior , and one ( 3.4% ) had a posterior sca ( fig .
one patient , who presented with sah , had a suspected sca aneurysm and no coexisting aneurysms on initial conventional angiogram .
rupture of a middle cerebral artery ( mca ) aneurysm was suggested in one patient with multiple aneurysms , of which the s1 aneurysm was not prominent on initial angiogram .
one patient , with a previous history of surgical clipping of another ruptured aneurysm and no definite sca aneurysm on initial angiogram , suffered from sah four years after surgery , with conventional angiography showing that the sca aneurysm had grown .
in contrast , residual neck was observed in one patient , residual sac in one patient , and the residual status was not evaluated in one patient who underwent simple brain ct due to a poor neurological condition .
nine patients underwent follow - up radiological evaluation with a mean of 33.3 months ( range 3 - 84 months ) .
causes of loss to radiological follow - up included poor neurological status in two patients and poor compliance in one patient .
all patients who underwent radiological follow - up had no evidence of aneurysm regrowth or recurrence
. clinical outcome at discharge in patients who underwent microsurgery included gos 5 in seven patients , gos 4 in one and gos 3 in four .
reasons for poor neurological outcome included 1 ) initially poor neurological condition without improvement ( n = 2 ) , 2 ) injury to the recurrent artery of heubner due to surgical clipping of coexisting anterior communicating artery aneurysm ( n = 1 ) , and 3 ) occipital infarction due to compromise of pca ( n = 1 ) . operation related morbidities included third nerve palsy in five patients , with most showing gradual recovery , huebner 's injury in one patient , and parent vessel compromise in one patient .
clinical follow - up was available for 11 patients at 3 to 84 months ( mean , 28.8 months ) .
clinical follow - up included gos 5 in eight patients , gos 4 in one , and gos 3 in two .
of the 21 patients treated endovascularly , 20 with saccular sca aneurysms showed occlusion of the aneurysm using a coil , whereas one patient with s2 - 3 dissecting aneurysm was treated by parent artery occlusion using a coil .
angiographic results of patients treated with embolization demonstrated complete occlusion in 12 patients ( 57.1% ) , residual neck in five ( 23.8% ) , and residual sac in four ( 19.0% ) .
twelve patients ( 60.0% ) underwent follow - up radiological evaluations at a mean 35.7 months after treatment ( range , 3 - 79 months ) .
reasons for loss to radiological follow - up included 1 ) clinical severe vasospasm - induced mortality ( n = 1 ) , 2 ) transfer to other hospitals ( n = 2 ) , 3 ) poor neurological status ( n = 2 ) , and 4 ) poor compliance ( n = 4 ) .
four patients with complete occlusion ( n = 2 ) or residual neck ( n = 2 ) were demonstrated coil compaction or further recanalization of the aneurysm .
additional embolization was performed in three patients with significant progressive coil compaction or recanalization of the aneurysm .
clinical outcomes at discharge in patients who underwent embolization included gos 5 in 12 patients , gos 4 in six , gos 3 in two , and gos 1 in one patient .
a 76-year - old female patient presenting with hunt - hess grade iv underwent external ventricular drainage and subsequent coil embolization .
another 64 year - old female patient presenting with hunt and hess grade i underwent surgical clipping of an mca aneurysm that was not ruptured on surgical assessment .
follow - up angiogram four days after microsurgery revealed growth of an s1 aneurysm ( size , 3 mm ) , previously thought not to be a definite aneurysm .
coil embolization resulted in pca and sca territory infarction opposite to embolization lesion , due mainly to a hypercoagulation period after microsurgery and partial coil exposure .
a 51 year - old female patient presented with hunt and hess grade ii underwent coil embolization and suffered from intractable clinical vasospasm resulting in death .
of these 20 patients , 17 ( 85% ) were available for clinical follow - up , at a mean of 32 months after treatment ( range , 3 to 79 months ) , with 14 showing gos 5 and three showing gos 4 .
the mean or median diameter of sca aneurysms according to classification was 1 ) lateral superior in 20 patients ( mean , 6.5 mm ) , 2 ) lateral horizontal in six ( median , 8.0 mm ) , and 3 ) lateral inferior in two ( median , 5.7 mm ) .
our classification of sca aneurysms , which has no clinical implications at present , may be helpful in the selection of microcatheters and may be a factor prognostic for the endovascular treatment of sca aneurysms . according to a general consensus on sca aneurysms , the presence of important perforating arteries and adjacent cranial nerves makes clipping of sca aneurysms and complications occur as basilar apex aneurysms .
however , perforators do not originate from the axillar of the p1 posterior cerebral artery ( pca ) or the shoulder of the sca , where the neck of an sca aneurysm is dissected.23 ) therefore , perforator infarctions , which may lead to morbidity at the basilar apex , may rarely develop during microsurgery of sca aneurysms .
we did not observe any perforator infarctions related to sca aneurysms , although the number of patients undergoing microsurgery was small .
since the proximal sca is intimately related to cranial nerves iii , iv , and v,19 ) aneurysms on the sca may present with palsies of these nerves.1)7)17 ) one patient , with a 2.5 mm sized lateral - superior type of sca aneurysm , showed third cranial nerve palsy , but this finding is relatively rare.1)7)17 ) cranial nerve palsy may be affected by the direction or growth pattern of the aneurysm rather than its size .
complete or incomplete third nerve palsies developed in five patients who underwent microsurgery but recovered completely in all patients available for clinical follow - up .
occlusion of the sca appears to be well tolerated.4)9 ) one of the 33 sca aneurysms was dissecting and was located in the s2 - 3 segment of the artery .
treatment of this aneurysm by complete trapping using a coil resulted in no neurological deficits .
collateral blood flow and a paucity of perforating arteries from the s1 and s2 segments may limit ischemic infarctions , usually resulting in good outcomes.4 - 6)8)18 ) sca aneurysms lie off the midline below the basilar bifurcation and are generally directed laterally .
this orientation may improve visualization in the trajectory of the trans - sylvian approach and coil embolization of the aneurysms relative to basilar apex aneurysms , resulting in low procedural morbidity and mortality rates .
procedural morbidities related to sca aneurysm were observed in one patient each treated with microsurgery or embolization and its rate was relatively low compared with rates in basilar apex aneurysms.12 ) it is generally recommended that unruptured aneurysms < 7 mm in size with no associated symptoms be managed conservatively.3 ) while a detailed natural history of unruptured sca aneurysms has not been reported due to their rare incidence , it may differ from that of aneurysms at other locations .
it may therefore be impossible to apply treatment guidelines for unruptured aneurysms of other locations to the treatment of sca aneurysms .
the mean diameter of the 22 sca aneurysms accompanied by sah was 7 mm ( range , 1.8 to 15.6 mm ) , whereas nine patients ( 41.0% ) had ruptured sca aneurysms < 5 mm .
poor clinical outcomes in three of four neurologically disabled patients resulted from initially poor clinical conditions .
no procedural morbidities related to sca aneurysms developed in any patient who underwent microsurgery or embolization of the non - ruptured aneurysms .
one patient with a previous history of surgical clipping of another ruptured aneurysm and pre - aneurysmal dilatation in the junction between the ba and sca on initial angiogram suffered from sah four years after surgery .
a conventional angiogram revealed the growth of the sca aneurysm with rupture ( diameter , 5.7 mm ) .
active guideline of treatment or tight image follow - up may be necessary in the unruptured sca aneurysms .
sca aneurysms , which differ from basilar apex aneurysms , have peculiar characteristics , including a paucity of perforators , abundant collateral supplies and off the midline location , accompanied by relatively low procedural morbidity and no procedural mortality without treatment modalities .
more active guidelines for the treatment of non - ruptured sca aneurysms may be required based on our clinical experience of sca aneurysms accompanied by sah . | objectivesuperior cerebellar artery ( sca ) aneurysms are regarded as being as difficult to treat surgically as posterior circulation aneurysms .
we describe here a series of 33 of these aneurysms treated with microsurgery or embolization.methodsbetween june 1997 and august 2007 , 33 patients ( 9 men , 24 women ; age , 29 to 76 years ) with sca aneurysms underwent microsurgical ( n = 12 ) or endovascular ( n = 21 ) treatment .
twenty two patients presented with subarachnoid hemorrhage .
thirty aneurysms were located in the junction between the sca and the basilar artery ( ba ) , two in the proximal sca ( s1 ) and one in the distal sca ( s2 - 3).resultsof the 29 sca aneurysms , located in the junction between the sca and ba , which were available on conventional angiography , 20 were lateral - superior , six lateral - horizontal , two lateral inferior , and one posterior type .
of the 12 patients treated microsurgically , eight had clinically excellent or good outcomes .
causes of poor outcomes included initial poor clinical status ( n = 2 ) , infarction due to parent artery compromise ( n = 1 ) , and artery of heubner injury due to surgery for a coexisting anterior communicating artery aneurysm ( n = 1 ) . of the 21 patients treated endovascularly , 17 had clinical good or excellent outcomes .
causes of clinically poor outcomes included initial poor clinical status ( n = 2 ) and infarction due to thrombosis of exposed coil mesh ( n = 1 ) .
one patient underwent embolization resulted in death due to vasospasm .
three patients required additional embolization for coil compaction.conclusionthere was no morbidity related to perforator injury , regardless of the treatment modality .
embolization or microsurgery is an effective modality , with relatively low procedural morbidity and mortality rates . |
metabolic syndrome refers to a collection of issues including visceral obesity , elevated blood glucose levels , dyslipidemia ( elevated fasting triglycerides and low high - density lipoprotein ( hdl ) cholesterol levels ) , and hypertension .
it leads to an increase in the risk of developing of cardiovascular disease ( cvd ) , type 2 diabetes , and cancer .
thus , effective strategies preventing metabolic syndrome are required to decrease the incidence of diseases and promote healthy aging .
the development of metabolic syndrome is primary caused by a sedentary lifestyle and overnutrition ; however , genetic characteristics are also involved to some extent .
daily physical activity directly influences obesity and metabolic syndrome associated with the metabolic function of skeletal muscle ( figure 1 ) .
thus , dietary exercise , along with adequate diet , is well known to be one of the major preventive therapies against metabolic syndrome . in the last few decades
, it has been shown , in epidemiological and experimental studies , that exercise reduces obesity , improves glucose tolerance , and decreases the risk of diabetes and cvd .
the effects of exercise are brought about by elevated energy consumption , improvement of insulin sensitivity , and a reduction in inflammation .
a single bout of exercise drastically changes various physiological parameters such as hormone production , blood flow , and the activity of the nervous system , in addition to altering the expression / activity of certain genes and proteins in the skeletal muscle .
this paper reviews evidence regarding the influence of exercise on the progress of metabolic syndrome along with its underlying molecular mechanisms .
it particularly focuses on the skeletal muscle , a major metabolic organ , and describes the benefits of functional food factors combined with exercise therapy .
sedentary behavior and persistent low levels of physical activity are known to induce progression toward metabolic syndrome , type 2 diabetes , and cvd [ 15 ] .
energy consumption depends on the intensity and amount of physical activity ; therefore , a sedentary life tends to result in a positive energy balance and leads to an accumulation of body fat .
adipose tissue secretes bioactive factors , adipocytokines , such as tumor necrosis factor alpha ( tnf- ) , plasminogen activator inhibitor , and resistin , into circulation .
it has been considered that there is aclose relationship between these adipocytokines and health problems , such as obesity and metabolic and cardiovascular disorders , as they cause insulin resistance , injury to the endothelium , and inflammation .
this decrease may be due to atrophy of skeletal muscle , a major energy - consuming tissue in the body .
it has been reported that the loss of fat - free mass with inactivity and age explains a reduction in the resting metabolic rate ( rmr ) .
muscle atrophy may be due to both muscle fiber atrophy and loss of complete muscle fibers [ 8 , 9 ] due to several factors including the apoptosis of muscle cells , decreased differentiation of satellite cells , and reduced protein levels as a result of decreased protein synthesis and increased protein degradation .
the activity of enzymes involved in aerobic metabolism and glucose uptake in muscle is also decreased by inactivity and aging .
laboratory studies have shown that significant protein degradation is seen within 2 days of muscle immobilization leading to loss of muscle mass within 1 week .
insulin - induced glucose uptake into the muscle is also reduced along with a reduction in its signaling pathway , within 2 days of immobilization . at the same time , the activity of lipoprotein lipase ( lpl ) , a protein important for controlling plasma triglyceride catabolism hdl cholesterol and other metabolic risk factors was lost .
consistent with the decrease in lpl function , the clearance of plasma triglyceride by skeletal muscle was significantly decreased and plasma hdl cholesterol concentration declined .
recently , it has been established that low - grade continuous inflammation and oxidative stress are associated with metabolic disorders and cvd [ 1618 ] .
low levels of physical inactivity lead to chronic inflammation and oxidative stress in the skeletal muscle , the circulatory system , and other tissues .
some adipocytokines , such as tnf- and interleukin-6 ( il-6 ) , which are secreted from accumulated visceral adipose tissue can cause this inflammation .
insulin - induced activation of the insulin receptor ( ir ) , phosphatidylinositol 3-kinase ( pi3k ) , and akt is prevented along with ib kinase ( ikk ) activation and degradation of ib in the muscle tissue [ 1921 ] .
furthermore , ikk- silencing prevents tnf--induced impairments in insulin action on akt phosphorylation and glucose uptake .
growing evidence suggests that additional adipocytokines including resistin , fatty - acid - binding protein ( fabp ) , and visfatin have also induced insulin resistance associated with inflammation [ 2325 ] .
in addition , a reduction of circulating adiponectin , an adipocytokine with anti - inflammatory properties , occurs with obesity and leads to insulin resistance in skeletal muscle and liver [ 2628 ] .
a recent study clearly showed that adiponectin directly improves glucose and lipid metabolism along with mitochondria biogenesis and activation of the key metabolic modulators via adiponectin receptor 1 ( adipor-1 ) in skeletal muscle .
this inflammation of skeletal muscle is caused by muscle inactivity even when body fat is low .
indeed , expression of tnf- in skeletal muscle is elevated with insulin resistance in human .
this indicates that tnf- generated from not only other cells but also myocytes disturbs insulin signaling .
an increased level of oxidation of lipids , dna , and proteins is also observed in muscles of sedentary subjects compared to that of active subjects [ 3134 ] .
furthermore , continuous activation of intracellular oxidative - stress - sensitive factors such as the nuclear factor - kappa b ( nf-b ) and mitogen - activated protein kinase is seen in the muscle of sedentary men [ 35 , 36 ] .
oxidative - stress is also strongly associated with development of insulin resistance in the skeletal muscle .
thus , oxidative - stress - induced insulin resistance in muscle leads to the initiation of diabetes and potentially late diabetic complications .
it is without doubt that insulin sensitivity is inversely correlated with the plasma levels of free radicals in diabetic patients [ 17 , 18 ] .
several studies have demonstrated that reactive oxygen species ( ros ) impair insulin - mediated glucose uptake and storage by disrupting signaling control points such as glycogen synthase kinase-3 , akt phosphorylation , and actin remodeling [ 3739 ] .
in addition , we have recently found that 3-nitrotyrosine modification of adenylate kinase 1 ( ak1 ) , a key enzyme in synthesis ; equilibration ; regulation of adenine nucleotides is elevated in older muscle and that the modification of ak1 is involved in the impairment of glucose uptake via inhibition of amp - activated protein kinase ( ampk ) .
furthermore , it has been suggested that metabolic regulation of adiponectin is associated with reduction of oxidative - stress in skeletal muscle .
inflammatory cytokines and ros are also associated with protein degradation via activation of the ubiquitin - proteasome pathway .
have revealed that the addition of oxidants and tnf- to myotubes increases protein degradation rates , ubiquitination of proteins such as myosin , and expression of the main components of the ubiquitin - proteasome pathway [ 4042 ] .
muscle ring finger 1 ( murf1 ) and atrogin-1 have been identified as the ubiquitin ligases whose activities increase during atrophy [ 43 , 44 ] .
nf-b can regulate the ubiquitin - proteasome proteolytic pathway through the induction of murf1 and proteasome expression [ 4547 ] .
furthermore , it has been shown that the 20s proteasome can selectively degrade oxidatively modified proteins without ubiquitination [ 48 , 49 ] .
these observations suggest that protein degradation could be the link between oxidative stress , inflammatory cascade , and muscle atrophy .
in fact , hyperactivity of nf-b and the ubiquitin - proteasome pathway has been identified as a major cause of aged - related muscle atrophy [ 50 , 51 ] .
many large cohort studies have found that higher level of physical activity is associated with reduced risk of developing diabetes and cvd [ 5258 ] .
one of the first major trials to examine the effect of physical activity was the university of pennsylvania alumni health study . in this study , the level of physical activity was found to be inversely related to the development of type 2 diabetes in 202 male subjects .
the incidence declined by 6% for each 500 kcal increment in energy expenditure from less than 500 to 3500 kcal .
in addition , the osaka health survey showed , in 444 men , that regular exercise , at least once a week , reduced the relative risk of type 2 diabetes to 0.75 compared with in those engaging in exercise less often .
subjects who engaged in intense exercise at least once a week , at weekends , exhibited further reduction of the multiple - adjusted relative risk of type 2 diabetes to 0.55 compared with sedentary subjects .
cardiorespiratory fitness is also protective against diabetes and metabolic syndrome [ 60 , 61 ] .
studies lasting for 312 months involving exercise sessions of 3060 min in a week have shown that regular exercise reduces fat mass and improves insulin sensitivity without dietary caloric restriction in overweight men and women [ 6266 ] . moreover ,
regular exercise decreases plasma levels of triglyceride and hdl cholesterol and lowers blood pressure [ 6769 ] .
consistent with the improvement , regular exercise reduces circulating adipocytokines , such as resistin , visfatin , and fabp , involving development in metabolic disorder and inflammation [ 7072 ] . on the other hand ,
several studies have suggested that the improvement in insulin sensitivity induced by regular exercise is not mediated by changes in plasma adiponectin [ 7375 ] .
however , the ratio of high - molecular - weight form to total adiponectin was increased by regular exercise and there was a positive correlation between the increase of the adiponectin ratio and the improvement of insulin sensitivity in older insulin - resistant adults .
in addition , it has been shown that muscle adipor-1 is elevated in response to physical exercise , which elevates metabolic signal transduction of adiponectin and then improves oxidative metabolism .
therefore , the regulation of these adipocytokines including adiponectin likely contributes to the prevention of metabolic syndrome by daily exercise .
aerobic training has traditionally been adopted as the main form of exercise therapy in epidemiological and laboratory studies . however ,
recently , the inclusion of resistance training as an integral part of an exercise therapy program has recently been endorsed by the american heart association , the american college of sports medicine , and the american diabetes association .
cross - sectional studies have shown that muscle mass is inversely associated with mortality and the prevalence of metabolic syndrome , independent of cardiorespiratory fitness levels . even in the elderly , resistance training increases muscle mass from , 7.4% to 10.0% , along with muscle strength after 1016 weeks [ 83 , 84 ] .
one study demonstrated that twice - weekly resistance training could prevent age - associated loss of lean body mass ( lbm ) and rmr , which is closely correlated to losses in lbm .
resistance training contributes to an elevation in rmr as a result of a greater muscle protein anabolism .
theoretically , a gain of 1 kg in muscle mass should result in an increase of approximately 21 kcal in rmr .
thus , resistance training , when sustained over years or decades , translates into clinically important differences in daily energy expenditure and can prevent age - associated fat gains .
however , resistance training can rather inhibit lbm loss when combined with dietary restriction in antimetabolic syndrome therapy .
in several randomized control trials , where obese men were randomly assigned to either a diet - only group or a diet with resistance training group , lbm was preserved by exercise training [ 8795 ] .
furthermore , there is a strong support for the notion that resistance training is at least as effective as aerobic training in reducing some major cvd risk factors
. findings from several studies demonstrate that resistance training significantly decreases glycosylated hemoglobin levels in people with an abnormal glucose metabolism and has a tendency to improve lipoprotein - lipid profiles [ 90 , 96 , 97 ] , independent of changes in body weight or composition .
in addition , several factors such as hormones , growth factors , oxidative stress , and heat stress also affect signaling .
numerous human and animal studies have shown that metabolic improvement due to exercise occurs along with changes in the expression / activity of muscle proteins and alterations in their mrna transcription ( figure 2 ) .
a single exercise bout improves glucose uptake in skeletal muscles via insulin - dependent and insulin - independent signal transduction mechanisms [ 98101 ] .
this effect is observed for several hours after exercise , often persisting until the next day .
the increase in glucose uptake is caused by the translocation of the glucose transporter 4 ( glut4 ) to the plasma membrane after activation of the ir / pi3k / akt signaling pathway [ 102 , 103 ] .
elevated ampk activity and intracellular calcium levels can also induce glut4 translocation independent of the insulin signaling pathway [ 104 , 105 ] .
in addition , a considerable amount of attention has been given to the peroxisome proliferator - activated receptor gamma coactivator-1 alpha ( pgc-1 ) as a target for the prevention or treatment of metabolic syndrome .
pgc-1 has been shown to have the central role in a family of transcriptional coactivators involved in aerobic metabolism and is activated by exercise .
activation of pgc-1 alters the metabolic phenotype through interaction with nuclear respiratory factor and the peroxisome proliferator - activated receptor [ 107 , 108 ] .
improved understanding of the activation of the pgc-1 protein by exercise has implications beyond improving athletic performance [ 109 , 110 ]
. it may be target for the treatment of various diseases such as the mitochondrial myopathies and diabetes [ 111113 ] .
the activity / expression of lpl , a protein important for controlling triglyceride catabolism and cholesterol levels in plasma , is also elevated for 3 to 22 hours after exercise [ 114 , 115 ] .
these beneficial adaptations persist in humans and animals who perform regular exercise , independent of the acute effects of exercise .
initiation of protein synthesis appears to be regulated by the akt / the mammalian target of rapamycin ( mtor ) signaling proteins .
akt phosphorylation regulates the catabolic pathway by preventing the induction of muscle - specific ubiquitin ligases such as atrogin-1 and murf1 and activates the anabolic pathway by phosphorylating mtor .
mtor then initiates translation via the activation of translation regulators p70 and the eukaryotic initiation factor-4e ( eif-4e ) complex , following phosphorylation of eif-4e - binding protein-1 , one of the main translational inhibitors .
in addition , regular exercise can inhibit the apoptotic signaling pathway ; this is associated with reducing oxidative stress and inflammation , and it results in the preservation of skeletal muscle fibers .
this inhibitory effect results from the upregulation of antiapoptotic mediators , such as b - cell lymphoma / leukemia , x - linked inhibitors of apoptosis , and heat shock protein 70 and from the downregulation of proapoptotic mediators such as caspase-3 and bax [ 119 , 120 ] .
a decrease in tnf- , a factor that accelerates the caspase cascade , may be involved in antiapoptotic signaling , as regular exercise blunts tnf- expression in aged muscle [ 121 , 122 ] .
furthermore , the inhibition of tnf- and oxidative stress would lead to a reduction in age - related muscle dysfunction , including prevention of protein degradation and impaired glucose uptake .
the study of the mechanisms underlying the effects of exercise has been enhanced by the analysis of the function of micrornas ( mirnas ) in recent years .
the mirnas are small noncoding sections of rna that regulate gene expression by degrading mrna molecules or , more frequently in mammalian cells , inhibiting their translation [ 123 , 124 ] .
it has been suggested that mirna - mediated gene regulation is a part of the fundamental mechanism of posttranscriptional regulation and may have diverse functional effects .
several of these mirnas have been suggested to have a role in a wide range of biological processes , including development , cell death , carcinogenesis , and response to stress [ 126129 ] .
some mirnas , including mir-1 , mir-133 , and mir-206 , which are referred to as myomirs , have also been suggested to act as modulators of skeletal muscle function [ 130 , 131 ] .
we along with other researchers have shown that physical activity elevates muscle metabolism associated with pgc-1 , via regulation of some mirnas [ 132 , 133 ] .
furthermore , there is growing evidence that secreted proteins derived from muscle , also known as myokines , are elevated in plasma in response to exercise and regulate various functions of other organs .
muscle - derived il-6 is well known as a representative myokine that is markedly elevated in muscle and secreted into plasma following muscle contraction [ 134 , 135 ] .
this myokine could mediate some of the exercise - induced metabolic changes and anti - inflammatory effects in other organs such as the liver , adipose tissue , and blood vessels .
subsequently , other muscle - derived proteins , il-15 , and fibroblast growth factor-21 have been reported to regulate nutrient metabolism in other organs .
furthermore , it has been shown that brain - derived neurotrophic factor is produced in skeletal muscle in response to contraction and has been suggested to increase fat oxidation in skeletal muscle in both an autocrine and paracrine fashion .
the potential effects of several food factors on muscle lipid metabolism in exercise have been investigated .
some of them have been found to accelerate lipid utilization ; however , their efficacy is still controversial . a rate - limiting step in lipid metabolism in myocytes is the entry of long - chain fatty acids into the mitochondria .
carnitine palmitoyltransferase i ( cpt i ) , located on the outer mitochondrial membrane , plays an important role in the entry of fatty acids into mitochondria .
we found that a novel antioxidant astaxanthin limits the oxidative modification of cpt i by hexanoyl lysine .
this causes , along with elevated cpt i activity during exercise , acceleration of the reduction in body fat due to exercise training .
ikeuchi et al . also showed that astaxanthin supplementation accelerates a catabolism in body fat along with a reduction of blood lactate .
catechin , one of the polyphenols contained in japanese green tea , accelerates the utilization of fatty acids as an energy source for skeletal muscle contraction during exercise .
it has been suggested that the effect of catechin is related to the enhancement of -oxidation activity and the level of fatty acid translocase / cd36 mrna in muscle .
intake of -lipoic acid , combined with endurance exercise training , further accelerates glucose uptake and activity of the insulin signaling pathway compared with training alone .
this leads to an increase in circulating free fatty acids and a further improvement in endurance .
capsaicin , obtained from hot red peppers , is likely to enhance fat metabolism by increasing lipolytic hormones and promoting fat oxidation in the skeletal muscle .
however , growing evidence indicates that a large dose of dietary antioxidants prevents the adaptation generally seen as a result of regular exercise .
vitamin c supplementation ( 1 g / day , for 6 weeks ) decreases the improvement of vo2 max associated with training .
antioxidant vitamins and n - acetylcysteine reduced mitochondria biogenesis associated with the expression of pgc-1 , a key modulator of aerobic metabolism in skeletal muscle cells [ 143 , 144 ] .
more recently , in a prospective randomized intervention study , a combination of vitamin c ( 1000 mg / day ) and vitamin e ( 400 iu / day ) has been shown to inhibit the improvement of insulin sensitivity and elevation of plasma adiponectin along with the cancellation of pgc-1 induction in response to exercise training in healthy men .
these observations suggest that the intake of antioxidants is not always beneficial in counteracting muscle dysfunction related with inactivity and that oxidative stress is involved in signal transduction of exercise adaptation .
it has been shown that the protein requirement for subjects performing resistance training is higher than that for sedentary individuals .
the daily recommended protein intake is estimated to be 1.41.8 g / kg for those performing resistance exercise when the intake of calories and carbohydrates is adequate .
however , it is not only the amount of protein , but also the timing of intake that is important for the efficient building of muscle . eating protein immediately after exercise is more effective , in terms of increasing protein synthesis , compared with several hours later .
the cross - sectional area of the quadriceps muscle after a 12-week resistance training program is greater .
additionally , protein synthesis in muscle can be promoted by intake of proteins combined with carbohydrates via the actions of insulin as this accelerates the increase in muscle mass and strength . in addition
, it has been reported that the intake of amino acids and peptides is beneficial .
amino acids are not only utilized in the synthesis of muscle protein but can also exert a variety of physiological effects .
attention has been focused on the effects of branched - chain amino acids ( bcaas ) , including valine , leucine , and isoleucine , which are known to be found in relatively high concentrations in both muscle proteins and food proteins .
therefore , when bcaas are not supplied in the diet , muscle protein is catabolized to obtain them . furthermore ,
dietary bcaas modulate muscle protein metabolism to promote the synthesis and inhibit the degradation of proteins .
it is the most abundant free amino acid in muscle tissue , and its intake leads to an increase in myocyte volume and results in the stimulation of muscle growth .
glutamine is also found at relatively high concentrations in many other human tissues and has an important homeostatic role .
therefore , during catabolic states such as exercise , glutamine is released from skeletal muscle into the plasma to be utilized for maintenance of the glutamine level in other tissues .
-hydroxy--methylbutyrate ( hmb ) is a metabolite of the branched - chain amino acid leucine .
it increases muscle mass by inhibiting the degradation of protein via its influence on the metabolism of branched - chain amino acids . a meta - analysis supported the use of hmb while performing resistance exercise to augment lbm and strength .
several studies have demonstrated that an intake of hmb for at least 4 weeks achieved a greater increase in lbm or muscle power output .
previously , numerous studies on prevention and development of metabolic syndrome have focused on effects of adipose tissue . on the other hand , because skeletal muscle plays an important role as a metabolic organ in the development of metabolic syndrome , recently these relationships have been established , as described in this paper .
daily exercise habit and physical fitness level are associated with a reduction in risk of metabolic syndrome , independently of body fat level .
therefore , although adequate diet is important for prevention and treatment of metabolic syndrome , regular exercise it needed for the therapies . | a sedentary lifestyle can cause metabolic syndrome to develop .
metabolic syndrome is associated with metabolic function in the skeletal muscle , a major consumer of nutrients . dietary exercise , along with an adequate diet , is reported to be one of the major preventive therapies for metabolic syndrome ; exercise improves the metabolic capacity of muscles and prevents the loss of muscle mass .
epidemiological studies have shown that physical activity reduces the risk of various common diseases such as cardiovascular disease , diabetes , and cancer ; it also helps in reducing visceral adipose tissue .
in addition , laboratory studies have demonstrated the mechanisms underlying the benefits of single - bout and regular exercise .
exercise regulates the expression / activity of proteins associated with metabolic and anabolic signaling in muscle , leading to a change in phenotype .
the extent of these changes depends on the intensity , the duration , and the frequency of the exercise .
the effect of exercise is also partly due to a decrease in inflammation , which has been shown to be closely related to the development of various diseases .
furthermore , it has been suggested that several phytochemicals contained in natural foods can improve nutrient metabolism and prevent protein degradation in the muscle . |
gastric cancer is the fourth most common malignancy worldwide , and most common malignancy in korea .
although survival time has been lengthened , the life expectancy of advanced disease still remains within one year . for patients with advanced gastric carcinoma or recurrent cancer , the response rate ( rr ) for first - line chemotherapy ranges from 33 - 55% , and
the survival time following chemotherapy is superior to that of patients given best supportive care . despite poorer survival benefit and tolerance of second - line chemotherapy ,
about 20% of patients with failure of first - line chemotherapy receive second - line chemotherapy .
asian studies have reported a rr of second - line chemotherapy as 5 - 25% and progression - free survival ( pfs ) of 5.2 - 8.8 months .
many clinicians consider second - line chemotherapy after failure of first - line chemotherapy for patients with advanced gastric carcinoma .
there is a controversy about the benefit of sequential salvage therapy further to second - line .
however , with prolonged survival time of patients and tolerable toxicity of drugs , there has been renewed interest in chemotherapy after failure of second - line treatments
. it has been our empiric observation that some patients maintain good performance after failure of second - line chemotherapy , and live longer with salvage chemotherapy than other patients with supportive care .
the aim of the present study was to clarify the relationship between the sequence of second- and third - line chemotherapy regimen including taxane and irinotecan with survival of patients , and to identify clinical characteristics of patients with better response to salvage chemotherapy than others .
we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 .
patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) .
exclusion criteria were the presence of other severe medical illness , central nervous system metastasis , another active malignancy , or history of anaphylaxis to drugs .
clinical information included gender , age at diagnosis , ecog performance status , pathologic differentiation , lauren classification , coexisting peritoneal or hepatic metastases , date of chemotherapy , and number of cycles .
in addition , laboratory data including hemoglobin , serum albumin , carcinoembryonic antigen ( cea ) , and cancer antigen ( ca ) 19 - 9 were also reviewed .
sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively .
patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens .
arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy .
patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days .
cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 mg / m / day ) for 2 days .
irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 .
response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected .
a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging .
tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd .
the rr was defined as proportion of the patients with responses of cr or pr out of all patients .
overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date .
time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date .
the kaplan - meier method was used for survival analysis , and survival curves were compared assessed using the log - rank test .
following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . for variables with a probability p - value0.3 in univariate analysis , multivariate analysis was also performed using the cox 's proportional hazard regression model ( two - sided ) .
we retrospectively evaluated the medical records of advanced or recurred gastric carcinoma patients who were diagnosed and treated at gangnam severance hospital between november 2004 and july 2010 .
patients who met the following criteria were eligible : histologically proven advanced gastric adenocarcinoma with metastatic or recurrent disease ; received third - line chemotherapy at gangnam severance hospital using folinic acid , 5-fluorouracil ( 5-fu ) and oxaliplatin ( folfox ) or 5-fu+cisplatin ( fp ) , folinic acid , 5-fu and irinotecan ( folfiri ) , paclitaxel or docetaxel with 5-fu , with or without epirubicin ; measurable lesion according to the response evaluation criteria in solid tumors ( recist ) or a non - measurable lesion , assessed using either computed tomography ( ct ) or magnetic resonance imaging ; 18 - 75 years of age ; eastern cooperative oncology group ( ecog ) performance status2 ; life expectancy of at least 3 months ; and adequate hematologic function , evident as absolute neutrophil count1500/l , platelet count100,000/l , hepatic function ( total bilirubin2 times the upper normal limit [ unl ] , serum transaminase level2 times the unl ) and renal function ( serum creatine1.5 times the unl ) .
exclusion criteria were the presence of other severe medical illness , central nervous system metastasis , another active malignancy , or history of anaphylaxis to drugs .
clinical information included gender , age at diagnosis , ecog performance status , pathologic differentiation , lauren classification , coexisting peritoneal or hepatic metastases , date of chemotherapy , and number of cycles .
in addition , laboratory data including hemoglobin , serum albumin , carcinoembryonic antigen ( cea ) , and cancer antigen ( ca ) 19 - 9 were also reviewed .
sequential chemotherapy was defined as chemotherapy using more than two different chemotherapeutic regimens performed consecutively .
patients were assigned to either arm a or arm b , according to the sequence of three chemotherapeutic regimens .
arm a comprised folfox or fp for first - line chemotherapy ; folfiri for second - line chemotherapy ; and docetaxel or paclitaxel , leucovorin , and 5-fu ( with or without epirubicin ) for third - line chemotherapy . in arm b , patients were treated with folfox or fp for first - line chemotherapy ; docetaxel or paclitaxel , leucovorin , and fu with or without epirubicin for second - line chemotherapy ; and folfiri for third - line chemotherapy .
patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days .
cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000
irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 .
response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected .
a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging .
tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd .
the rr was defined as proportion of the patients with responses of cr or pr out of all patients .
patients received oxaliplatin ( 100 mg / m in 500 ml normal saline ) or 5% dextrose water over 2 hours followed by leucovorin ( 100 mg / m ) over 2 hours on day 1 and continuous infusion of 5-fu 1,000 mg / m / day for 2 days .
cisplatin ( 70 mg / m in 500 ml normal saline ) or 5% dextrose water was intravenously administered over 2 hours on day 1 followed by continuous infusion of 5-fu ( 1,000 mg / m / day ) for 2 days .
irinotecan ( 150 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours on followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
docetaxel ( 75 mg / m in 500 ml normal saline ) or paclitaxel ( 175 mg / m in 500 ml normal saline ) was intravenously administered over 2 hours followed by leucovorin ( 100 mg / m over 2 hours ) on day 1 and 5-fu ( 1,000
for some patients , epirubicin ( 50 mg / m in 100 ml normal saline ) was intravenously administered over 30 minutes on day 2 .
response was assessed after two cycles of chemotherapy and when disease progression was clinically suspected .
a measurable lesion was defined as a lesion with the longest diameter 10 mm in any dimension assessed by spiral ct imaging .
tumor response was evaluated according to the recist guidelines as follows : complete response ( cr ) if all target lesions disappeared , partial response ( pr ) if the sum of diameters of the target lesions decreased by at least 30% , progressive disease ( pd ) if the sum of the longest diameters of the target lesions increased by at least 20% and one or more new lesions appeared , and stable disease ( sd ) for responses that were neither pr nor pd .
the rr was defined as proportion of the patients with responses of cr or pr out of all patients .
overall survival ( os ) was defined as the interval from the first date of chemotherapy to death or the last follow - up date .
time to progression ( ttp ) was defined as the time from the first date of chemotherapy to the time of progression of the disease or the last follow - up date .
the kaplan - meier method was used for survival analysis , and survival curves were compared assessed using the log - rank test .
following variables were included in univariate analysis : age at diagnosis , gender , performance status by ecog criteria , pathologic differentiation , coexisting peritoneal or hepatic metastases , type of chemotherapeutic regimen , hemoglobin , serum albumin , cea , ca19 - 9 , and ttp of first- or second - line chemotherapy . for variables with a probability p - value0.3 in univariate analysis , multivariate analysis was also performed using the cox 's proportional hazard regression model ( two - sided ) .
between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital .
the median age was 52.5 years ( range , 29 to 70 years ) , and 34 patients ( 68% ) were male .
forty subjects ( 80% ) had good performance status ( ecog 0 - 1 ) .
seven patients had diffuse type , 13 patients had interstitial type , and the other two patients had mixed type adenocarcinoma . no significant difference except for peritoneal carcinomatosis was observed .
fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered .
median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig .
1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy .
the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively .
1 ) . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) .
for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) .
each ttp of the two groups were 2.2 months in arm a and 2.7 months in arm b ( p=0.297 ) .
in addition , we analyzed clinicopathologic characteristics of the patients . patients with poorly - differentiated adenocarcinoma or signet ring cell feature showed poorer prognosis than others . furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) .
the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy .
in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) .
in second - line chemotherapy , dcr of arm a was 38.9% , and that of arm b was 53.1% ( p=0.333 ) .
in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) .
twenty - one patients did not show any response ( pr or cr ) to any of the three sequential chemotherapies .
the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders .
between november 2004 and july 2010 , 50 patients with gastric carcinoma were treated with a sequential chemotherapy protocol , based only on folfox or fp , folfiri , and paclitaxel or docetaxel , at gangnam severance hospital .
the median age was 52.5 years ( range , 29 to 70 years ) , and 34 patients ( 68% ) were male .
forty subjects ( 80% ) had good performance status ( ecog 0 - 1 ) .
seven patients had diffuse type , 13 patients had interstitial type , and the other two patients had mixed type adenocarcinoma . no significant difference except for peritoneal carcinomatosis was observed .
fifty patients received median fourth - line chemotherapy ( range , 3 to 10 ) , and a median of 18 cycles ( range , 4 to 60 cycles ) were administered .
median os of all patients was 16.0 months ( 95% confidence interval [ ci ] , 13.6 to 18.3 months ) ( fig .
1 ) , and median ttp was 5.0 months ( 95% ci , 3.9 to 6.1 months ) in first - line chemotherapy , 2.4 months ( 95% ci , 1.6 to 3.2 months ) in second - line chemotherapy , and 2.5 months ( 95% ci , 1.8 to 3.3 months ) in third - line chemotherapy .
the median os time of arm a and arm b was 17.1 months ( 95% ci , 10.7 to 23.6 months ) , and 15.4 months ( range , 13.8 to 17.1 months ) , respectively .
1 ) . ttp of arm a and arm b with first - line chemotherapy was 5.5 months ( 95% ci , 1.2 to 9.8 months ) and 4.8 months ( 95% ci , 3.8 to 5.8 months ) , respectively ( p=0.064 ) .
for the second - line chemotherapy , ttp of arm a and arm b was 2.7 months and 2.2 months , respectively ( p=0.850 ) .
each ttp of the two groups were 2.2 months in arm a and 2.7 months in arm b ( p=0.297 ) .
patients with poorly - differentiated adenocarcinoma or signet ring cell feature showed poorer prognosis than others .
furthermore , elevated cea level and shorter ttp of first - line chemotherapy were significantly related with shorter survival times ( table 2 ) .
the rr was 50% for first - line chemotherapy , 8% for second - line chemotherapy , and 8.5% for third - line chemotherapy .
in second - line chemotherapy , rr was 11.1% in arm a and 6.3% in arm b. in third - line chemotherapy , rr was 6.3% in arm a and 9.7% in arm b. the disease control rate ( dcr ) which was determined by the disease status ( cr , pr , or sd ) controlled by treatment , of first - line chemotherapy was 88.9% in arm a and 78.1% in arm b ( p=0.342 ) . in second - line chemotherapy , dcr of arm
in third - line chemotherapy , dcr was 25% in arm a and 41.9% in arm b ( p=0.252 ) .
twenty - one patients did not show any response ( pr or cr ) to any of the three sequential chemotherapies .
the median os was 20.0 months ( 95% ci , 2.4 to 37.5 months ) in the good - responders and 13.9 months ( 95% ci , 12.0 to 15.8 months ) in the poor - responders .
it has generally been accepted that palliative chemotherapy can significantly prolong survival of patients with advanced gastric carcinoma , longer than best supportive care .
platinum - based chemotherapy is widely - used as first - line chemotherapy for advanced gastric cancer .
given the reported tolerance and survival benefits of second - line chemotherapy , studies have sought to identify the benefit and adequate regimen of second - line chemotherapy .
the current study evaluated two groups of gastric cancer patients who received third - line chemotherapy .
previous studies reported the median pfs of 2.5 - 3.3 months and the median os of 5.3 - 8.7 months in gastric cancer patients who received second - line chemotherapy [ 6,8 - 10 ] . in this study ,
the median pfs was 2.4 months ( 95% ci , 1.6 to 3.2 months ) for patients treated with second - line chemotherapy , similar to the previous report .
the median os was 16.0 months ( 95% ci , 13.6 to 18.3 months ) , and 32.0% of patients survived after 2 years .
these times were slightly longer than that of historical control . we suggest that this is due to sequential treatment of the patients .
this study included a restricted population with a performance status that permitted the use of third - line chemotherapy after failure of second - line chemotherapy .
therefore , this result was subject to limitation in comparison with other survival data of patients without third - line chemotherapy . nevertheless , based on 2.5 months of median ttp in third - line chemotherapy ,
the authors suggest that third - line chemotherapy would provide a survival benefit for patients with favorable performance status .
in many phase ii clinical trials of second - line chemotherapy for advanced gastric cancer , rr was about 20.8% , and dcr was about 51% .
in the present study , rr for second - line chemotherapy was 8% and dcr was 48% .
there is still little information to support the provision of a second - line chemotherapeutic regimen after failure of platinum - based first - line chemotherapy .
reported that sequential chemotherapy appears to be both a feasible and effective treatment for advanced gastric cancer .
their sequential protocol was s-1 based chemotherapy for first - line chemotherapy , a low dose irinotecan / cisplatin for second - line chemotherapy , and paclitaxel for third - line chemotherapy .
thirty - two gastric cancer patients showed relatively long median survival ( 17.4 months ; 95% ci , 323 to 723 days ) .
this result shows that a consecutive use of chemotherapy has a substantial impact on os of gastric cancer patients .
these facts also demonstrate that second- and third - line chemotherapy is necessary to treat advanced gastric cancer .
many clinicians consider the folfiri regimen and paclitaxel or docetaxel with 5-fu regimen as second - line chemotherapy after failure of platinum - based first line chemotherapy .
numerous physicians suggested that folfiri is tolerable and has a modest effect as a second - line chemotherapy for gastric carcinoma . in an asian study ,
the median pfs was 3.2 months , and the median os was 9.1 months . in another study ,
rr was 18.2% , median os was 5.1 months , and median ttp was 2.3 months . on the other hand , the tolerance and effectiveness of taxane - based chemotherapy apparent in several studies ( median ttp , 2.6 - 3.9 months ; median os , 7.2 to 10.1 months ) warrants its use as second - line chemotherapy in advanced gastric carcinoma .
kim et al . reported that overall rr and dcr for patients with docetaxel / cisplatin followed by folfiri and patients with folfiri followed by a docetaxel / cisplatin regimen showed no significant difference .
however , to the best of our knowledge , survival benefits according to sequence of regimens of second - line and third - line chemotherapy after failure of first line chemotherapy have not been reported . in this study , we analyzed 50 patients treated with either folfox or fp chemotherapy followed by folfiri , and paclitaxel or docetaxel , or folfox or fp chemotherapy followed by paclitaxel or docetaxel , and folfiri regimen .
however , we found some patients responded better to chemotherapy , and their os was longer than the others .
previous investigators suggested that low hemoglobin level , low serum albumin level , high cea level , poor performance status [ 8 - 10,16,17 ] , shorter ttp of first - line chemotherapy , shorter ttp of second - line chemotherapy , and poor differentiation of tumor are poor prognostic factors for patients with gastric carcinoma under sequential chemotherapy .
we analyzed the previously reported factors and found some differences in survival according to histologic differentiation , elevated cea level , and ttp of first line chemotherapy in this study .
there has been a controversy regarding the influence of the histological feature of signet ring cell on the prognosis of gastric carcinoma .
some researchers reported poorer prognosis of advanced gastric carcinoma with signet ring cell histology , while better prognosis of early gastric carcinoma with signet ring cell histology has been reported . in this study , gastric adenocarcinoma with
poorly differentiated or signet ring cell features had poorer response to sequential chemotherapy , and had shorter median os than well or moderately differentiated ones .
concerning cea level , some investigators reported the relationship of serum cea level with tumor invasion , lymph node metastasis , and higher recurrence rate .
herein , we suggest that differences in gene and protein expressions are other prognostic values that should be taken into account . in a recent study ,
we suppose that there are some genetic differences in diffuse gastric adenocarcinoma and that the different genetic defect influences a poorer response to sequential chemotherapy . furthermore , cea is a widely used tumor marker , and the cea gene has been widely - used as a tumor - specific promoter and target of treatment over the last decade .
we propose that the difference in response could also be attributed to the differences in gene expression profiles between the two groups . in recent years , it has become clear that a pharmacogenic approach is a potential tool for optimizing treatment for several human tumors .
the ability of genetic polymorphisms to influence pharmacogenetics of 5-fu , platinum derivatives , anthracyclines , irinotecan , and docetaxel in gastric cancer has been reported .
the use of multiple gene analyses in the development of individualized gastric cancer chemotherapies could be helpful in selecting patients who are more likely to benefit better to a particular therapy . the possible prediction of cancer outcome from gene expression classifiers , sets of genes , or signatures associated with prognosis together with classification rules has been suggested . limitations of this study include the retrospective study design and small patient pool .
nevertheless , the results of this study provides evidence that third - line chemotherapy , including folfox or fp , folfiri , paclitaxel or docetaxel regimen , helps to improve os of patients , regardless of the sequence of use of taxane and irinotecan .
in addition , os seems to be poorly affected by poorly differentiated or signet ring cell feature of adenocarcinoma , elevated cea level , and shorter ttp of first - line chemotherapy .
different chemotherapy - responses to locally advanced or metastatic gastric carcinoma treated with second and third - line chemotherapy using irinotecan or taxane could be attributed not to sequence of chemotherapeutic regimen , but to patients ' different clinical features underlying different gene expression profiles .
future studies will be required to investigate the impact of genetic difference on response to sequential chemotherapy . | purposethe aims of this study are to find out whether the sequence of chemotherapeutic regimens including second- and third - line taxane and irinotecan influences the survival of patients with unresectable gastric carcinoma and to identify clinical characteristics of patients with improved response.materials and methodsfifty gastric carcinoma patients who were treated by third - line sequential chemotherapy between november 2004 and july 2010 were enrolled in this study . their overall survival ( os ) and time to progression ( ttp )
were set up as primary and secondary end points . for the sequence of chemotherapy regimen , two arms were used .
arm a was defined as 5-fluorouracil ( 5-fu)+cisplatin ( fp ) or folinic acid , 5-fu and oxaliplati ( folfox ) , followed by folinic acid , 5-fu and irinotecan ( folfiri ) , and paclitaxel or docetaxel plus 5-fu , with or without epirubicin .
arm b was defined as fp or folfox , followed by paclitaxel or docetaxel plus 5-fu , and folfiri.resultsthe median os of all patients was 16.0 months ( 95% confidence interval , 13.6 to 18.3 months ) , which is longer than historical control of patients who did not receive third - line chemotherapy .
the sequence of second and third - line regimen , including irinotecan and taxane , did not present significant difference in os or ttp after failure of 5-fu with platinum chemotherapy . in survival analysis of patients ' clinicopathologic characteristics
, poor prognosis was shown in patients with poorly differentiated histologic features , elevated serum carcinoembryonic level , and shorter ttp of first line chemotherapy.conclusionit is possible for patients to respond differently to chemotherapy due to differences in clinical features and underlying gene expression profiles .
development of individualized chemotherapy regimens based on gene expression profiles is warranted . |
dental erosion is defined as the loss of tooth structure resulted from chemical dissolution , which was produced by non - bacterial produced acid.1 the etiology of dental erosion is multi - factorial in origin .
it can be produced by intrinsic or extrinsic acid exposure based upon the patient history .
the intrinsic factor of dental erosion is the attacking of gastric acid on the teeth in the case of vomiting and gastro - esophageal reflex.2 the extrinsic origin is caused by consumption of acidic beverages , medication , or the occupational environment.3 it has been reported that the exposure of the tooth surface to acidic beverages with ph below 5.5 can cause enamel dissolution .
its progression can be accelerated by the combination of frequent acidic diet consumption and exposure to potentially high erosive environments.4 previous clinical reports showed that poorly chlorinated swimming pools have been a contributing factor to dental erosion among competitive swimmers .
an insufficient buffering system of the swimming pool resulted in low ph values of the pool water that could cause erosion of teeth .
the maxillary and mandibular anterior teeth are commonly affected by dental erosion caused by swimming .
the characteristics of the typical lesion are severe loss of enamel creating diastema , definite margins of the lesion resembling anterior veneer preparation , wear of the incisal edges resulting in the reduction of clinical crown height , and tooth hypersensitivity.1,5,6 definitive treatment for this type of lesion aims to eliminate hypersensitivity , replace lost tooth structure , and establish proper esthetics .
several treatment modalities can be performed depending on the severity of the tooth surface loss such as direct composite resin restorations , laminate veneers , and complete coverage crowns.6 to gain desirable esthetic outcomes , metal - free restorative materials are usually recommended to restore the extensively damaged anterior teeth.7 the objective of this case report is to describe the clinical appearance and treatment approach in a case of severe dental erosion caused by swimming in a poorly maintained swimming pool with low ph pool water .
a 48-year - old man presented with a chief complaint of hypersensitivity and unacceptable esthetic appearance of his anterior teeth .
the patient went to see a dentist at a private clinic to receive dental treatment for his anterior teeth erosion .
the dentist performed history taking and found no obvious intrinsic or extrinsic causes of the erosive lesion .
thus , the patient was referred to the prosthodontic clinic at the faculty of dentistry , prince of songkla university , thailand for consultation and joint management .
medical and dental history taking were performed to verify whether the patient was exposed to any cause of dental erosion or not .
the patient mentioned that he rarely consumed acidic foods or beverages and had no sign of acid regurgitation presented .
apparently , it was found that the patient had been routinely swimming for 3 months . since then
he stopped swimming 2 weeks ago because of the extreme sensitivity of the anterior teeth .
the patient was asked to collect a sample of pool water from the swimming pool that he routinely went to swim in .
the sample of pool water was evaluated for a ph value using a ph meter ( precisa , ph900 , precisa gravimetrics ag , dietikon , switzerland ) .
the ph value for the pool water was 4.5 , which was lower than the critical value for enamel dissolution.8 moreover , the patient reported that he spent more than an hour swimming each time , which meant that he had been exposed to low ph pool water for more than an hour every day over 3 months . intra - oral examination
revealed loss of enamel on the labial and incisal surface of the maxillary anterior teeth .
four mandibular incisor teeth presented with loss of enamel on the facial surface and there was excessive reduction of the incisal edges .
diastemas appeared between most of the teeth , and the incisal plane was uneven ( fig .
the patient 's esthetic appearance was affected by the irregular incisal edge position of the remaining tooth structure .
the erosive lesion appeared with well - defined finish lines along the free gingival margins resembling veneer preparation ( fig .
2 ) . there was no dental caries which existed and his periodontal health was fine .
the occlusal function and vertical dimension of occlusion appeared to be normal since the patient had adequate and stable posterior stops .
a significant finding in this case was the maxillary - mandibular relationship , which appeared to have anterior open bite due to the loss of tooth structure .
however , the patient had no difficulty in pronouncing an " s " and " f " sound during function . during the comprehensive oral examination , the patient complained that his teeth were extremely sensitive to air blow .
the diagnosis in this case was severe anterior tooth surface loss , resulting from long - term exposure to low ph in swimming pool water .
the treatment goals in this case were to restore the damaged tooth structure and reestablish the esthetics of the smile .
definitive treatment modalities for excessive tooth erosion include direct composite resin restorations , porcelain laminated veneers , and complete coverage crowns . since the remaining tooth structures were insufficient to support composite resin restorations and porcelain - laminated veneers and the optimal clinical crown height needed to be restored , complete coverage metal - free crown restorations were the treatment of choice in this case for long - term durability and esthetic satisfaction .
irreversible hydrocolloid impressions were performed for fabricating diagnostic casts . a face - bow transfer was made and the casts were mounted in maximum intercuspation on a semi - adjustable articulator ( denar mark ii , whip mix , louisville , ky , usa ) .
diagnostic wax - up was made on the casts with the optimal tooth proportion and with 1 mm horizontal and vertical overlap .
the wax - up was transferred to the patient 's mouth using the direct intra - oral mock - up procedure with bis - acryl provisional material ( protemp 4 , 3 m espe , seefeld , germany ) .
3 ) was adjusted to obtain desirable esthetics , phonetics and function , and it was subsequently duplicated for using as a guideline for fabricating provisional restorations .
the splinted provisional restorations were made with heat - polymerized polymethyl methacrylate ( namilon , american tooth industries , oxnard , ca , usa ) prior to the tooth preparation .
it was decided to use a novel hybrid ceramic ( vita enamic , vita , zahnfabrik , germany ) for constructing metal - free restorations in this case .
vita enamic is an interpenetrating phase composite material that combines the properties of ceramic and polymer .
this material consists of a fine microstructure of feldspathic ceramic matrix and an acrylate - based polymer network . as a result ,
wear characteristics , flexural properties , and elasticity of this material are similar to dentin.9,10 the minimum thickness required to ensure clinical success of restorations made from vita enamic is at least 0.8 mm circumferentially .
therefore , the amount of tooth reduction required is less than tooth preparation designs for other all - ceramic materials.10 maxillary incisor and canine teeth were prepared with a 1 mm wide chamfer finish line and mandibular incisor teeth with a 0.8 mm wide chamfer finish line .
internal line angles were rounded in all prepared teeth . a double cord gingival retraction was performed and
final impressions were taken using a polyether impression material ( impregum soft , 3 m espe , seefeld , germany ) .
splinted provisional restorations were relined and temporarily cemented ( temp - bond ne , kerr , orange , ca , usa ) .
the impressions were poured in type iv stone ( silky rock , whip mix , louisville , ky , usa ) and pindexed .
laboratory prescription for vita enamic crowns were completed and sent to an outside laboratory ( t dental lab , bangkok , thailand ) .
4 ) . clinical try - in and adjustment were performed prior to permanent cementation .
the occlusion was assessed to eliminate occlusal interferences , especially on the maxillary and mandibular canines .
the edge - to - edge occlusal relationship was established in this case to avoid excessive coronal crown height in both maxillary and mandibular anterior teeth .
the intaglio surfaces of the restorations were treated with 5% hydrofluoric acid ( vita ceramics etch , vita , zahnfabrik , germany ) for 60 seconds according to the manufacturer 's instruction .
the etching gel was completely removed using water spray and the restorations were dried for 20 seconds .
self - adhesive resin cement ( rely x unicem , 3 m espe , seefeld , germany ) was used to cement restoration following the manufacturer 's recommendations .
excess cement was removed completely and the special care instructions for ceramic restorations were given to the patient ( fig .
since there have been insufficient clinical report regarding using hybrid ceramic in this type of case , long - term follow - up will be performed for at least 3 years . further long - term clinical performance will be provided .
previous clinical case reports and in vitro studies demonstrated that low ph swimming pool water was a contributing factor of enamel erosion on the anterior teeth.5,6 this patient had a significant habit of frequent swimming in an improperly chlorinated swimming pool .
the clinical findings in this case fitted with the clinical characteristics of dental erosion caused by exposure to poor - chlorinated pool water .
full coverage restorations were selected to achieve desirable esthetics , function , and long - term durability of the restorations .
a novel hybrid ceramic ( vita enamic ) was used as a material of choice to restore this patient .
it was chosen because the material requires less tooth preparation and it is more conservative than tooth preparation for other all - ceramic materials .
also , this material consists of a complex interconnected polymer - ceramic microstructure that helps in resistance to chipping.9,10 the restorations were cemented with adhesive resin cement since the hybrid ceramic , which contains glassy matrix , can adhesively bonded to the resin cement by using silane solution .
previous study showed that adhesively cemented glass - containing restorations exhibited higher fracture loads compared to the restorations , which were cemented with conventional cements.11 the esthetic outcome of hybrid ceramic restorations appeared to be satisfying for the patient .
also , the patient was instructed to understand the significant influence of swimming in a poorly - maintained swimming pool which could cause severe tooth surface loss , and therefore would further damage his teeth .
the diagnosis and treatment procedure of a patient with severe anterior teeth erosion from swimming have been described .
hybrid ceramic restorations were fabricated to overcome extreme sensitivity and obtain desirable esthetics , phonetics , and function . | this clinical report presents the clinical appearance and treatment approach in a case of excessive anterior teeth erosion resulted from swimming in a poorly - chlorinated swimming pool .
clinical findings revealed tooth sensitivity , severe enamel erosion resembling veneer preparations , and the presence of anterior open bite . a novel hybrid ceramic ( vita enamic ) was chosen for fabricating full - coverage crowns for this patient .
after 6-months follow - up , the tooth sensitivity disappeared and the patient was satisfied with esthetic outcome .
the hybrid ceramic restorations can be recommended with no complications . |
to explore the molecular subtypes of leiomyosarcoma ( lms ) , paraffin blocks of 99 lms cases from 1991 to 2012 from nine hospitals ( stanford hospital , brigham and women 's hospital , mckay - dee hospital center , st .
luke 's hospital , baptist health medical center , ingalls hospital , vancouver general hospital , hospital de la santa creu i sant pau and alta bates summit medical center ) , were collected with irb approval and a waiver of consent due to the archival nature of the specimens .
the total rna was extracted for these cases and subsequently analyzed by 3 end rna sequencing .
consensus clustering was used to determine the optimal number of subtypes and silhouette analysis was then performed to measure the confidence of subtype assignment per case . to test the reproducibility of molecular subtype classification from the 3seq dataset
, the expression profiles ( rnaseq data ) of 82 additional lms cases were downloaded from the cancer genome atlas ( tcga ) database and analyzed in an identical way to the 3seq data .
the lms cases used in this study were formalin - fixed , paraffin - embedded ( ffpe ) tissues .
after lms ffpe blocks were obtained , two experienced pathologists ( one from stanford university and one from brigham and women 's hospital ) assessed and circled the regions comprised of lms tumor cells .
multiple 2 mm - diameter cores from the areas circled were re - embedded longitudinally into new paraffin blocks , and were sectioned and re - evaluated for the second time by h&e staining to ensure the purity of the samples . only cores with 90% of tumor cells were processed for subsequent rna extraction .
the total rna of the lms cases was extracted using recoverall total nucleic acid isolation kit ( ambion , cat # 1975 ) .
the quality of total rna was assessed by agarose gel electrophoresis and used to determine the amount of time necessary for shearing of the total rna by heat in first strand buffer ( invitrogen , cat # 18080 - 044 ) for subsequent 3seq library construction .
the 3seq library construction included the following steps ; first strand cdna synthesis with superscript iii reverse transcriptase ( invitrogen , cat # 18080 - 044 ) , second strand cdna synthesis with e
coli dna ligase ( invitrogen , cat # 18052 - 019 ) and e. coli dna polymerase i ( new england biolab , cat # m0209l ) , followed by the addition of
a to the 3 end of double strand dna fragments with klenow exo ( 3 to 5 exo minus , new england biolab , cat # m0212l ) , ligation of adapters ( illumina , cat # 1001782 oligo mix ) with dna ligase ( new england biolab , cat # m2200l ) and pcr amplification using 2 phusion pcr master mix ( new england biolab , cat # f-531s ) , , , , , .
the detailed protocol of 3seq library construction can be accessed using the following link , http://med.stanford.edu/labs/vanderijn-west/documents/3endrnaseqlibraryconstruction_update_11_4_2014.doc .
the 3seq libraries were sent to the stanford center for genomics and personalized medicine to be sequenced directionally ( 36 bp ) from 5 end of mrna fragments towards their poly(a ) ends using illumina ga iix and hiseq 2000 machines ( illumina , inc .
the gene expression profiling data ( 3seq ) have been deposited in the gene expression omnibus ( geo ) and are publicly accessible through gse45510 .
sequence reads ( fastq format ) , first filtered for read quality , were re - filtered by fastx ( fastx_artifacts_filter - v - q 33 , http://hannonlab.cshl.edu/fastx_toolkit/index.html ) , and mapped to the transcriptome ( refmrna , downloaded from the ucsc genome browser , http://www.genome.ucsc.edu/ ) using soap2 , allowing at most two mismatches .
the total numbers of sequence reads for each gene symbol from the transcriptome mapping were determined and used to create the gene - expression profile matrix ( 22,144 genes ) .
read counts from each library were normalized to transcripts per million reads ( tpm ) .
a custom perl script was used to run the 3seq data processing and is publicly available . to determine the optimal number of molecular subtypes of leiomyosarcoma , the expression matrix of genes with the most variant expression levels , filtered with a standard deviation greater than 100 across all 99 lms cases ( 1300 genes ) , were transformed by log2 and gene - based centering .
this analysis was run over 1000 iterations with the settings of distance ( 1 pearson correlation ) , 80% sample resampling , 80% gene resampling , maximum evaluated k of 12 , and agglomerative hierarchical clustering algorithm
expression profiling data of the 82 additional lms cases by rnaseq was downloaded from the tcga database . to compare with 3seq data
, the tcga data were normalized into tpm and analyzed with consensusclusteringplus , as was done for the 3seq data .
to measure the reproducibility of the lms subtypes , the cases from both datasets ( 3seq and tcga rnaseq ) were evaluated using silhouette analysis , where an lms case was defined as a core case upon assignment of a positive silhouette value .
subclass mapping was performed to determine the common lms subtypes based on these core cases identified in the 3seq and tcga rnaseq datasets . in order to discover subtype - specific genes , samseq
was performed on both datasets ( 3seq and tcga rnaseq ) between each subtype and all other subtypes with a fdr of 0.05 , and significantly differentially expressed genes from the samseq analysis was referred to identify the diagnostic biomarker for each lms subtype .
to explore the molecular subtypes of leiomyosarcoma ( lms ) , paraffin blocks of 99 lms cases from 1991 to 2012 from nine hospitals ( stanford hospital , brigham and women 's hospital , mckay - dee hospital center , st .
luke 's hospital , baptist health medical center , ingalls hospital , vancouver general hospital , hospital de la santa creu i sant pau and alta bates summit medical center ) , were collected with irb approval and a waiver of consent due to the archival nature of the specimens .
the total rna was extracted for these cases and subsequently analyzed by 3 end rna sequencing .
consensus clustering was used to determine the optimal number of subtypes and silhouette analysis was then performed to measure the confidence of subtype assignment per case . to test the reproducibility of molecular subtype classification from the 3seq dataset
, the expression profiles ( rnaseq data ) of 82 additional lms cases were downloaded from the cancer genome atlas ( tcga ) database and analyzed in an identical way to the 3seq data .
the lms cases used in this study were formalin - fixed , paraffin - embedded ( ffpe ) tissues .
after lms ffpe blocks were obtained , two experienced pathologists ( one from stanford university and one from brigham and women 's hospital ) assessed and circled the regions comprised of lms tumor cells .
multiple 2 mm - diameter cores from the areas circled were re - embedded longitudinally into new paraffin blocks , and were sectioned and re - evaluated for the second time by h&e staining to ensure the purity of the samples . only cores with 90% of tumor cells were processed for subsequent rna extraction .
the total rna of the lms cases was extracted using recoverall total nucleic acid isolation kit ( ambion , cat # 1975 ) .
the quality of total rna was assessed by agarose gel electrophoresis and used to determine the amount of time necessary for shearing of the total rna by heat in first strand buffer ( invitrogen , cat # 18080 - 044 ) for subsequent 3seq library construction .
the 3seq library construction included the following steps ; first strand cdna synthesis with superscript iii reverse transcriptase ( invitrogen , cat # 18080 - 044 ) , second strand cdna synthesis with e
coli dna ligase ( invitrogen , cat # 18052 - 019 ) and e. coli dna polymerase i ( new england biolab , cat # m0209l ) , followed by the addition of
a to the 3 end of double strand dna fragments with klenow exo ( 3 to 5 exo minus , new england biolab , cat # m0212l ) , ligation of adapters ( illumina , cat # 1001782 oligo mix ) with dna ligase ( new england biolab , cat # m2200l ) and pcr amplification using 2 phusion pcr master mix ( new england biolab , cat # f-531s ) , , , , , .
the detailed protocol of 3seq library construction can be accessed using the following link , http://med.stanford.edu/labs/vanderijn-west/documents/3endrnaseqlibraryconstruction_update_11_4_2014.doc .
the 3seq libraries were sent to the stanford center for genomics and personalized medicine to be sequenced directionally ( 36 bp ) from 5 end of mrna fragments towards their poly(a ) ends using illumina ga iix and hiseq 2000 machines ( illumina , inc .
the gene expression profiling data ( 3seq ) have been deposited in the gene expression omnibus ( geo ) and are publicly accessible through gse45510 .
sequence reads ( fastq format ) , first filtered for read quality , were re - filtered by fastx ( fastx_artifacts_filter - v - q 33 , http://hannonlab.cshl.edu/fastx_toolkit/index.html ) , and mapped to the transcriptome ( refmrna , downloaded from the ucsc genome browser , http://www.genome.ucsc.edu/ ) using soap2 , allowing at most two mismatches .
the total numbers of sequence reads for each gene symbol from the transcriptome mapping were determined and used to create the gene - expression profile matrix ( 22,144 genes ) .
read counts from each library were normalized to transcripts per million reads ( tpm ) .
a custom perl script was used to run the 3seq data processing and is publicly available .
to determine the optimal number of molecular subtypes of leiomyosarcoma , the expression matrix of genes with the most variant expression levels , filtered with a standard deviation greater than 100 across all 99 lms cases ( 1300 genes ) , were transformed by log2 and gene - based centering .
this analysis was run over 1000 iterations with the settings of distance ( 1 pearson correlation ) , 80% sample resampling , 80% gene resampling , maximum evaluated k of 12 , and agglomerative hierarchical clustering algorithm .
expression profiling data of the 82 additional lms cases by rnaseq was downloaded from the tcga database . to compare with 3seq data , the tcga data were normalized into tpm and analyzed with consensusclusteringplus , as was done for the 3seq data . to measure the reproducibility of the lms subtypes , the cases from both datasets ( 3seq and tcga rnaseq ) were evaluated using silhouette analysis , where an lms case was defined as a core case upon assignment of a positive silhouette value .
subclass mapping was performed to determine the common lms subtypes based on these core cases identified in the 3seq and tcga rnaseq datasets . in order to discover subtype - specific genes , samseq
was performed on both datasets ( 3seq and tcga rnaseq ) between each subtype and all other subtypes with a fdr of 0.05 , and significantly differentially expressed genes from the samseq analysis was referred to identify the diagnostic biomarker for each lms subtype . | leiomyosarcoma ( lms ) is a malignant neoplasm with smooth muscle differentiation .
little is known about its molecular heterogeneity and no targeted therapy currently exists for lms .
we performed expression profiling on 99 cases of lms with 3 end rna sequencing ( 3seq ) and demonstrated the existence of 3 molecular subtypes in this cohort .
we consequently showed that these molecular subtypes are reproducible using an independent cohort of 82 lms cases from tcga .
two new formalin - fixed , paraffin - embedded ( ffpe ) tissue - compatible diagnostic immunohistochemical markers were identified for two of the three subtypes : lmod1 for subtype i lms and arl4c for subtype ii lms .
subtype i lms and subtype ii lms were associated with good and poor prognosis , respectively . here , we describe the details of lms diagnosis , rna isolation , 3seq library construction , 3seq sequencing data analysis and molecular subtype determination .
the 3seq data produced in this study was deposited into gene expression omnibus ( geo ) under gse45510 . |
a 22-year - old , right - handed , unmarried woman was admitted to the psychiatric unit due to her selective retrograde amnesia that occurred after a severe stressful event .
she could remember nothing of the autobiographical events for the preceding four years , that is , her university life period .
she was aware only of a hallucinatory vision of a parking lot in which she was kidnapped and violated by a masked robber .
however , she could recount in detail the events of her high school life and also the events of her life before that .
there were no neurological signs , no suspicion of head trauma and no epileptic discharge on the electroencephalography .
her brain mri was normal . to examine the underlying brain functional disturbances associated with psychogenic amnesia
the patient viewed three kinds of face photographs : recognizable faces of familiar high school friends , unrecognizable faces of familiar university friends , and unfamiliar control faces .
the patient was instructed to try to recognize the faces that were shown to her .
all faces were presented to the patient through a mirror located at the top of the head coil that received the face photographs from outside of the magnetic room during the fmri procedure .
the t1-weighted images ( time to repeat / echo time / flip angle = 500 ms/50 ms/90 ) were obtained for anatomical localization by using a 1.5 t mri scanner ( ge medical system , horizon milwaukee , wi , usa ) .
the bold - contrast fmri images were acquired from 11 slices that covered the limbic areas with using gradient - echo epi ( time to repeat / echo time / flip angle = 3000 ms/50 ms/90 ) .
post - processing of images was performed with matlab ( mathworks , natick , ma , usa ) and spm99 ( statistical paramatric mapping software ; wellcom department of cognitive neurology , london , uk ) .
the spm99 software was used for image realignment , normalization , smoothing and to create the statistical maps of the significant relative changes in the regional blood oxygenation level - dependent ( bold ) response . the blocked design fmri paradigm , which was preceded by three dummy scans to allow the magnetic resonance signal to reach a steady state , was comprised of seven repetitions with nine seconds of rest ( fixation cross ) , and six repetitions of a nine seconds activation state ( three kinds of photograph that were randomly comprised of three seconds of recognizable faces of high school friends , three seconds of unrecognizable faces of university friends , and three seconds of unfamiliar control faces ) .
a correlation coefficient algorithm was used to correlate the time course data sets with the periodic boxcar function .
three different subsets of the activation periods were independently compared with those of the rest periods .
the bold signals were estimated for the comparison of whole brain signals across all the experimental conditions .
specific effects were tested for by applying appropriate linear contrasts to the parameter estimates for each condition , and this resulted in a z - score for each voxel .
the voxels were identified as being significant only if they passed a height threshold of z = 3.5 and they belonged to a cluster of at least 30 activated voxels .
the data were analyzed for the main effects of each stimulation : the recognizable faces of high school friends , the unrecognizable faces of university friends and the unfamiliar control faces .
for the recognizable condition , different patterns of brain activation were found as compared with the unrecognizable condition .
the functional maps of the representative brain slices demonstrated differences for activation in the limbic areas : the amygdala , hippocampus , parahippocampal gyrus and insula .
especially , the activation of the amygdala and hippocampus was significantly greater during the stimulation with recognizable faces of high school friends ( fig .
1 ) . however , the activation patterns stimulated with the unrecognizable faces of university friends and the unfamiliar control faces were similar to each other .
they showed significantly less activation in the limbic area than the recognizable faces ( fig .
in our case , we found significantly different patterns of activation in the limbic system , especially in the amygdala and hippocampus areas , during the stimulation with recognizable faces of high school friends and the unrecognizable faces of university friends .
well - known faces have the potential to elicit retrieval of affective responses and personal episodic incidents , and they may evoke activation of the limbic system , especially the amygdala ( 7 ) . the amygdala is located in the anterior medial portion of the temporal lobe , and it is connected with the hippocampal formation and with further cortical and subcortical structures . the amygdala is primarily involved in the acquisition and expression of emotional memories ( 8) . presumably , this patient has an abnormality for the expression of emotional memories during the amnesic period . emotional overflow could have resulted in the memory impairments .
for instance , traumatic stress may lead to remarkable encoding and retrieval disturbance , and this is probably due to the increased release of stress - related hormones such as glucocorticoids .
the intense release of glucocorticoids may result in morphologic and functional alterations of the limbic structures , especially in mediotemporal regions .
these can cause broad changes in the neurotransmitter systems of the brain . in a recent study
, the relation between the traumatic stress and functional brain alterations was described in terms of " psychogenic amnesia " ( 9 ) .
the hippocampal region is concerned with explicit judgments that are related to the identification of faces .
the lack of activation in the hippocampal region might be related to an abnormality in the explicit components of the face judgment ( 10 ) .
, this fmri study could give objective evidence and illustrate that a psychogenic amnesic patient has an abnormality in the retrieval of emotional memories during the amnesic period .
these findings suggest that changes in the limbic functions are related to the symptoms of psychogenic amnesia . | we present here a case in which functional mr imaging ( fmri ) was done for a patient who developed retrograde psychogenic amnesia for a four year period of her life history after a severe stressful event .
we performed the fmri study for a face recognition task using stimulation with three kinds of face photographs : recognizable familiar faces , unrecognizable friends ' faces due to the psychogenic amnesia , and unfamiliar control faces .
different activation patterns between the recognizable faces and unrecognizable faces were found in the limbic area , and especially in the amygdala and hippocampus . |
the great auricular nerve is a cutaneous branch of the cervical plexus originating from the c2 and c3 spinal nerves .
it innervates the skin over the external ear , the angle of the mandible and the parotid gland .
we present a case of a 41-year - old female with paroxysmal ear pain accompanied by dysautonomia , tingling in the tongue , dysphagia , dysarthria and abdominal symptoms .
a combined approach using this block with facet block of c2 and c3 induced a more pronounced and prolonged benefit .
great auricular neuralgia is not often encountered in practice and can be accompanied by symptoms originating from the ansa cervicalis network .
the great auricular nerve ( gan ) is a cutaneous branch of the cervical plexus originating from the c2 and c3 spinal nerves.1 it innervates the skin over the external ear , the angle of the mandible and the parotid gland.2 here , we present a case of unusual ear pain with dysautonomia and gastrointestinal manifestations .
treatment may include a combination of peripheral nerve blocks targeting the gan itself and the c2c3 cervical roots .
the patient is a 41-year - old female who presented with a 1-year history of paroxysmal left ear pain and tingling sensation in her tongue . the pain in her ear radiated to the ipsilateral side of her face as well as posteriorly in the distribution of the lesser occipital nerve .
the pain worsened at the end of the day to a severe intensity , at which time there was red discoloration of the ear .
the patient alternated between a constant dull aching pain at 5 out of 10 and periods of paroxysmal pain reaching 8 out of 10 on a numerical rating scale and lasting between 1 and 5 consecutive days . during exacerbations ,
there was a history of sports - related mild traumatic head injury as a young adult and a whiplash involving subluxation of the c4c5 cervical vertebrae .
past medical history also included mononucleosis , two previous superficial lower extremity venous thromboses postpartum and heterozygous factor v leiden thrombophilia .
examination of her cranial nerves revealed diffuse hypoesthesia to touch , pinprick and temperature in the left trigeminal distribution .
there was decreased sensation to touch and pinprick in the area of the greater and lesser occipital nerves .
lateral neck flexion to the right was limited with diffuse tenderness on palpation of the left paracervical muscles .
neck compression testing for radiculopathy and facet syndrome using spurling s maneuver was able to provoke the patient s red ear discoloration after a 15-second compression .
the patient s workup included a 3 t cerebral angio - magnetic resonance imaging with contrast , constructive interference in steady state sequence imaging of the circle of willis and magnetic resonance imaging of the cervical spine .
a contact between the superior cerebellar artery and the cisternal portion of the left trigeminal nerve was seen , but without evidence of compression .
gastrointestinal workup , including food allergy testing , gastroscopy and colonoscopy with biopsies were negative .
drug trials included acetaminophen , naproxen , tricyclic antidepressant , gabapentinoids , carbamazepine , topical analgesics , bupropion , muscle relaxant , tapentadol , morphine , hydromorphone and methadone .
in addition , she developed a multiple drug intolerance syndrome , including a probable stevens
she was evaluated by an allergist . due to the serious nature of side effects ,
all pain medications had been stopped at least 2 weeks prior to the ultrasound - guided infiltrations described below . a diet at the patient s own initiative proved slightly successful in reducing her postprandial abdominal pains .
the diagnosis was later revised to great auricular neuralgia , given the distribution of the pain .
we used a ge logiq e ultrasound machine equipped with a 12 mhz high - frequency transducer .
the gan was identified at its emergence from the posterior border of the sternocleidomastoid muscle , at the level of the cricoid cartilage located by palpation.3,4 once the hypoechoic round shaped nerve was visualized , a 25 g , 50 mm needle was introduced in plane with the ultrasound probe , and we injected a mix of 1 ml 2% xylocaine , 1 ml 0.5% bupivacaine and 1 ml of triamcinolone ( 40 mg ) .
this technique proved successful in relieving the patient s paroxysmal earaches for over 1 month with reduction of pain intensity of 60% .
we combined the gan block with ultrasound - guided facet blocks of the c2 and c3 spinal nerves as well as lesser occipital nerve block .
the combination of these blocks reduced not only the intensity of her paroxysmal ear pain , but also the duration of exacerbations from 1 to 5 days to < 24 hours and the background pain by > 50% .
the patient is a 41-year - old female who presented with a 1-year history of paroxysmal left ear pain and tingling sensation in her tongue . the pain in her ear radiated to the ipsilateral side of her face as well as posteriorly in the distribution of the lesser occipital nerve .
the pain worsened at the end of the day to a severe intensity , at which time there was red discoloration of the ear .
the patient alternated between a constant dull aching pain at 5 out of 10 and periods of paroxysmal pain reaching 8 out of 10 on a numerical rating scale and lasting between 1 and 5 consecutive days . during exacerbations ,
there was a history of sports - related mild traumatic head injury as a young adult and a whiplash involving subluxation of the c4c5 cervical vertebrae .
past medical history also included mononucleosis , two previous superficial lower extremity venous thromboses postpartum and heterozygous factor v leiden thrombophilia .
examination of her cranial nerves revealed diffuse hypoesthesia to touch , pinprick and temperature in the left trigeminal distribution .
there was decreased sensation to touch and pinprick in the area of the greater and lesser occipital nerves .
lateral neck flexion to the right was limited with diffuse tenderness on palpation of the left paracervical muscles .
neck compression testing for radiculopathy and facet syndrome using spurling s maneuver was able to provoke the patient s red ear discoloration after a 15-second compression .
the patient s workup included a 3 t cerebral angio - magnetic resonance imaging with contrast , constructive interference in steady state sequence imaging of the circle of willis and magnetic resonance imaging of the cervical spine .
a contact between the superior cerebellar artery and the cisternal portion of the left trigeminal nerve was seen , but without evidence of compression .
gastrointestinal workup , including food allergy testing , gastroscopy and colonoscopy with biopsies were negative .
drug trials included acetaminophen , naproxen , tricyclic antidepressant , gabapentinoids , carbamazepine , topical analgesics , bupropion , muscle relaxant , tapentadol , morphine , hydromorphone and methadone .
in addition , she developed a multiple drug intolerance syndrome , including a probable stevens
she was evaluated by an allergist . due to the serious nature of side effects ,
all pain medications had been stopped at least 2 weeks prior to the ultrasound - guided infiltrations described below . a diet at the patient s own initiative proved slightly successful in reducing her postprandial abdominal pains .
the diagnosis was later revised to great auricular neuralgia , given the distribution of the pain .
we used a ge logiq e ultrasound machine equipped with a 12 mhz high - frequency transducer .
the gan was identified at its emergence from the posterior border of the sternocleidomastoid muscle , at the level of the cricoid cartilage located by palpation.3,4 once the hypoechoic round shaped nerve was visualized , a 25 g , 50 mm needle was introduced in plane with the ultrasound probe , and we injected a mix of 1 ml 2% xylocaine , 1 ml 0.5% bupivacaine and 1 ml of triamcinolone ( 40 mg ) .
this technique proved successful in relieving the patient s paroxysmal earaches for over 1 month with reduction of pain intensity of 60% .
we combined the gan block with ultrasound - guided facet blocks of the c2 and c3 spinal nerves as well as lesser occipital nerve block .
the combination of these blocks reduced not only the intensity of her paroxysmal ear pain , but also the duration of exacerbations from 1 to 5 days to < 24 hours and the background pain by > 50% .
the gan arises from the ventral rami of c2c3 roots , along with two other sensory branches , the lesser occipital nerve and the transverse cervical nerve.5 the fourth cervical cutaneous nerve and the supraclavicular nerve arise from c3c4.6 the gan has anastomoses with the auricular branch of the vagus nerve , the posterior auricular branch of the facial nerve , the lesser occipital nerve and the auriculotemporal nerve of the trigeminal nerve.7,8 the ansa cervicalis , also known as goose s neck or ansa hypoglossi , is a loop of nerves formed by the ventral rami of c1 to c4 which join the hypoglossal nerve via a branch of c1 ( figure 2).5,9 it is also involved in the innervation of infrahyoid muscles , which are important for speech modulation and swallowing.10 the case presented above illustrates this complex network of connections and suggests a dysfunction at a more proximal level than the peripheral branches .
however , the mechanical disturbance and trigger at the neck , along with the presence of digestive symptoms prompt us to believe that the primary dysfunction is proximal to the peripheral cutaneous branches of the cervical plexus , most likely within the ansa cervicalis.5,9,10 however , a central dysregulation of the brainstem trigeminal - autonomic circuits can not be ruled out .
contrary to cases of red ear syndrome described in the literature , this patient did not have another primary headache disorder , such as migraine.11 history and physical examination revealed pain of neuropathic origin as per the douleur neuropathique 4 questionnaire , but the patient suffered serious adverse reactions to usual neuropathic pharmacotherapy .
our nerve blocks targeted the gan peripherally in addition to its spinal nerve root origins at c2 and c3 .
the combination of these blocks provided more relief to the patient in terms of intensity , duration and frequency of paroxysmal earaches as well as her constant dull background pain , in comparison to blocking the gan peripherally alone .
advantages to this technique versus fluoroscopic guidance included radiation - free imaging in a young female patient and ability to identify blood vessels in needle trajectory of a highly vascularized region , all in real time.12 adverse events were not reported , but rare and serious complications could include injury to the vertebral artery , spinal radicular arteries , nerve roots and spinal cord direct or indirect trauma with leakage of anesthetic into the spinal canal causing motor and sensory blockade.13,14 minor potential events could include local hematoma , bruising , soreness , transient pain exacerbation and vasovagal reactions.15 a technique aiming for the ansa cervicalis itself could be more successful , as we suspected this was the primary structure of dysfunction that could explain our patient s constellation of symptoms .
we are not aware of any such case reports and this kind of technique is not yet described .
however , a better understanding of the anatomical source guided our choices for better targeted nerve blocks that were able to provide our patient with symptomatic relief .
great auricular neuralgia is an uncommon cause of facial pain that can be accompanied by other symptoms related to the cervical plexus .
cases refractory to usual pain medication could be treated by a combination of ultrasound - guided nerve blocks .
future potential therapeutic options would involve medial branch blocks of c2c3 , followed by radiofrequency ablation with or without peripheral neuromodulation of the gan.16 | backgroundthe great auricular nerve is a cutaneous branch of the cervical plexus originating from the c2 and c3 spinal nerves .
it innervates the skin over the external ear , the angle of the mandible and the parotid gland .
it communicates with the ansa cervicalis .
great auricular neuralgia is rarely diagnosed in clinical practice and can be refractory .
we present a new approach using ultrasound - guided nerve blocks.casewe present a case of a 41-year - old female with paroxysmal ear pain accompanied by dysautonomia , tingling in the tongue , dysphagia , dysarthria and abdominal symptoms .
no significant findings were found on cervical and brain imaging .
the patient responded partially to a great auricular nerve block .
a combined approach using this block with facet block of c2 and c3 induced a more pronounced and prolonged benefit.conclusiongreat auricular neuralgia is not often encountered in practice and can be accompanied by symptoms originating from the ansa cervicalis network .
a combined approach of nerve blocks can be considered in refractory cases . |
all known organisms belonging to the trypanosomatidae , a family of protists within the order kinetoplastida , are parasites of vertebrates , invertebrates , or plants .
species of two trypanosomatid genera , trypanosoma and leishmania , are responsible for several serious diseases of humans and domestic animals in the tropical and subtropical regions of the world .
trypanosoma brucei is the causative agent of human sleeping sickness , a disease that threatens millions of mainly impoverished people in 36 countries of the african continent .
this parasite is transmitted by several species of the genus glossina , insects commonly known as tsetse flies , which are endemic in the affected sub - saharan countries .
the two human infective subspecies of t. brucei , t. b. rhodesiense , and t. b. gambiense , live extracellularly , multiplying as so - called long - slender trypanosomes in the bloodstream . by a population - density - dependent mechanism
trypanosomes are passed on to the insect vector when a tsetse fly takes blood from an infected host .
once in the tsetse fly gut , the stumpy parasites differentiate into so - called procyclic - form trypanosomes , which subsequently undergo several successive differentiations while migrating from the gut via the proboscis to the salivary glands . from there , the parasites , then called metacyclic forms , are transmitted to a new host [ 1 , 4 ] . without treatment , human sleeping sickness is considered fatal . vaccines or adequate drugs for effective treatment of this disease are currently not available .
trypanosoma cruzi is responsible for chagas ' disease in south and central america , a form of trypanosomiasis that threatens 15 million people on the american continent .
this parasite is mainly transmitted to humans via the feces of infected triatomine bugs ; however , transfusions with infected blood , organ transplantations , and use of contaminated food are also important ways of transmission .
t. cruzi is an intracellular parasite of humans and many other vertebrates ; the parasite is dedicated to invade cardiac and digestive tissues , where infective trypomastigotes differentiate into replicative amastigote forms .
the amastigotes differentiate again into trypomastigotes in the cytosol before they burst from the infected host cell , thus liberating parasites that perform a new round of cell infection .
blood trypomastigotes , taken up again by triatomine bugs when they feed on an infected host , differentiate then into replicative epimastigotes in the insect 's intestine .
the parasites are transmitted again to the vertebrate host when the infected bug takes a blood meal , depositing the contaminated feces on the skin of the new host .
people infected with t. cruzi can only be effectively treated when such treatment is applied shortly after the infection has occurred .
several species of the genus leishmania are the etiological agents of leishmaniasis , a disease characterized by clinical manifestations with a wide range of symptoms
. the disease is distributed in 88 countries of tropical and subtropical areas around the world .
the parasite is transmitted by the bite of a female phlebotomine sand fly , which feeds from vertebrate blood .
leishmaniasis can occur as three different forms : cutaneous leishmaniasis is the most common and mild form of the disease characterized by the presence of ulcers that appear at the places of parasite inoculation ; mucocutaneous leishmaniasis is manifested by destruction of the mucosa membranes of mouth , nose , and throat after parasite spreading ; visceral leishmaniasis is the most dangerous form of the disease in which organs such as liver and spleen are compromised .
leishmania species multiply mainly inside host macrophages as amastigote forms ; this form of the parasite is present in the phagolysosome where the parasite divides until they burst from the host cells . subsequently ,
the parasite is taken up by the sand fly when a female phlebotomine feeds on an infected host .
the amastigotes then differentiate into proliferative metacyclic promastigote forms in the sand fly gut before migrating to the salivary glands in order to be transmitted to a new host .
cutaneous and mucocutaneous leishmaniases are disabling diseases that affect the quality of life of infected people ; however , visceral leishmaniasis is a fatal disease when no treatment is applied .
although several drugs are available to be used for sleeping sickness , chagas ' disease and leishmaniasis , they do not offer adequate treatment .
none has been rationally designed to specifically treat these diseases . as a consequence , toxic side effects ,
sometimes very serious , are associated with the use of such drugs , and increasing resistance has been reported against some of them .
additionally , the application of some of the treatments requires highly qualified staff and medical equipment , which are usually not available in the regions where most of the people affected by these diseases live .
consequently , there exists an urgent need to develop new drugs that will inhibit the viability of the parasites while causing minimal side effects to the infected people and which can be easily administered and will be affordable for these people usually living in resource - poor areas of the world . in that context , efforts have been made in recent decades to identify , in the parasites , crucial metabolic pathways or cellular structures with features sufficiently different from those in the host in order to exploit them as chemotherapeutic targets .
a unique feature of all kinetoplastids is that the majority of glycolytic and gluconeogenic enzymes are compartmentalized in unique peroxisome - like organelles called glycosomes [ 6 , 7 ] .
several lines of evidence have shown that this compartmentalization is absolutely essential for survival of the organisms .
metabolic studies on t. brucei , t. cruzi , and leishmania spp . have shown that in the human - pathogenic stages of each of these parasites glycolysis and/or gluconeogenesis are crucial or at least major processes in the provision of free energy and carbon and consequently essential for parasite viability . moreover ,
the peculiar compartmentalization of these pathways in kinetoplastids has endowed the enzymes with specific structural and functional properties that offer potential for development of trypanocidal drugs .
trypanosomatid parasites are also equipped with several potent mechanisms that allow them to evade the immune response by the host , as well as to migrate through several tissue barriers to reach their final destination .
t. brucei is present in the bloodstream of the mammalian host during the initial phase of the infection .
a sophisticated mechanism of antigenic variation at the level of their surface coat allows the parasites to escape continuously from the host 's immune defense system . at a second stage of the disease ,
the parasites reach the central nervous system by crossing the blood - brain barrier via a multistep mechanism of which the details remain to be established . in contrast , t. cruzi lives only briefly in the bloodstream of the vertebrate host before invading the cardiac muscle and cells of the digestive system .
it is known that metacyclic trypomastigotes initially adhere to the host cell through several proteins exposed on the parasites ' cell surface that function as ligands and adhering molecules .
then , the parasites control their invasion by exploiting phagocytic and nonphagocytic mechanisms that lead to their internalization into the parasitophorous vacuole . once there , the parasites differentiate into amastigotes , which excrete potent pore - forming peptides and transsialidases / neuraminidases that mediate the vacuolar membrane 's disintegration to let the parasites free in the cytosol in order to multiply .
are exposed during a very short time to the lytic factors present in the bloodstream .
surviving parasites are able to adhere through different mechanisms to the host cell , which belongs to the monocyte / macrophage family .
the presence on the parasites ' surface of lipophosphoglycans , metalloproteases , and specific ligands for macrophage receptors allows the parasites to invade the host cells , as well as migrate through the extracellular matrix .
once in the phagolysosome , promastigotes differentiate into amastigotes that are better equipped with molecules that provide resistance against the stress conditions encountered in the phagolysosome , such as the presence of hydrolases and the acidic ph .
the surface molecules of the amastigotes play an important role in inhibiting the mechanism that triggers the macrophage 's activation as well as the proper antigen presentation through the mhci and ii complexes , which eventually would activate a host protective th1-cell response .
studies of the metabolic pathways essential for parasite survival and the mechanisms by which the trypanosomatids establish an infection in the human host are important both for future chemotherapy and vaccination .
unique or at least highly different from host molecules that are interesting as candidate drug targets and the subsequent discovery of selective inhibitors of these molecules to combat the parasitic diseases , as well as molecules that could be exploited as potential antigens to induce immunoprotection against the trypanosomiases and leishmaniases . in this paper
, we will argue that enolase , a protein involved both as enzyme in glycolysis and gluconeogenesis , metabolic processes essential for t. brucei , t. cruzi , and leishmania spp . , and probably also as a virulence factor in the pathogenesis caused by leishmania spp .
, t. cruzi and possibly also t. brucei , represents both an interesting drug target and vaccine candidate .
enolase is the enzyme responsible for the reversible conversion of d-2-phosphoglycerate ( 2pga ) and phosphoenolpyruvate ( pep ) in glycolysis and gluconeogenesis , two metabolic pathways that are often vital for cellular function .
enolase is a highly conserved protein found in archaea , bacteria , and eukaryotes with catalytic properties that are similar among divergent organisms .
the ubiquitous presence of the enzyme and the sequence homology between enolases from extant organisms belonging to different phyla indicate that an enolase gene has already been present in the common ancestor and diversified by speciation of organisms and gene duplication within organisms . since enolase catalyzes the reversible conversion of 2pga into pep , the forward reaction being the ninth step of glycolysis and the reverse reaction being the first one of gluconeogenesis , an organism , tissue , or cell may need to optimize the properties of this enzyme in such a way that the flux in either one or the other direction is facilitated , dependent on whether it should adapt its metabolism to performing the catabolic or anabolic process . indeed , in many organisms ( vertebrates , saccharomyces cerevisiae , toxoplasma gondii ) the presence of different enolase isoforms has been reported , often with kinetic properties favouring a flux in the glycolytic or gluconeogenic direction . in vertebrates
, it has been shown that expression of the different enolase isoforms , formed by homodimers and heterodimers of , , and subunits , is regulated in a developmentally and/or tissue - specific manner [ 13 , 15 ] . in yeast ,
the expression of the two enolase isoforms is dependent on the carbon source used to propagate the cells , glucose , or a nonglycolytic substrate ; however , the kinetic properties of homodimeric enzymes of both isoforms are very similar . in the case of t.
gondii , the specific expression of enolase genes is linked to life - stage conversion .
while enolase 1 is expressed during encystations where anaerobic glycolysis is promoted , enolase 2 is expressed in the actively dividing and invasive tachyzoite .
some highly conserved proteins perform multiple functions that are different from their classical , well - known activities , and , therefore , are called moonlighting functions .
these latter functions are often revealed when these proteins are found at cellular localizations different from where the primary function is exerted . a considerable number of glycolytic enzymes , including enolase , exhibit nonglycolytic functions in several organisms , both bacteria and eukaryotes .
enolase is located mainly in the cytosol of all eukaryotic cells where , besides its function in glycolysis and gluconeogenesis , it is also involved in regulation of cell morphology and material trafficking by interacting with the cytoskeleton system .
enolase has also been detected in the nucleus of mammalian cells where it participates in transcriptional regulation of genes involved in morphological transformation and cell proliferation .
bacterial enolase has been reported as a main component of the degradosome where it functions , in a still poorly understood manner , in the regulation of mrna stability .
the mammalian -enolase is also found as a main structural component of the eye lens , where it is found as an inactive monomer .
additionally , a considerable number of reports described that enolase can be expressed at the cell surface of several nonpathogenic and pathogenic organisms .
the enzyme has been found at the cell wall of s. cerevisiae and also at the surface of the pathogenic yeast candida albicans where it constitutes an immunodominant antigen during invasive candidiasis .
in some bacterial pathogens , such as bacillus anthracis , streptococcus pneumoniae , and streptococcus mutans [ 2224 ] , surface enolase has been highlighted as an important virulence factor . in apicomplexan parasites like plasmodium spp .
additionally , in entamoeba invadens , enolase expression is induced by environmental signals , and it shows association with cytoplasmic vesicle - like structures that transport the protein to the cyst wall where it plays an essential but so far unknown function .
human and animal parasites of the genus schistosoma have been reported to express enolase also on the surface of the adult worms [ 27 , 28 ] , where it could exert an important role in the inhibition of clot formation during host infection .
enolase may also be excreted to the extracellular environment where it mediates degradation of host tissues and immune evasion , such as has been observed for the human pathogenic streptococcus pyogenes and the insect parasite aphidius ervi [ 29 , 30 ] .
african trypanosomes living in their mammalian host are entirely dependent on glucose , abundantly available in the blood .
metabolic studies performed on bloodstream - form t. brucei have shown that glycolysis represents the only process through which atp is synthesized by the parasite .
axenically cultured amastigotes have an essentially glycolytic metabolism , fermenting glucose to succinate and acetate , strongly suggesting that also in these cells enolase might be a candidate drug target .
glycolysis is probably also essential in the trypomastigote form of these parasites , as it lives in a glucose - rich bloodstream where it shows a highly upregulated glucose uptake activity .
interestingly , the pathogenic stage of leishmania , the amastigotes living intracellularly in phagolysosomes of macrophages , utilize amino sugars as a source of carbon and energy , indicating that glycolysis is important , while even during hexose uptake gluconeogenesis has been shown to be an essential pathway for the synthesis of glycoconjugates and -mannan , both required for the virulence of the parasite [ 35 , 36 ] ; therefore , enolase could also be considered as vital , and thus a drug target , in this organism .
the fact that even during uptake of ( amino ) sugars gluconeogenesis is required has been interpreted as an indication that the sugar levels in the phagolysosome are generally too low to sustain fluxes through essential pathways such as n - glycosylation , the pentose - phosphate pathway , inositol synthesis , and the catabolism of major carbohydrate reserve material . in these three parasitic trypanosomatid species , as in all kinetoplastids , the majority of the glycolytic enzymes
dependent on the life - cycle stage , the 6 or 7 enzymes from hexokinase to glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate kinase , converting glucose into 1,3-bisphosphoglycerate or 3-phosphoglycerate , respectively are compartmentalized in peroxisome - like organelles called glycosomes [ 6 , 37 , 38 ] .
only the last enzymes of the pathway phosphoglycerate mutase , enolase , and pyruvate kinase were not found to be associated with these organelles .
more detailed studies specifically devoted to enolase have confirmed that its activity is exclusively present in the cytosol of t. brucei bloodstream forms , t. cruzi epimastigotes ( w. quiones , and j. l. concepcin , unpublished results ) , and l. mexicana promastigotes . in t. cruzi , the enzyme was detected by peptide mass fingerprinting in the different developmental stages with a higher expression in trypomastigotes and amastigotes compared to epimastigotes .
in contrast , mass - spectrometric analysis of differentiating l. mexicana showed that the glycosomal glycolytic enzymes were 1.4-fold upregulated during axenic differentiation of promastigotes to amastigotes whereas the cytosolic glycolytic enzymes including enolase were 2-fold downregulated . in bloodstream - form
t. brucei , enolase has been genetically validated as a drug target by rna interference ( rnai ) . upon induction of rnai ,
enolase activity gradually decreased during the first 24 h to 16% , leading to growth arrest followed by trypanosome death after two days .
the genes coding for enolases of t. brucei , t. cruzi , l. mexicana , and l. major have been identified in the genome sequence database ( tritrypdb : http://tritrypdb.org/tritrypdb/ ) for a variety of african trypanosomes ( t. brucei , t. gambiense , t. congolense , t. vivax ) , the american trypanosome t. cruzi , and several leishmania species ( l. major , l. mexicana , l. infantum , l. braziliensis ) . in each case , a single gene copy per haploid genome has been found , except in t. gambiense where two tandemly linked genes coding for identical proteins were found , clearly the result of a recent gene duplication in this species .
they all encode polypeptides of 429 amino acids ( including the initiator methionine ) with relative molecular masses of 46 kda .
only the predicted t. congolense enolase polypeptide has 430 residues due to the presence of an additional lys residue at the c - terminus .
comparison of the three isoenzymes of the parasite 's human host and trypanosomatid enolases reveals between 60 and 63% identity .
residues essential for substrate binding , catalytic activity , as well as those constituting the binding sites of substrates , and two mg ions are invariably present in all sequences ( see the multiple sequence alignment in figure 1 ) .
the protein has been more extensively studied in t. brucei and l. mexicana [ 39 , 40 , 44 ] .
both enzymes have been purified from the parasites and expressed as n - terminally his - tagged proteins in escherichia coli .
the kinetic properties of both the natural and bacterially expressed enzymes were determined for the forward ( glycolytic ) and reverse ( gluconeogenic ) reactions . in each case , the enzyme displayed standard michaelis - menten kinetics for both substrates with very similar km and vmax values ( table 1 ) .
mg is essential for the activity of all enolases ( with ka values varying between 0.26 and 0.45 mm ; optimal concentration for activity between 1 and 2 mm ) , from trypanosomatids and other organisms , but inhibits the enzymes at high concentrations .
the ki values of the trypanosomatid and yeast enzymes for mg are similar ( 4350 mm ) , but the mammalian enzyme is more susceptible to inhibition ; the inhibition of this latter enzyme , but not that of trypanosomatids and yeast , is ph dependent and seems to involve two processes with ki values of 7.5 ( 40% inhibition ) and > 100 mm .
furthermore , other divalent metals such as co , mn , and cu inhibit all enolases tested .
enolases are also generally inhibited by the monovalent ions li and na ; rabbit muscle enolase is activated by k , but the trypanosomatid and yeast enzymes are not .
furthermore , the l. mexicana enzyme is inhibited by fluoride with ki values of 1.9 mm ( versus 2pga ) and 6.3 mm ( versus pep ) for the natural enzyme and 13.0 mm ( versus 2pga ) and 10.0 mm ( versus pep ) for the bacterially expressed enzyme , in agreement with reports about other enolases . similarly , enolase purified from t. cruzi epimastigotes
is inhibited by fluoride with a ki value of 2.6 mm ( versus 2pga ; l. gonzlez - gonzlez , w. quiones , and j. l. concepcin , unpublished results ) .
the l. mexicana enolase is also inhibited by inorganic pyrophosphate ( ppi ) : ki = 0.232 mm ( versus 2pga ) and 0.238 mm ( versus pep ) for the natural enzyme and ki = 0.127 mm ( versus 2pga ) and 0.260 mm ( versus pep ) for the recombinant enzyme . for the natural t. cruzi enzyme , inhibition by ppi with a ki = 0.21 mm ( versus 2pga )
was found ( l. gonzlez - gonzlez , w. quiones , and j. l. concepcin , unpublished results ) .
it should be noted that , previously , several other leishmania and t. cruzi glycolytic enzymes have been shown to be inhibited by ppi [ 45 , 46 ] whereas no such inhibition was found for t. brucei enzymes .
crystal structures of enolase the enzyme in its apo - form and with bound ligands are available for a variety of organisms : s. cerevisiae , lobster , and e. coli .
the structural analysis of these different enolases has shown that the enzyme usually folds as a homodimer , although , for enolase of some bacteria , an octameric quaternary structure has been reported .
the overall structure and catalytic site are highly conserved , explaining the very similar kinetic properties between the enzymes from highly different organisms , as described in the previous section .
each subunit of the dimer is composed of two domains , a small n - terminal domain containing three helices and four sheets and a c - terminal domain which folds as an atypical eight - fold / barrel at the end of which the active site is found .
although the catalytic site lies at the dimer interface , each site involves residues of only a single subunit , explaining that the monomeric form is catalytically active , despite earlier claims of the contrary .
the early studies , particularly with the yeast enzyme , had shown that the enzyme is naturally activated by two divalent metal ions that bind to each monomer in two distinct metal - binding sites ( i and ii ) near the active site .
these studies also revealed the importance of sequential binding of a first metal ion , the substrate and a second metal ion , and associated local conformational changes , notably movements of three loops near the active site , for catalysis to occur .
the loops define open , closed , and incompletely closed active - site structures .
structures of the apo - enzyme and the enzyme in complex with its substrate or product indicate that , in the open
when the substrate or product and the divalent ions are bound , an important conformational change occurs , bringing the loops one ( comprising residues 3744 ) , two ( 150166 ) , and three ( 248272 ) to the catalytic active site producing the closed enzyme conformation .
the catalytic mechanism of the reaction implies an acid - base chemistry involving a lys - glu dyad .
it had also already initially been found that enolase activity is inhibited by high metal concentrations , which suggested the presence of a third inhibitory metal binding site . notwithstanding
the agreement about the kinetic data , no structural data were available to support this hypothesis .
more recently , seven structural determinations have been performed for the t. brucei enzyme in ligation states with substrates , inhibitors , sulphate , phosphate , and metal ions and in various conformations [ 49 , 50 ] . a subunit structure of t. brucei enolase
all these structures together have provided a profound insight into the structure - function relationship and the catalytic mechanism of the enzyme , and they solved some questions raised by the earlier structure determinations .
first , the different structures of the t. brucei enzyme and complementary molecular dynamics simulations revealed a strikingly larger conformational variability for the catalytic loops than initially thought .
secondly , in the studies of the parasite 's enzyme , also an unexpected diversity of metal binding was found , and two additional metal - binding sites were detected near the active site .
iii , explained the inhibitory effect of mg at high concentrations ; the contribution of site iv to this inhibition remains to be determined .
importantly , all available structures together , from trypanosomes and other organisms , and a sequence comparison of trypanosomatid and human enolases have revealed that , despite the high similarity ( approximately 60% positional amino - acid identity ) , similar overall fold and active - site architecture and identical catalytic residues , potentially important differences exist between parasite and host enzymes that might be exploited for drug discovery .
first , each of the trypanosomatid enolases contains three unique residues , absent from the enolases of mammals and almost all other organisms studied ( figure 1 ) , which might be targets for irreversible reaction with active - site inhibitors .
a pair of cysteine residues , numbered 147 and 241 ( see figure 3 ) , is present in a water - filled cavity near the active site . despite their proximity , no disulphide bond between these residues was observed in the structure , nor is expected to be formed in vivo because of the low redox potential in the cytosol .
one of these residues , and probably both , could be chemically modified by iodoacetamide with concomitant loss of enzyme activity indicating their accessibility to potentially reactive ligands .
substrate analogues extended with reactive groups reaching either one of these cys residues underneath the catalytic site may thus result in trypanosomatid - enzyme specific , irreversible inhibitors . in addition , a unique lys , at position 155 , was located on one of the active - site loops ( figure 3 ) and could also potentially interact with ligands in the active site .
although the side chain of this lys residue points away from the catalytic site in original t. brucei enolase crystal structures , later structures , backed up by molecular dynamics simulations , show the lys to approach the catalytic site more closely ; it may , therefore , be available for irreversible binding of active - site inhibitors . despite the closed catalytic site being small
, the studies showed that occupation of the site by ligands does not need to lead to full closure , leaving open a tunnel near which lys 155 is closely located .
this offers thus a possibility for the design of enlarged ligands that interact with this potentially modifiable residue and inhibit the enzyme irreversibly .
a study of enolase localization in l. mexicana by differential centrifugation detected this protein , as expected from its function in glycolysis / gluconeogenesis , mainly in the cytosol .
however , some enolase was also present in the microsomal fraction that contains predominantly the plasma membrane .
similar results were obtained upon selective permeabilization of cells with digitonin ; western blot analysis showed that enolase was present in both the supernatant and pellet of each cell suspension , independent of the digitonin concentration added , indicating that the detergent was unable to fully release the enzyme from the permeabilized cells .
the presence of enolase at the plasma membrane was further assessed by immunofluorescence experiments using nonpermeabilized parasites .
these experiments not only confirmed the plasma - membrane localization of enolase but also showed that it is associated to the external surface of the parasite . in agreement with these results
is the recent visualization of enolase in the proteome of the plasma membrane from leishmania chagasi , prepared by two different approaches : live cell biotinylation followed by streptavidin affinity purification and octyl glucoside extraction .
prior to these studies , subcellular fractionation of l. infantum with digitonin followed by proteomic analysis of the different fractions had predicted that enolase could have an additional localization , apart from the cytosol , also in this species .
measurement of the enolase activity in the fractions obtained by differential centrifugation of an l. mexicana lysate showed that it was all present in the cytosolic fraction ; no activity whatsoever was found in the microsomal fraction , indicating that the enolase on the parasite surface is inactive .
this was corroborated by the digitonin solubilization experiment : all enolase activity was released with the cytosolic marker and no activity was detected in the pellets .
another indication for the lack of activity of this surface enolase was recently provided by the report that its localization in l. donovani and l. major is regulated by oligopeptidase b ( opb ) , a serine - protease that is highly expressed in the amastigote form .
although loss of this peptidase in opb parasites resulted in the accumulation of enolase at the cell surface of the parasites , no increased enolase activity was detected in total cell lysates .
the authors hypothesized that oligopeptidase b causes clearance of surface enolase , once in the macrophage , to avoid macrophage activation .
enolase is also present at the cell surface of several mammalian cells [ 5456 ] .
in addition , enolase can be found at the cell surface of numerous prokaryotic and eukaryotic pathogenic organisms including , among others , group a streptococci , s. pneumoniae , l. mexicana [ 40 , 53 , 59 ] , c. albicans , schistosoma bovis , and plasmodium falciparum . in the majority of these cases ,
plasminogen is the zymogenic form of the serine - protease plasmin that is part of the fibrinolytic system .
this molecule possesses five kringle domains that provide lysine - binding sites responsible for its binding to protein substrates and cell surfaces .
surface - located enolases of mammalian cells and some pathogens bind plasminogen through a c - terminal lysine that is recognized by the lysine - binding sites of the plasminogen molecule .
however , in the amino - acid sequence of enolase from s. pneumoniae , an internal motif ( fydkerkvyd ) was identified as being responsible for the plasminogen recognition .
similar internal motifs were identified in the enolases of l. mexicana ( aydaerkmy ) and aeromonas hydrophila ( fydaekkey ) [ 59 , 64 ] .
enolases from other trypanosomatids ( figures 1 and 2 ) as well as from other pathogenic organisms have similar internal motifs at the same position that could contribute to plasminogen binding .
a similar motif has been shown to be responsible for plasminogen binding by another l. protein , the parasite 's homologue of receptors for activated c - kinase .
in addition to its localization on the membrane surface , enolase is secreted by several pathogenic microorganisms , such as the bacteria s. pyogenes , s. mutans , and lactobacillus crispatus . in leishmania parasites
, enolase has been found as a part of the secretome / exosome of l. donovani [ 68 , 69 ] , l. major , and l. braziliensis .
enolase has also been found in the secretome of t. brucei [ 71 , 72 ] and as a protein secreted by other parasites : eimeria tenella , fasciola hepatica , echinostoma caproni , giardia lamblia , and schistosoma japonicum .
different exosome proteomes from mammalian cell types indicate that enolase is commonly present in this structure .
enolase does not contain a detectable secretion signal or membrane anchor region that can explain its membrane localization .
studies with s. pneumonia showed that soluble enolase can bind to the pneumococcal surface , suggesting that one possibility for the surface localization of enolase in this bacterium is the reassociation of secreted enolase with the plasma membrane .
one may thus speculate that , in the case of leishmania parasites , enolase , after its secretion via exosomes , might bind to the cell surface .
further studies are necessary to elucidate the mechanism of translocation of enolase to the cell surface in these parasites .
the binding of plasminogen to the cell surface has been demonstrated for several mammalian cells [ 54 , 63 ] including metastatic tumor cells .
equally , over the last 15 years , a steadily increasing number of reports have been published about plasminogen binding by both prokaryotic and eukaryotic pathogens , and even viruses .
this interaction has been implicated as part of their mechanisms of invasion and establishment in the host . however , in contrast to the large number of reports about plasminogen binding by mammalian cells and unicellular organisms , only few analyses have been reported thus far that provide evidence for the importance and physiological role of this interaction .
evidence for the physiological role and importance in infection and pathogenesis of the plasminogen - pathogen interaction has been provided by studies of animal models infected with group a streptococci , yersinia pestis , borrelia spp . [
the binding of plasminogen through their plasminogen - binding proteins and its activation made the pathogens acquire a potent protease for their own purpose by kidnapping the fibrinolytic system of the host .
plasmin is involved in several physiological processes , such as the degradation of fibrin and other extracellular matrix proteins .
acquisition of this host protease allows the pathogens to invade and disseminate in the host . in the case of the streptococci , staphylococcus aureus , and y. pestis ,
the bound plasminogen can also be activated by the host plasminogen activators , the tissue plasminogen activator ( t - pa ) , and urokinase ( u - pa ) .
once the plasminogen is activated , the surface - bound plasmin can not be inhibited by the host plasmin inhibitors ( for reviews on bacteria - plasminogen interaction see [ 30 , 8589 ] ) . besides l. mexicana , surface plasminogen
binding has also been demonstrated in several other parasites : t. cruzi , trichomonas vaginalis , s. bovis [ 28 , 61 ] , and s. japonicum . in most of them
, enolase has been proposed as the plasminogen receptor . for l. mexicana , it was demonstrated that plasminogen and plasmin bind to both the promastigote and amastigote forms of this parasite [ 92 , 93 ] .
the kd for this interaction in promastigotes is 2.4 m for plasminogen and 1.2 m for plasmin .
these values are consistent with an in vivo interaction since the concentration of plasminogen in plasma and the extracellular milieu is around 2 m . moreover , enolase seems to contribute for 60% to this binding , suggesting that it is the principal plasminogen receptor on the cell surface .
this parasite does not appear to have its own plasminogen activator , thus requiring an exogenous source of activators similar to several other plasminogen - binding pathogens such as borrelia burgdorferi .
t - pa , urokinase , and streptokinase are able to activate the bound plasminogen of l. mexicana .
indeed , the activation of plasminogen by t - pa is facilitated in the presence of the parasite ( up to nine - fold enhancement of this activation was observed ) .
interestingly , there are different plasminogen - binding capacities among the morphotypes present in axenic cultures of l. mexicana promastigotes .
although the role of the interaction with plasminogen is considered to be important in bacterial pathogenesis , it is not clear what the function of this interaction is in the case of leishmania parasites , particularly because this organism thrives within macrophages .
however , this parasite can find plasminogen , as promastigotes , at the moment of host inoculation or as amastigotes when it is liberated from a macrophage to infect other cells .
a clue about a possible function of the interaction of leishmania with plasminogen was provided by studies of cutaneous lesions caused by l. mexicana in plasminogen - deficient mice . in the lesion of plg ,
mice , the parasites were limited to isolated foci in contrast to the plg mice lesion where a scattered pattern was observed .
the parasites were less infectious in the plasminogen - deficient mice . in the lesion of plg ,
these deposits are present in some organs in plasminogen - deficient mice [ 97 , 98 ] .
thus , in natural conditions , fibrin could be limiting the encounter between leishmania and macrophages .
fibrin is part of the host defense mechanism providing a barrier limiting invasion and dissemination and is also linked to inflammatory response [ 81 , 99 ] .
in addition , fibrin is one of the most important targets for plasmin in vivo .
the inflammatory response initiated by leishmania parasites may increase capillary permeability , a process associated with inflammation , resulting in the deposition of fibrin matrix within the lesion .
this fibrin matrix would provide , together with other extracellular matrix proteins , the medium for parasite - macrophage interaction . activated plasminogen on the parasite surface , or on secreted plasminogen - binding proteins , might thus help to degrade this fibrin matrix favoring the encounter with macrophages . in the latter case ,
secreted plasminogen - binding proteins would act as torpedo mines mediating clearance of fibrin . indeed , secreted proteins of l. mexicana are able to enhance the plasminogen activation by t - pa .
the plasmin formed on these cells , together with metalloproteases , mediate macrophage migration during the inflammatory process [ 104 , 105 ] .
it is known that plasmin can have a cytoprotective effect on macrophages by inhibiting apoptosis in these cells and could so support more infection by the parasite .
it is clear , however , that more information is needed to assess the function of the interaction of leishmania parasites with plasminogen . equally , more studies of the role and dynamics of fibrin in the leishmaniasis lesion are needed as well as of the effect of this molecule and plasminogen on the inflammation process within the lesion
although fibrinolysis seems to be the most important function of plasmin , one may not discard the possibility that other known functions of this enzyme may play a role in the case of leishmania infection and pathogenesis .
since both leishmania parasites and macrophages have plasminogen receptors , it is tempting to speculate that plasminogen might act as a bridge between these two cells .
moreover , it has been demonstrated previously that the pathogen mycoplasma fermentans is able to adhere and invade hela cells in a plasminogen - dependent manner .
although the real function of the interaction plasminogen - leishmania is not known , this interaction suggests that plasminogen - binding proteins , such as enolase , could be virulence factors .
as described above , enolase may have one or more of three important functions in the different trypanosomatid parasites , in glycolysis , in gluconeogenesis , and/or as plasminogen receptor . in bloodstream - form
t. brucei , enolase is involved in glycolysis , a process crucial for the atp supply of the parasites . in intracellular leishmania amastigotes , amino sugars
are utilized as carbon and energy source , while also gluconeogenesis is an essential pathway for the synthesis of glycoconjugates and -mannan , both required for virulence of the parasite ; therefore , enolase should also be important for the parasitic life of this organism .
less information exists about the nature of the carbohydrate and energy metabolism of the human - pathogenic stages of t. cruzi , but the available data indicate that axenically cultured trypomastigotes and amastigotes are highly dependent on glucose catabolism , strongly suggesting that , also in these cells , enolase fulfills an essential function in metabolism . therefore
, in each of these trypanosomatid species , enolase may be considered as a validated or at least a highly likely candidate drug target .
in addition to this essential metabolic role played by enolase in the cytosol of the parasites , the enzyme was also shown to be present , in a catalytically inactive form , at the outer surface of l. mexicana and t. cruzi ( w. quiones , and j. l. concepcin , and l. avilan , unpublished results ) , where it probably acts as a plasminogen receptor .
although pathogens may have also other means to bind plasminogen , in l. mexicana , enolase was proposed to be the predominant receptor for this zymogen , responsible for 60% of its total binding .
this plasminogen binding , and the subsequent activation of the plasminogen into the protease plasmin , could be important for the parasite 's virulence .
several moonlighting functions are known for the glycolytic / gluconeogenic enzyme enolase ( discussed in section 1 ) . in some cases
, organisms have multiple enolase isoforms , and some moonlighting functions have been attributed to specific isoforms . in each of the trypanosomatids , except in t. gambiense
, only a single enolase gene copy is present , thus the encoded protein must be responsible for both the glycolytic / gluconeogenic activity and the plasminogen - binding function .
the absence of activity of the surface - associated plasminogen - binding form may be due to processing of the enzyme during or after its translocation across the plasma membrane .
how the enzyme arrives at the surface , and is bound to the membrane , is not clear yet . the protein does not possess a predicted transmembrane region or glycosylphosphatidylinositol ( gpi ) anchor site , nor a detectable n - terminal transit peptide , suggesting that it is not transported via the classical secretion pathway .
moreover , enolase was detected in the secretome of l. donovani , and also in that of t. brucei , both bloodstream - form and procyclic cells [ 71 , 72 ] .
strong experimental evidence has been provided for both leishmania and trypanosoma that these parasites use multiple nonclassical secretion pathways , including active exocytosis via microvesicles or exosomes .
this suggests that the enolase is first secreted and subsequently retrieved by leishmania to associate in a still unknown manner with its outer surface .
in addition to a gene coding for a typical enolase , some trypanosomatids possess genes coding for hypothetical proteins with regions homologous to those of authentic enolases . it is unlikely that these proteins , which are usually considerably longer than typical enolases , possess enolase activity , because some catalytically important residues seem to be missing . their location in the cell and their function a receptor function or something different
enolases from a variety of pathogenic organisms have the ability to bind plasminogen via a c - terminal lys residue .
however , in s. pneumoniae enolase an internal peptide located in a loop at the surface of the protein was identified as the primary site for interaction with plasminogen . for l.
mexicana that , like almost all other trypanosomatid enolases ( except t. congolense ) , lacks a c - terminal lys , a peptide was found with high sequence similarity to , and at the corresponding position as a plasminogen - binding peptide of , s. pneumoniae enolase .
the plasminogen binding of leishmania enolase and the involvement of this peptide in this interaction , remain to be proved by biochemical and/or structural studies .
the peptide is also well conserved in the enolase of t. cruzi as well as , intriguingly , in that of all species of african trypanosomes .
the necessity of t. cruzi to recruit host plasminogen can be understood in view of the fact that this parasite , like leishmania spp . , invades tissues and enters cells and may require extracellular proteases for these activities .
however , the conservation of the candidate zymogen - binding peptide and the presence of enolase in the secretome of the extracellularly living t. brucei raises the question as to whether it plays also a role as plasminogen - receptor in african trypanosomes , and if so , what could be the function of recruiting a host protease .
it should be noted that , whereas in l. mexicana the enolase is bound to the cell surface , it has not been detected in the proteome of the t. brucei plasma membrane .
the ability of african trypanosomes to degrade fibrinogen and fibrin has already been reported several decades ago .
it may be that the infection is accompanied with an increased synthesis of fibrinogen by the host and formation of microthrombi that have to be dealt with by the parasites .
alternatively , the parasites may recruit plasminogen from the host to enable them to cross the blood - brain barrier .
this crossing is a still poorly understood process , but may occur in a paracellular way . in this case ,
plasmin , as a host - derived protease , can help to degrade adherens junctions and tight junctions in the trypanosome - induced blood - brain barrier dysfunction model as proposed by grab et al . .
the facts that enolase plays an essential function in the metabolism of probably all trypanosomatids and is probably an important virulence factor for , at least , the intracellular parasites offer two possibilities for using this protein to combat or prevent an infection .
first , the presence of the plasminogen - receptor form of enolase could be used in two different ways to interfere with the virulence of the parasites .
compounds may be developed as drugs that prevent or disrupt the receptor - plasminogen interaction .
alternatively , the trypanosomatid enolase , or unique parts of its sequence , may be used either as an antigen for an infected person to boost his immune system in combating the parasites , or it may be administered as a vaccine to protect people at risk of being infected . secondly , as discussed above , the availability of the crystal structure of t. brucei enolase has opened the possibility to design parasite - enzyme selective inhibitors .
substrate analogues may be synthesized that exploit unique physicochemical and/or steric features of the catalytic site of the parasite 's enzyme and , therefore , provide high affinity and selectivity . moreover , incorporating substitutions that interact with the reactive lys and cys residues ( figure 3 ) specific of the trypanosomatid enolases will not only increase the selectivity but also render the inhibition irreversible .
irreversible and uncompetitive inhibitors are particularly attractive because , contrary to the much more often observed competitive inhibition , the increase of substrate concentration , resulting from this process , will not overcome the inhibition but may rather lead to an increase of metabolic intermediates to toxic levels . because of the high similarity between the enolases of the different trypanosomatids , it is likely that inhibitors developed in this way for the t. brucei enzyme may also affect the activity of the enzymes of t. cruzi and leishmania spp .
alternatively , the availability of systems for the easy production of purified bacterially expressed enolases of all three trypanosomatids allows high - throughput screening of large libraries of drug - like compounds .
any hits obtained by such screenings will subsequently be optimized through successive cycles of structure - activity relationship ( sar ) analysis .
the next steps in the development of drug candidates against these diseases will involve the selection of compounds with potent and highly selective inhibitory activity on cultured parasites versus human cells , and the evaluation of their efficacy , bioavailability , and toxicity in infected animal models .
finally , lead compounds thus obtained may serve for their further development to trypanocidal or leishmanicidal drugs . | glycolysis and glyconeogenesis play crucial roles in the atp supply and synthesis of glycoconjugates , important for the viability and virulence , respectively , of the human - pathogenic stages of trypanosoma brucei , trypanosoma cruzi , and leishmania spp .
these pathways are , therefore , candidate targets for antiparasite drugs .
the glycolytic / gluconeogenic enzyme enolase is generally highly conserved , with similar overall fold and identical catalytic residues in all organisms .
nonetheless , potentially important differences exist between the trypanosomatid and host enzymes , with three unique , reactive residues close to the active site of the former that might be exploited for the development of new drugs . in addition
, enolase is found both in the secretome and in association with the surface of leishmania spp . where it probably functions as plasminogen receptor , playing a role in the parasite 's invasiveness and virulence , a function possibly also present in the other trypanosomatids .
this location and possible function of enolase offer additional perspectives for both drug discovery and vaccination . |
leiomyoma , a benign smooth muscle tumor , is classified by the world health organization ( who ) into three types on the basis of histopathology : i ) solid leiomyoma , ii ) vascular leiomyoma ( angiomyoma , angioleimyoma ) , and iii ) epithelioid leiomyoma .
although leiomyoma may occur anywhere in the body , leiomyoma most commonly arises in the muscular layer of the gut and the body of the uterus1 .
it rarely affects the oral cavity23 and such cases account for only 0.4% of all neoplasms of the soft tissue and 0.06% of all leiomyoma lesions .
leiomyoma lesions normally have presented as angioleiomyoma at rates ranging from 64.0% to 66.2% of all the reported cases4 .
a 25-year - old male patient presented to department of periodontology , faculty of dentistry , mustafa kemal university ( hatay , turkey ) with a complaint of painless gingival swelling that had persisted for one year . on clinical examination we observed a well - demarcated , firm swelling in the region of the lower left first molar vestibular gingiva.(fig .
2 ) in addition to general gingival health , the periodontal condition of the mandibular left first molar was healthy .
the mass had an ulcerative surface and was measured to be 55 mm in diameter .
as a result of the narrow periodontal probing depth , we chose blunt dissection for the excision of the mass . during excision of the mass ,
the excised lesion was placed in 10% buffered formalin solution and sent for histopathological assessment .
amoxicillin 1 g/12 hr per oral was administered for the following 7 days , while ibuprofen was prescribed as an analgesic along with chlorhexidine gluconate rinse ( 0.12% ) .
we confirmed the diagnosis of angioleiomyoma through histological examination via hematoxylin - eosin and smooth muscle actin ( sma ) staining . in the histopathological report
, the angioleiomyoma was composed of spindle shaped smooth muscle cells ( smc ) nested in dilated vascular structures with prominent endothelial cells .
3 ) the postoperative recovery of the patient was uneventful and his sutures were removed after 7 days .
a 55-year - old female patient was referred to department of oral and maxillofacial surgery , faculty of dentistry , mustafa kemal university for a routine dental examination . upon examination , a 1520 mm diameter , painless gingival swelling was detected in the maxillary first molar region .
an elliptical incision was made to fully enucleate the lesion along with the overlying mucosa .
the excised lesion was placed in 10% buffered formalin solution and sent for histopathological assessment .
6 ) during the postoperative period the patient received amoxicillin 1 g/12 hr per oral for 7 days , dexketoprofen as an analgesic and chlorhexidine gluconate rinse ( 0.12% ) .
the surgical site healed uneventfully without recurrence over a period of 4-month follow - up .
a 25-year - old male patient presented to department of periodontology , faculty of dentistry , mustafa kemal university ( hatay , turkey ) with a complaint of painless gingival swelling that had persisted for one year . on clinical examination we observed a well - demarcated , firm swelling in the region of the lower left first molar vestibular gingiva.(fig .
2 ) in addition to general gingival health , the periodontal condition of the mandibular left first molar was healthy .
the mass had an ulcerative surface and was measured to be 55 mm in diameter .
as a result of the narrow periodontal probing depth , we chose blunt dissection for the excision of the mass . during excision of the mass ,
the excised lesion was placed in 10% buffered formalin solution and sent for histopathological assessment .
amoxicillin 1 g/12 hr per oral was administered for the following 7 days , while ibuprofen was prescribed as an analgesic along with chlorhexidine gluconate rinse ( 0.12% ) .
we confirmed the diagnosis of angioleiomyoma through histological examination via hematoxylin - eosin and smooth muscle actin ( sma ) staining . in the histopathological report
, the angioleiomyoma was composed of spindle shaped smooth muscle cells ( smc ) nested in dilated vascular structures with prominent endothelial cells .
3 ) the postoperative recovery of the patient was uneventful and his sutures were removed after 7 days .
a 55-year - old female patient was referred to department of oral and maxillofacial surgery , faculty of dentistry , mustafa kemal university for a routine dental examination . upon examination , a 1520 mm diameter , painless gingival swelling was detected in the maxillary first molar region .
an elliptical incision was made to fully enucleate the lesion along with the overlying mucosa .
the excised lesion was placed in 10% buffered formalin solution and sent for histopathological assessment .
6 ) during the postoperative period the patient received amoxicillin 1 g/12 hr per oral for 7 days , dexketoprofen as an analgesic and chlorhexidine gluconate rinse ( 0.12% ) .
the surgical site healed uneventfully without recurrence over a period of 4-month follow - up .
leiomyoma occurs in all age groups , but its greatest prevalence is in adults 40 to 59 years of age is more common in females .
leiomyomas in the oral region are usually found on the lips , tongue , palate , and buccal mucosa .
brooks et al.6 reported that the most frequently affected site was the lip ( 48.6% ) , followed by the palate ( 21.1% ) , buccal mucosa and tongue ( each 9.2% ) , mandible ( 8.3% ) , and buccal sulcus , labial sulcus , floor of mouth , and gingiva ( each 0.9% ) .
the who classifies leiomyoma into three groups including angioleiomyoma ( 74% ) , solid leiomyoma ( 25% ) , and epithelioid leiomyoma ( 1% ) .
both of the cases described in this report involved gingival angioleiomyoma , which is one of the most common forms of leiomyoma .
oral angioleiomyoma is rare due to the deficiency of smooth muscles in this area found associated with the tunica media layer of blood vessels , circum vallate papillae of the ductus lingualis , and heterotrophic smooth muscle4 . in histologic sections , the walls of small blood vessels stained positively with hematoxylin - eosin and sma .
therefore , we conclude that the angioleiomyomas , which were localized in the gingiva , originated from the smooth muscle of blood vessels due to the absence of smooth muscles except for blood vessels in the gingiva and their immediate vicinity .
the differential diagnosis in similar cases should include benign lesions such as fibroma , neurofibroma , myofibroma , fibrous histiocytoma , mucocele , schwannoma , and malignant tumors such as leiomyosarcoma8 .
due to the similarities between these tumors , the diagnosis of leiomyomas can only be made histopathologically4 . in such cases ,
histopathological sma positivity may be used to rule out the tumors that do not originate from the smooth muscle .
although the malignant transformation of angioleiomyoma is very rare9 there are several case studies describing angioleiomyosarcoma in the literature ( added ) . in the majority of reported cases ,
angioleiomyomas are characterized by slow growing masses , firm to the touch with color depending on depth and vascularity .
they average in size between 1 to 2 cm and typically present with a history of less than one year1011 .
both the masses in our cases were positioned in the buccal gingiva and of normal size , sessile , and caused slight soreness .
although ulceration is not observed very often in leiomyoma12 , one of our cases exhibited ulceration on the surface of the mass that may have arisen from trauma to the lesion during eating and mastication .
when tumors exhibit ulceration and are located in the gingiva or palate adjacent to the gingival sulcus they may be confused with periodontal lesions . in one of our cases ,
however , neither case exhibited pus , bone loss , or deep periodontal pockets typical of periodontal abscesses .
clinically , 55.9% of angioleiomyomas are red in color , while the remainder are gray , white , or normal mucosal color .
in addition , bone loss was not detected on radiological examination , and the bone exposed during surgery was healthy and normal in appearance .
although these lesions are vascular in origin , during excision there is typically no excessive bleeding4 . in our cases ,
the recurrence of this tumor after surgery is extremely rare , with recurrence within two weeks after the surgical incision indicating incomplete resection14 .
thus , it is important to ensure total resection with a border of healthy tissue to avoid recurrences . | leiomyoma is a type of benign smooth muscle neoplasm that is a common neoplasm of the uterus and gastrointestinal tract but rarely affects the head and neck region and is especially unlikely to affect the oral cavity .
the diagnosis of leiomyoma is mainly determined by histopathological studies due to variation in its clinical appearance and symptoms . in the present paper
we report two rare cases of gingival angioleiomyoma in the posterior maxilla and mandible .
after total excision , hematoxylin - eosin and smooth muscle actin staining confirmed the diagnosis of angioleimyoma . |
effective management of chronic pain has become a crucial consideration in medical care due to its prevalence and far - ranging effects.1 chronic pain is defined as pain that lasts for at least 3 months and is often associated with diseases such as cancer and diabetes,2 but may occur at any point across a patient s lifetime.3 in japan , addressing chronic pain particularly low back pain ( lbp ) is becoming an important issue in medical care .
lbp is a common condition in japan and is the fifth most widely cited reason for outpatient medical visits.4 a recent study found that the prevalence of chronic musculoskeletal pain in japan was 15.4% , with the lower back being reported as one of the most common sites for pain.5 another study assessing the societal burden of chronic pain in japan also found that back pain was one of the most common pain types and was reported by more than half of the respondents with chronic pain.6 chronic lbp ( clbp ) can lead to severe activity impairment and physical limitations.7 in addition to its physical impact , various studies have found that individuals with clbp also have significantly greater levels of depression , anxiety , and sleep impairment when compared with matched controls without clbp.8 clbp is also associated with higher use of health care services and greater work impairment.8,9 one study found that the total direct medical costs for those with clbp were more than twice as high when compared with controls who did not have clbp.8 another study found that workers with back pain had work impairment levels between 1.54 and 1.77 times the levels of employees who had no back pain.10 in spite of its documented impact , clbp has not been adequately studied in many populations.1 in particular , there is a dearth of literature exploring the relationship between the severity of clbp and mental and physical health outcomes , health care costs , and work productivity of patients suffering from this condition in japan .
research from sweden has found that both medical costs and productivity losses were significantly correlated with severity of clbp.11 similar results have been found in research conducted in other countries.12,13 the relationship between increasing severity of pain and worse outcomes is not unique to clbp but has been noted in other chronic pain conditions as well .
for example , osteoarthritis and painful diabetic peripheral neuropathy demonstrate a similar relationship between increasing pain severity levels and poorer outcomes in terms of increased health care costs , greater impairment at work , and impairment in performing daily activities.1315 chronic pain conditions not only can have an adverse impact on daily lives of patients but can also affect the people around them,16 further underscoring the importance of effective management of chronic pain conditions , including clbp .
identifying poorer outcomes at varying severity levels can inform the selection of appropriate management strategies and treatment options.7 this study was conducted to document the relationship between the severity of pain and a range of patient outcomes among japanese patients with clbp .
outcomes of interest included health - related quality of life ( hrqol ) , depression and anxiety symptoms , work productivity , activity impairment , and health care resource use .
data were provided by the 2012 japan national health and wellness survey ( nhws ; kantar health , new york , ny , usa ) , an annual internet - based survey of the general population aged 18 years and older in japan . the nhws was designed as a general health questionnaire and includes items regarding experience and diagnosis of a variety of health conditions , demographics , and several standard outcome measures , which are described in more detail in the following sections .
potential respondents were selected from an opt - in survey panel through stratified random sampling , with strata organized by age and sex that were specified to match the population .
in addition to membership in the survey panel , respondents were required to read and write japanese , be at least 18 years old , and provide informed consent .
the protocol and questionnaire for the nhws were reviewed and approved by essex institutional review board , inc .
this analysis was focused exclusively on respondents with clbp , which is defined in the measures section .
the flow of respondents from sampling through inclusion in the analyses is depicted in figure 1 .
clbp : respondents were considered to have clbp if they experienced lbp in the past month and had also been diagnosed by a physician with lbp for at least 3 months .
respondents who also reported pain due to broken bones , cancer , or surgery were excluded , as were respondents with other types of pain , which they rated as more severe than they rated their lbp on the 3-point scale mentioned in the following sections .
pain severity : severity of pain in the past week was reported on numeric rating scale ( nrs ) ranging from 0 ( no pain ) to 10 ( pain as bad as you can imagine ) .
patients also rated the severity of their clbp as well as other types of pain on a 3-point scale as mild , moderate , or severe , separately for each type of pain .
hrqol : the revised medical outcomes study 36-item short - form survey instrument ( sf-36v2 ) was used to measure the hrqol.17 this is a multipurpose , generic hrqol instrument .
two summary scores calculated from this measure were used : the physical component summary ( pcs ) , an index of overall physical functioning , and the mental component summary ( mcs ) scores , which is an index of mental and emotional health .
scores can be interpreted relative to the us population average of 50 with a standard deviation of 10 , with higher scores indicating better hrqol .
responses to the sf-36v2 were also used to generate health state utilities according to the short - form 6d ( sf-6d ) algorithm , a preference - based , single index measure for health using general population values.18 the sf-6d index has interval scoring properties and yields summary scores on a theoretical 01 scale ( with an empirical floor of 0.3 ) .
higher scores indicate better ( more preferred ) health status , with 1 being equivalent to perfect health .
depression symptoms : depression symptoms / severity of depression over the last 2 weeks was assessed using the patient health questionnaire-9,19 a validated scale used to screen for depression and to assess its severity .
the total scale score was calculated , and the severity of depression was assigned according to the standard cutoff categories : minimal ( 04 ) , mild ( 59 ) , moderate ( 1014 ) , moderately severe ( 1519 ) , and severe ( 20 or greater ) . anxiety symptoms : symptoms of anxiety were measured using the generalized anxiety disorder-7 scale.20 the total score was analyzed both as a continuous measure and according to the standard categories : minimal ( 04 ) , mild ( 59 ) , moderate ( 1014 ) , and severe ( 15 or greater ) .
work productivity and activity impairment : work productivity was assessed using the general health version of the work productivity and activity impairment questionnaire , which assesses absenteeism ( work time missed ) , presenteeism ( impairment while at work ) , overall work productivity impairment ( a combination of absenteeism and presenteeism ) , and activity impairment ( impairment in daily activities ) due to health problems over the prior 7 days.21 all are reported as percentages , with higher numbers indicating greater impairment .
only respondents who reported being employed full - time , part - time , or self - employed provided data for absenteeism , presenteeism , and overall work impairment . all respondents provided data for activity impairment .
health care resource use : health care resource use was assessed in the nhws as all - cause health care provider ( hcp ) visits , emergency room ( er ) visits , and hospitalizations in the 6 months prior to the survey .
hcp visits were calculated by summing the self - reported number of visits to specific types of hcps ( eg , general internist , psychiatrist , allergist , dentist , and nurse).statistical analysis respondents who reported a physician diagnosis of clbp were first described with univariate statistics .
bivariate spearman correlations were then calculated to assess the strength of the relationship between severity of pain in the past week and health outcomes .
a series of generalized linear models were conducted to assess the relationship between the severity and clbp and outcomes while accounting for potential confounders . because the rating of pain severity in the past week was modeled as a continuous variable , means at the first quartile , median , and the third quartile value of severity of pain in the past week were estimated to demonstrate outcomes associated with mild , moderate , and severe pain , respectively .
an additional set of generalized linear models were conducted using self - ratings of clbp as mild , moderate , or severe as dummy - coded categories to ensure the pattern of results was consistent using a rating of severity specific to lbp .
the specifications of the models varied based on the outcome being modeled . health status and the sf-6d
were modeled using linear models , while work productivity and activity impairment and health care visits were modeled using negative binomial distributions and log link functions .
covariates included duration of clbp , age , sex , charlson comorbidity index calculated from self - reported diagnoses,22 annual household income , marital status , completion of a university degree , body mass index category , smoking status , alcohol use , and exercise in the prior month .
an alpha error level of 5% ( two - tailed ) was used for all significance tests .
data were provided by the 2012 japan national health and wellness survey ( nhws ; kantar health , new york , ny , usa ) , an annual internet - based survey of the general population aged 18 years and older in japan . the nhws was designed as a general health questionnaire and includes items regarding experience and diagnosis of a variety of health conditions , demographics , and several standard outcome measures , which are described in more detail in the following sections .
potential respondents were selected from an opt - in survey panel through stratified random sampling , with strata organized by age and sex that were specified to match the population .
in addition to membership in the survey panel , respondents were required to read and write japanese , be at least 18 years old , and provide informed consent .
the protocol and questionnaire for the nhws were reviewed and approved by essex institutional review board , inc .
this analysis was focused exclusively on respondents with clbp , which is defined in the measures section .
the flow of respondents from sampling through inclusion in the analyses is depicted in figure 1 .
clbp : respondents were considered to have clbp if they experienced lbp in the past month and had also been diagnosed by a physician with lbp for at least 3 months . respondents who also reported pain due to broken bones , cancer , or surgery
were excluded , as were respondents with other types of pain , which they rated as more severe than they rated their lbp on the 3-point scale mentioned in the following sections .
pain severity : severity of pain in the past week was reported on numeric rating scale ( nrs ) ranging from 0 ( no pain ) to 10 ( pain as bad as you can imagine ) .
patients also rated the severity of their clbp as well as other types of pain on a 3-point scale as mild , moderate , or severe , separately for each type of pain .
hrqol : the revised medical outcomes study 36-item short - form survey instrument ( sf-36v2 ) was used to measure the hrqol.17 this is a multipurpose , generic hrqol instrument .
two summary scores calculated from this measure were used : the physical component summary ( pcs ) , an index of overall physical functioning , and the mental component summary ( mcs ) scores , which is an index of mental and emotional health .
scores can be interpreted relative to the us population average of 50 with a standard deviation of 10 , with higher scores indicating better hrqol .
responses to the sf-36v2 were also used to generate health state utilities according to the short - form 6d ( sf-6d ) algorithm , a preference - based , single index measure for health using general population values.18 the sf-6d index has interval scoring properties and yields summary scores on a theoretical 01 scale ( with an empirical floor of 0.3 ) .
higher scores indicate better ( more preferred ) health status , with 1 being equivalent to perfect health .
depression symptoms : depression symptoms / severity of depression over the last 2 weeks was assessed using the patient health questionnaire-9,19 a validated scale used to screen for depression and to assess its severity .
the total scale score was calculated , and the severity of depression was assigned according to the standard cutoff categories : minimal ( 04 ) , mild ( 59 ) , moderate ( 1014 ) , moderately severe ( 1519 ) , and severe ( 20 or greater ) .
anxiety symptoms : symptoms of anxiety were measured using the generalized anxiety disorder-7 scale.20 the total score was analyzed both as a continuous measure and according to the standard categories : minimal ( 04 ) , mild ( 59 ) , moderate ( 1014 ) , and severe ( 15 or greater ) .
work productivity and activity impairment : work productivity was assessed using the general health version of the work productivity and activity impairment questionnaire , which assesses absenteeism ( work time missed ) , presenteeism ( impairment while at work ) , overall work productivity impairment ( a combination of absenteeism and presenteeism ) , and activity impairment ( impairment in daily activities ) due to health problems over the prior 7 days.21 all are reported as percentages , with higher numbers indicating greater impairment .
only respondents who reported being employed full - time , part - time , or self - employed provided data for absenteeism , presenteeism , and overall work impairment .
health care resource use : health care resource use was assessed in the nhws as all - cause health care provider ( hcp ) visits , emergency room ( er ) visits , and hospitalizations in the 6 months prior to the survey .
hcp visits were calculated by summing the self - reported number of visits to specific types of hcps ( eg , general internist , psychiatrist , allergist , dentist , and nurse).statistical analysis
bivariate spearman correlations were then calculated to assess the strength of the relationship between severity of pain in the past week and health outcomes .
a series of generalized linear models were conducted to assess the relationship between the severity and clbp and outcomes while accounting for potential confounders . because the rating of pain severity in the past week was modeled as a continuous variable , means at the first quartile , median , and the third quartile value of severity of pain in the past week were estimated to demonstrate outcomes associated with mild , moderate , and severe pain , respectively .
an additional set of generalized linear models were conducted using self - ratings of clbp as mild , moderate , or severe as dummy - coded categories to ensure the pattern of results was consistent using a rating of severity specific to lbp .
health status and the sf-6d were modeled using linear models , while work productivity and activity impairment and health care visits were modeled using negative binomial distributions and log link functions .
covariates included duration of clbp , age , sex , charlson comorbidity index calculated from self - reported diagnoses,22 annual household income , marital status , completion of a university degree , body mass index category , smoking status , alcohol use , and exercise in the prior month .
an alpha error level of 5% ( two - tailed ) was used for all significance tests .
743 indicated a doctor diagnosis of lbp , with 356 indicating they experienced lbp for 3 or more months .
ten patients were excluded due to report of broken bones , cancer , or surgery as a source of comorbid pain , and a further 56 for having concomitant painful conditions with more severe pain than their lbp , resulting in 290 patients with clbp included in this analysis .
the sample had a mean age of 56 years ; 41% were females and 56% were employed .
most of the sample rated their lbp as moderate ( 56% ) , and the average severity of pain on the nrs was 4.9 out of 10 ; the 25th , 50th ( median ) , and 75th percentile values for pain were 3/10 , 5/10 , and 7/10 , respectively .
the great majority of respondents had been diagnosed by an orthopedist ( 85% ) , and 41% were taking prescription medications for pain , primarily nonsteroidal anti - inflammatory drugs ( 68% of those using a prescription ) . increasing severity of pain in the past week
more pain was associated with lower hrqol , with correlations ranging from rs = 0.25 to 0.47 .
employed respondents impairment at work was highly correlated with the level of pain , with pain level accounting for 27% of the variance in overall work impairment ( rs = 0.52 ) . likewise ,
hcp and er visits were related to severity of pain , though the relationship was not strong .
almost all the relationships between increasing severity of pain and worse outcomes remained significant after taking into account covariates ( table 4 ) . a change in pain from the 25th to the 75th percentile level of pain
was associated with a 3.9-point drop in mcs , a 5.5-point drop in pcs , and a 0.07-point drop in health utility .
the differences in depression and anxiety were modest in size but statistically significant . among employed respondents ,
the same increase in pain was associated with a 5% absolute increase in absenteeism , a 25% absolute increase in presenteeism , and a 29% absolute increase in overall work impairment .
the level of impairment to nonwork activities at the 75th percentile level of pain was more than double that of the 25th percentile level .
as pain is reduced from the 75th percentile level to the 25th percentile , the number of hcp visits during a 6-month period was reduced by 3.5 , though no differences were observed in emergency department visits or hospitalizations .
743 indicated a doctor diagnosis of lbp , with 356 indicating they experienced lbp for 3 or more months .
ten patients were excluded due to report of broken bones , cancer , or surgery as a source of comorbid pain , and a further 56 for having concomitant painful conditions with more severe pain than their lbp , resulting in 290 patients with clbp included in this analysis .
the sample had a mean age of 56 years ; 41% were females and 56% were employed .
most of the sample rated their lbp as moderate ( 56% ) , and the average severity of pain on the nrs was 4.9 out of 10 ; the 25th , 50th ( median ) , and 75th percentile values for pain were 3/10 , 5/10 , and 7/10 , respectively .
the great majority of respondents had been diagnosed by an orthopedist ( 85% ) , and 41% were taking prescription medications for pain , primarily nonsteroidal anti - inflammatory drugs ( 68% of those using a prescription ) .
increasing severity of pain in the past week was correlated with worse outcomes ( table 3 ) . more pain was associated with lower hrqol , with correlations ranging from rs = 0.25 to 0.47 .
employed respondents impairment at work was highly correlated with the level of pain , with pain level accounting for 27% of the variance in overall work impairment ( rs = 0.52 ) . likewise ,
hcp and er visits were related to severity of pain , though the relationship was not strong .
almost all the relationships between increasing severity of pain and worse outcomes remained significant after taking into account covariates ( table 4 ) . a change in pain from the 25th to the 75th percentile level of pain
was associated with a 3.9-point drop in mcs , a 5.5-point drop in pcs , and a 0.07-point drop in health utility .
the differences in depression and anxiety were modest in size but statistically significant . among employed respondents ,
the same increase in pain was associated with a 5% absolute increase in absenteeism , a 25% absolute increase in presenteeism , and a 29% absolute increase in overall work impairment .
the level of impairment to nonwork activities at the 75th percentile level of pain was more than double that of the 25th percentile level .
as pain is reduced from the 75th percentile level to the 25th percentile , the number of hcp visits during a 6-month period was reduced by 3.5 , though no differences were observed in emergency department visits or hospitalizations .
the current study revealed significant relationships between the severity of pain among patients with clbp and a range of health outcomes relating to hrqol in terms of both physical and mental health outcomes , work productivity , activity impairment , and use of health care resources among japanese adults .
as pain severity increased from the first quartile ( 3/10 ) to the third ( 7/10 ) , the differences observed in hrqol surpassed the minimally important differences for these measures , which are 3 points for the mcs and pcs scores,17 and 0.03 points for the sf-6d index.23 this demonstrates that meaningful decreases in hrqol are associated with increasing pain severity among patients with clbp and builds on earlier research conducted in five medical centers that estimated that diagnosed lbp ( not necessarily chronic ) led to a loss of 947,000 quality - adjusted life years in japan.24 severity of pain was also associated with increased levels of anxiety and depression among these patients , consistent with other evidence indicating that psychological issues can be associated with the manifestations and progression of clbp.24 as pain becomes more severe , patients may need greater support addressing the psychological effects of the condition , in addition to the physical effects . furthermore , higher severity of pain was correlated with impairment in both nonwork activities and worker productivity , demonstrating the far - ranging effects of pain on various aspects of daily life . as the intensity of pain increased from 3/10 to 7/10 on the nrs , both nonwork activity impairment and overall work impairment more than doubled .
this is in line with other research that found more patients with severe clbp had to switch their job roles compared with patients who had lower pain severity.9 the increase in overall work impairment among nhws respondents was mainly due to higher rates of presenteeism , though absenteeism also rose 5% , which would equate to 2 hours per worker per 40-hour workweek .
although interventions that support timely work return and reduce productivity loss for patients with clbp could result in considerable cost savings,25 targeting individuals who have the most severe pain levels might offer the greatest savings .
these results also demonstrate an opportunity for employers to offer worksite wellness programs that treat clbp and ensure that those diagnosed with clbp do not progress in their pain severity levels .
pain at the 75th percentile was associated with approximately seven additional hcp visits per year .
this is consistent with findings from studies in other countries that assessed use of outpatient care and found that direct medical costs increased proportionately with worsening pain severity among patients with clbp.11,12 ensuring individuals who experience lbp are offered appropriate treatment and support in an effort to reduce or at least halt the progression of their pain may help contain such costs .
the number of er visits and the number of hospital admissions were not significantly related to pain severity ; however , the sample size of the current study was too small to detect relationships between severity and relatively rare events such as these .
overall , the current study provides further evidence of increased health care use and contributes to existing literature indicating increased direct medical costs among these patients.11,12 this analysis does have potential limitations . although other studies found that severity of clbp was associated with significant medication use,7 this study did not look at medication costs or the charges associated with health care resource use , only the resource use itself .
the analysis was based on an internet survey , which may limit the findings generalizability by including only the study respondents who use the internet and computers .
another limitation is the use of patient - reported data , particularly with measures of physical functioning .
research suggests that patients with clbp may be more likely to underestimate their activity,26 so future research should confirm the relationship with measures of activity limitation that do not rely on self - report .
additionally , in order to be included in the current analysis , respondents had to identify pain as one of the medical conditions they experienced in the prior 12 months .
this may have resulted in the identification of fewer patients than if all respondents to the nhws had been asked their level of pain without having to identify pain as a medical condition that they experienced and may partially account for the disparity between the prevalence in previous survey results and those reported here .
part of the disparity is also likely due to the numerous exclusion criteria adopted here in order to ensure other pain was not misattributed to clbp , particularly other types and sites of pain .
indeed , 2,226 of the 29,997 respondents ( 7.4% ) reported experiencing lbp in the past month , but most were excluded for lack of diagnosis or comorbidities .
however , focusing on respondents with relatively pure clbp allows the current study to demonstrate the relationship between pain severity and outcomes with the fewest potential confounders .
this analysis does have potential limitations . although other studies found that severity of clbp was associated with significant medication use,7 this study did not look at medication costs or the charges associated with health care resource use , only the resource use itself .
the analysis was based on an internet survey , which may limit the findings generalizability by including only the study respondents who use the internet and computers .
another limitation is the use of patient - reported data , particularly with measures of physical functioning .
research suggests that patients with clbp may be more likely to underestimate their activity,26 so future research should confirm the relationship with measures of activity limitation that do not rely on self - report . additionally , in order to be included in the current analysis , respondents had to identify pain as one of the medical conditions they experienced in the prior 12 months
this may have resulted in the identification of fewer patients than if all respondents to the nhws had been asked their level of pain without having to identify pain as a medical condition that they experienced and may partially account for the disparity between the prevalence in previous survey results and those reported here .
part of the disparity is also likely due to the numerous exclusion criteria adopted here in order to ensure other pain was not misattributed to clbp , particularly other types and sites of pain . indeed , 2,226 of the 29,997 respondents ( 7.4% ) reported experiencing lbp in the past month , but most were excluded for lack of diagnosis or comorbidities .
however , focusing on respondents with relatively pure clbp allows the current study to demonstrate the relationship between pain severity and outcomes with the fewest potential confounders .
this study provides a valuable addition to the literature by examining the relationship between the severity of pain and a range of outcomes specifically among patients with clbp in japan , which has been understudied relative to other areas of the world .
our results demonstrate that more severe pain among patients with clbp is significantly related to reduced physical functioning , mental health , worker productivity , and increased health care use in japan .
this underscores the importance of not only targeting and treating back pain but also assessing patients severity levels in order to develop effective treatments and interventions . due to the impact of clbp on hrqol , health care use , worker productivity , and daily activities
, we suggest that patients , insurers , and employers can all benefit from better treatment and management strategies addressing this condition.11 | objectivethe aim of this study was to quantify the impact of pain severity on patient - reported outcomes among individuals diagnosed with chronic low back pain in japan.methodsdata were provided by the 2012 japan national health and wellness survey ( n=29,997 ) , a web - based survey of individuals in japan aged 18 years .
this analysis included respondents diagnosed with low back pain of 3-month duration .
measures included the revised medical outcomes study 36-item short - form survey instrument , the patient health questionnaire-9 , the generalized anxiety disorder-7 scale , the work productivity and activity impairment : general health questionnaire , and self - reported all - cause health care visits ( 6 months ) .
generalized linear models were used to assess the relationship between outcomes and severity of pain in the past week as reported on a numeric rating scale ranging from 0 ( no pain ) to 10 ( pain as bad as you can imagine ) , controlling for length of diagnosis , sociodemographics , and general health characteristics.resultsa total of 290 respondents were included in the analysis ; mean age was 56 years , 41% were females , and 56% were employed .
pain severity was 3/10 for the first quartile , 5/10 for the median , and 7/10 for the third quartile of this sample .
increasing severity was associated with lower scores for mental and physical component summaries and short - form 6d health utility , higher depression ( patient health questionnaire-9 ) and anxiety ( generalized anxiety disorder-7 ) scores , greater absenteeism and presenteeism , greater activity impairment , and more health care provider visits ( all p<0.0001).conclusionthe impact of chronic low back pain on health - related quality of life , depression and anxiety symptoms , impairment to work and daily activities , and health care use increases with the severity of pain .
interventions reducing the severity of pain may improve numerous health outcomes even if the pain can not be eliminated . |
. there are no absolute anatomical landmarks for reference and the foramen can not be clinically visualized or palpated . prior to surgery ,
one of the inadvertent complications that occur during implant placement in anterior mandible is a neurosensory alteration in the chin and lower lip due to improper identification and protection of the mental foramen and anterior mental loop .
on radiograph the mental foramen presents as a single circular or elliptical radiolucent area bilaterally in the premolar region .
several studies comparing different radiographic methods to assess the location of the mental foramen have suggested that digital panoramic radiographs show least error in the imaging of the mental foramen .
green et al . demonstrated clear racial differences in the position of the mental foramen . in the present study
, the change in position of mental foramen in different age groups and gender related variations were evaluated using digital panoramic radiography .
the study group comprised a total of 250 patients who required panoramic radiography for various dental treatments .
the age groups were 6 - 12 years , 13 - 18 years , 19 - 30 years , 31 - 50 years , and above 50 years .
each age group was divided into two subgroups based on gender having 25 subjects in each subgroup .
digital panoramic radiographs were taken using the digital panoramic radiography machine , planmeca promax ( helsinki , finland ) with a total filtration of 2.5 mm aluminium ( al ) equivalent .
each radiograph was standardized while taking the radiograph by tilting the patient 's head 5 downward with reference to frankfurt 's horizontal plane as suggested by dharma et al .
position of mental foramen ( x ) was fixed in relation to three reference points [ figure 1 ] : parameters measured in the study : alveolar crest ( a ) , base of mandible ( b ) , median reference line ( c ) , and position of mental foramen ( x )
alveolar bone crest ( a)base of mandible ( b)a midline reference point ( c ) ( a line is drawn from the anterior nasal spine to the most inferior portion of mentis ) .
alveolar bone crest ( a ) a midline reference point ( c ) ( a line is drawn from the anterior nasal spine to the most inferior portion of mentis ) .
measurements in millimeters were taken on both sides of the mandible according to the following parameters :
distance from the reference line to mental foramendistance from the alveolar crest to mental foramendistance from the base of mandible to mental foramen .
distance from the reference line to mental foramen distance from the alveolar crest to mental foramen distance from the base of mandible to mental foramen .
there was no statistical difference between the positions of the mental foramen on either side of a patient and so the mean was taken as a single measurement per sample .
the data collected were subjected to statistical analysis using paired student 's t - test .
the mean distance of position of mental foramen from reference line , alveolar bone crest , and the base of mandible of each age group are shown in figure 2 .
comparisons between male and female patients for each age group are given in tables 15 .
graph showing the mean distance of mental foramen position in each age group comparison between males and females in the age group 6 - 12 years comparison between males and females in the age group 13 - 18 years comparison between males and females in the age group 19 - 30 years comparison between males and females in the age group 31 - 50 years comparison between males and females in the age group above 50 years in the 6 - 12 years age group : statistical correlation showed a highly significant increase in the distance of mental foramen from base of mandible in female patients ( p < 0.01 ) , and a marginally statistically significant increase in the mean distance of mental foramen from reference line in the female patients ( p < 0.05 ) when compared with the male patients . in the 13 - 18 years ,
19 - 30 years , and 31 - 50 years age group : statistical correlation showed a significant increase in the distance of mental foramen from base of the mandible , reference line , and alveolar crest in the male patients ( p < 0.01 ) when compared with the female patients . in the above 50 years age group : statistical correlation showed a significant increase in the distance of mental foramen from reference line and alveolar crest in the male patients ( p < 0.01 ) when compared with the female patients .
both male and females patients showed no statistically significant difference in the distance of the mental foramen from the base of mandible ( p 0.5 ) .
anatomically the mental foramen lies midway between the free alveolar border and the border of the mandible closer to the latter .
it varies in position according to the age and sex as substantiated in several previous studies .
the aim of our study was to determine the radiographic position of mental foramen in relation to anatomical structures in different age groups in males and females .
the age groups were determined according to different stages of growth and development as prepubertal , pubertal , adolescent , middle aged , and elderly people .
panoramic radiographs have advantages like bilateral visualization of anatomical structures , broader visualization of hard tissue areas , and the ability to visualize adjacent areas , thus allowing for a more standardized localization of mental foramen .
mental foramen position may not be revealed in a periapical radiographic film , if the position of mental foramen falls below the edge of the film , previous studies comparing digital panoramic images and conventional panoramic images showed that the measurement errors was less in the former .
the variation in position of the mental foramen between females and males in group 1 may be attributed to the hormonal changes induced in growth spurts . in 1927 , hellman identified gender differences in the timing , extent , velocity , and intensity of facial growth and recognized that these changes result in an increase in size and a change in the proportions of the face .
this spurt in the peak of incremental growth has been found to occur approximately 2 years earlier in females than males , at mean ages of 12 years and 14 years , respectively .
the study by ochoa and nanda et al . showed that the female subjects tended to have the greatest skeletal changes between 10 years and 14 years of age .
the male patients had the greatest changes between 12 years and 16 years of age and even up to 18 years of age . in groups 2 - 5
, male patients showed a statistically significant increase in the mean distance of mental foramen position from the midline and alveolar crest .
this change in the mean distance of mental foramen position could be due to the growth spurt favoring males after the age of 12 and lag of growth spurt in females .
the female patients demonstrated a relative slow down of growth after 14 years of age in mandibular length , whereas in the male patients jaws continued to grow significantly until 16 years of age .
the first and fifth groups showed a reduced mean distance of mental foramen position when compared to second , third , and fourth groups . in the fifth age group ,
the mean distance of mental foramen position from the alveolar bone crest showed the least value when compared to other groups .
this may be due to bone remodeling as age advances . in a study conducted by gershenson in 1986
, he found that in children before tooth eruption , the mental foramen is somewhat closer to the alveolar margin ; during the eruption period , the mental foramen descends to half way between the margins and in adults with the teeth preserved , the mental foramen is somewhat closer to the inferior border .
with loss of teeth and bone resorption , the mental foramen moves upward closer to the alveolar border .
the mental foramen position is not constant and changes as age advances . apart from this ,
there is a gender - related variation in the position of mental foramen in each age group we have studied .
these variations in the position of the mental foramen have been reported to exhibit a clear trend in the pattern of their occurrence in relation to both age and gender .
an accurate assessment of position of mental foramen is very useful for the prevention of postsurgical neurovascular complications and also holds the potential of contributing as an aid for forensic identification .
| objective : comparative evaluation of the location of mental foramen in different age groups .
determine the variation in position of mental foramen with gender using digital panoramic radiography.materials and methods : digital panoramic radiographs of 250 patients were reviewed .
the study population was divided into five age groups with 50 patients each .
radiographic position of mental foramen was evaluated in each radiograph based on three parameters .
measurements were taken in each radiograph using planmeca dimaxis pro version 4.4.0 ( helsinki , finland ) .
the collected data were subjected to statistical analysis using paired student 's t-test.results:the mean distance of position of mental foramen showed a significant variation within the five age groups . in the first group , female patients showed an increase in mean distance of mental foramen position in relation to three parameters . from the second to fifth groups , male patient showed an increase in the mean distance of mental foramen position .
the first and fifth group showed a reduced mean distance of mental foramen position when compared to other age groups.conclusion:this study concluded that the position of mental foramen varies with age .
there was a gender - related variation in position of mental foramen within the population too . |
the collagenous fibroma ( cf ) , is a slow - growing , benign , soft tissue lesion that was first described as recent as 1995 by evans , under the rubric ,
as the term fibroblastoma may be construed as an immature or potentially malignant neoplasm , nielsen et al proposed the name
collagenous fibroma as being not only more descriptive of its histological composition but also more reflective of its benign prognosis .
approximately 78 additional cases have been reported since then , describing the lesion better.[27 ] miettinen and fetsch have till date discussed 63 cases , the largest series ever .
the results obtained from all reports indicate clearly that cf has a propensity to occur in males in the fifth to sixth decades of life , presenting as subcutaneous or intramuscular lesions , having a wide anatomic distribution .
mesquita et al have recently claimed that their case was the first such cf seen intraorally .
the cf presents as a slow - growing neoplasm of more than 6 months duration , measuring anywhere between 1 cm and 20 cm .
clinically , the lesion is seen as a firm , well - circumscribed , round to oval , or lobulated mass , which on cut surface appears fibrous with a glistening gray - to - white aspect .
microscopically , the overlying surface epithelium of cf is normal , although fat or skeletal muscle may be seen either entrapped between the rete pegs or in the center of the lesion .
the neoplasm consists of sparsely distributed , medium to large , spindle- to stellate - shaped fibroblastic cells , interspersed among abundant intercellular collagen fibers . in dense fibrotic areas , cellularity is scarce and the cells do not exhibit any mitotic activity , nor is there any necrosis .
a few cells may show a positive immunoreactivity for muscle - specific and alpha - smooth muscle actin .
sciot et al studied two cfs and found that both exhibited an abnormality of the same band of the long arm of chromosome 11 , more specifically , a rearrangement of 11 q 12 . because the fibroma of the tendon sheath showed the same chromosomal aberration as that of the oral lesion
a 58-year - old female patient was referred to the department of oral and maxillofacial surgery , at nims dental college , jaipur , rajasthan , due to a complaint of a firm , slow - growing , pedunculated lesion measuring 2 cm 1 cm , in the lingual gingival region in relation to the permanent mandibular left 3 molar since 3 months .
intraoral examination showed a well - circumscribed , firm mass , covered by normal mucosa .
the related teeth were not mobile , ruling out underlying bone involvement , and intraoral radiographs confirmed the same .
the lesion was completely excised under local anesthesia and the specimen was sent to the department of oral pathology , nims dental college , jaipur , rajasthan , for histopathological evaluation .
5m sections were made and stained with hande , and 3 m sections were made for immunohistochemical analysis , by subjecting the sections to the streptavidin method .
the immunohistochemical analysis was performed by using primary antibodies against vimentin ( 1:200 ) ( biogenex , san ramon , california ) .
antigen retrieval for vimentin was accomplished by microwave treatment ( 3 times for 5 minutes ) at 700 w in citric acid ( 10 mmol / l , ph 6.0 ) .
the above - mentioned immunohistochemical analysis was performed in the department of histopathology at santokba durlabhji memorial hospital , jaipur , rajasthan .
the tissue received after the excisional biopsy was white in color , firm , shiny on cut surface with a soft yellowish area in the center of the cut surface .
sections stained with hande were observed under 4 , 10 , and 40 to reveal a surface mucosa which was normal , overlying a dense , homogenous , eosinophilic connective tissue . the connective tissue consisted of dense collagen bundles
in which were seen scarcely distributed spindle to stellate fibroblastic cells [ figure 1 ] . multinucleated giant cells were also observed .
a peculiar , yet unique feature , seen in our case , was an area in the center of the section , consisting of fat cells [ figure 2 ] .
sections were also subjected to the masson 's trichrome stain which revealed areas positive for collagen ( blue - green ) and some areas positive for muscle ( red ) .
the cytoplasm of the lesional cells took up a red stain [ figure 3 ] .
scant distribution of lesional fibroblastic cells in dense collagenous matrix ( h and e , 10 ) fat cells in centre of section ( h and e , 4 ) section shows collagen ( blue - green ) and muscle ( red ) ( masson 's trichrome , 4 )
immunohistochemically , the lesional fibroblastic cells were strongly positive for vimentin [ figure 4 ] .
the collagen bundles were seen as long and circular thick bundles , distributed in a random manner throughout the section .
in the oral cavity , the differential diagnosis for cf should include a wide range of soft tissue lesions , especially inflammatory fibrous hyperplasia , traumatic fibroma , and giant cell fibroma .
other oral soft tissue lesions that are seen less frequently , e.g. , soft tissue myofibroma , nodular fasciitis , fibromatosis , and neurofibroma , can also be considered in the differential diagnosis for oral cf .
the clinical and histopathological features of our case comply with the criteria for the diagnosis of cf reported by evans and nielsen et al who delineated desmoplastic fibroblastoma and cf , respectively .
clinically , our case appeared somewhat similar to the previously described cases . on gross examination ,
the lesion was seen as a firm mass , which on cut surface appeared shiny and included a central area of soft , yellowish tissue , which on histopathological evaluation turned out to be fatty tissue .
a similar feature has been described by miettinen and fetsch who also observed entrapment of fat in 51% of their series of 63 cases .
a doubt still persists as to the true nature of cf . according to evans and nielsen et al
, cf is a neoplasm , as it does not have a preceding traumatic or inciting cause .
our patient also did not give any history of trauma or any other triggering event .
the nature of the neoplastic cells in cf has been described by evans and nielsen et al , which have features of both fibroblasts and myofibroblasts .
in the oral cavity , the differential diagnosis should include giant cell fibroma as well as a whole range of soft tissue lesions such as , inflammatory fibrous hyperplasia and the simple fibroma .
the last mentioned lesion , i.e. , the simple fibroma is of great significance , as we feel , that a large number of cf may have been diagnosed in the past as the simple fibroma . in cf ,
the collagen presents as long , thick , and wavy bundles with a random distribution .
the fibroma also displays thick and wavy bundles of collagen with a random distribution , but the bundles are shorter than those seen in cf .
moreover , entrapment of adjacent fat and muscle appears to be a unique feature of cf .
our aim was to draw the attention of all oral pathologists to review cases in the past diagnosed as fibromas , which could possibly be cf / desmoplastic fibroblastomas instead .
the current treatment of cf is complete surgical excision as the prognosis is good thereafter . | collagenous fibroma ( desmoplastic fibroblastoma ) is a distinctive yet uncommon fibrous soft tissue tumor .
these tumors are rather nondescript in their morphological appearance and have been diagnosed as fibromas or some other benign mesenchymal lesions for years .
the most common sites are the upper extremities , followed by the lower extremities .
rare lesions arise in the head and neck region .
we report a rare case in the oral cavity and present its unique histopathological features ( central fat entrapment ) besides others , and diffusely strong vimentin immunopositivity . |
a human oral buccal epithelial cell line ho-1-n-1 was provided by the japanese collection of research bioresources ( osaka , japan ) .
ho-1-n-1 is an immortalized epithelial cell - like cell line that originated from a squamous cell carcinoma located on the buccal mucosa .
the epithelial cells were grown in tissue culture flasks with dulbecco 's modified eagle 's medium - f12 ( invitrogen , carlsbad , ca , usa ) supplemented with 10% fetal calf serum ( hyclone , logan , ut , usa ) , 2% antibiotics ( penicillin , streptomycin and fungizone , sigma , st .
louis , mo , usa ) in a humidified atmosphere at 37c with 5% co2 .
all p. gingivalis strains were grown anaerobically in brain heart - infusion ( bd difco , le pont de claix , france ) broth enriched with hemin ( 5 mg / l ) and menadione ( 1 mg / l ) until mid - log phase . in case of mutant selection and growth , 50 g / ml of erythromycin ( em ) or 1 g /
bacterial cultures were washed twice with dulbecco 's phosphate - buffered saline ( dpbs , invitrogen ) and resuspended in keratinocyte serum - free medium ( sfm ; invitrogen ) at od690 0.1 ( 510 cfu / ml ) .
experiments were performed with either viable or heat - inactivated bacteria , in which case the bacterial cultures were heated at 60c for 60 min .
p. gingivalis strains used in this study the influence of lps from p. gingivalis was tested by adding pure lps from p. gingivalis to the epithelial cells or by blocking lps in the cell wall of p. gingivalis .
ultrapure lps from p. gingivalis was purchased ( invivogen , toulouse , france ) and used in the assay in a concentration of 10 g / ml . to exclude the influence of lps , polymyxin b ( sigma aldrich , st .
louis , mo ) , an lps inhibitor , was added to 510 cfu / ml heat - inactivated p. gingivalis atcc 33277 in a concentration of 50 g / ml . besides using gingipain mutants from two wild - type strains , the influence of gingipain activity was also studied by using gingipain inhibitors .
kyt-1 ( an arginine gingipain inhibitor ) and kyt-36 ( a lysine gingipain inhibitor ) peptides ( 15 ) ( peptide institute , osaka , japan ) were added in the assay to viable p. gingivalis w50 at a concentration of 110 mol / ml .
cell cultures were trypsinized and seeded in 24-well plates at 310 to 510 cells / ml . when confluency was reached , a scratch with a sterile 1-ml pipet point was made in each well .
the wells were washed three times with dpbs and challenged with viable or heat - inactivated bacteria or lps from p. gingivalis . in control wells , only sfm was added .
bacteria were added at a multiplicity of infection ( moi ) of 1,000 ( 1,000 bacteria vs. 1 epithelial cell ) , 100 , and 10 .
most p. gingivalis cells remained viable and metabolically active until the end of the experiment .
images were taken of the middle of the well immediately after adding the bacteria and 17 h later with an inverted digital phase - contrast microscope ( advanced microscopy group , evos , fl ) .
the surface of the scratch was calculated with photoshop cs4 ( version 11.0.1 , adobe ) .
after 17 h , closure of the wound under control conditions varied between 60 and 80% .
in addition , several images were taken to analyze migration of epithelial cells over time and to construct a time - lapse movie ( supplemental file s2 ) .
the percentage of closure of the scratch was calculated as : 100 [ ( surface area of the scratch at time 17 h / surface area of the scratch at time 0)100 ] . to account for differences in wound closure between experiments , the closure of the scratch relative to control ( relative closure ) was calculated by dividing the percentage of closure of the scratch with the average percentage of closure of the scratch under control conditions ( sfm)100% .
relative closure between 1 and 100% indicated inhibition of wound closure , with 0% indicating complete inhibition .
each treatment was performed in five different wells per experiment , and each experiment was completed on three separate occasions .
non - parametric tests were performed since not all assumptions for parametric testing were met .
differences in relative closure between the different mois of specific bacterial strains and control were tested for significance with the kruskal
whitney u tests were performed as post hoc tests , and was corrected for multiple testing .
since three treatments were compared , and the number of pairwise comparisons was three , for the mann
differences between different mois of the same strain were tested with the wilcoxon signed rank test ( =0.05 ) .
spss version 21.0 ( ibm spss , chicago , il ) was used to perform the analyses .
a human oral buccal epithelial cell line ho-1-n-1 was provided by the japanese collection of research bioresources ( osaka , japan ) .
ho-1-n-1 is an immortalized epithelial cell - like cell line that originated from a squamous cell carcinoma located on the buccal mucosa .
the epithelial cells were grown in tissue culture flasks with dulbecco 's modified eagle 's medium - f12 ( invitrogen , carlsbad , ca , usa ) supplemented with 10% fetal calf serum ( hyclone , logan , ut , usa ) , 2% antibiotics ( penicillin , streptomycin and fungizone , sigma , st .
louis , mo , usa ) in a humidified atmosphere at 37c with 5% co2 .
all p. gingivalis strains were grown anaerobically in brain heart - infusion ( bd difco , le pont de claix , france ) broth enriched with hemin ( 5 mg / l ) and menadione ( 1 mg / l ) until mid - log phase . in case of mutant selection and growth , 50 g / ml of erythromycin ( em ) or 1 g /
bacterial cultures were washed twice with dulbecco 's phosphate - buffered saline ( dpbs , invitrogen ) and resuspended in keratinocyte serum - free medium ( sfm ; invitrogen ) at od690 0.1 ( 510 cfu / ml ) .
experiments were performed with either viable or heat - inactivated bacteria , in which case the bacterial cultures were heated at 60c for 60 min .
the influence of lps from p. gingivalis was tested by adding pure lps from p. gingivalis to the epithelial cells or by blocking lps in the cell wall of p. gingivalis .
ultrapure lps from p. gingivalis was purchased ( invivogen , toulouse , france ) and used in the assay in a concentration of 10 g / ml . to exclude the influence of lps , polymyxin b ( sigma aldrich , st .
louis , mo ) , an lps inhibitor , was added to 510 cfu / ml heat - inactivated p. gingivalis atcc 33277 in a concentration of 50 g / ml .
besides using gingipain mutants from two wild - type strains , the influence of gingipain activity was also studied by using gingipain inhibitors .
kyt-1 ( an arginine gingipain inhibitor ) and kyt-36 ( a lysine gingipain inhibitor ) peptides ( 15 ) ( peptide institute , osaka , japan ) were added in the assay to viable p. gingivalis w50 at a concentration of 110 mol / ml .
cell cultures were trypsinized and seeded in 24-well plates at 310 to 510 cells / ml . when confluency was reached , a scratch with a sterile 1-ml pipet point was made in each well .
the wells were washed three times with dpbs and challenged with viable or heat - inactivated bacteria or lps from p. gingivalis . in control wells , only sfm was added .
bacteria were added at a multiplicity of infection ( moi ) of 1,000 ( 1,000 bacteria vs. 1 epithelial cell ) , 100 , and 10 .
most p. gingivalis cells remained viable and metabolically active until the end of the experiment .
images were taken of the middle of the well immediately after adding the bacteria and 17 h later with an inverted digital phase - contrast microscope ( advanced microscopy group , evos , fl ) .
the surface of the scratch was calculated with photoshop cs4 ( version 11.0.1 , adobe ) .
after 17 h , closure of the wound under control conditions varied between 60 and 80% .
in addition , several images were taken to analyze migration of epithelial cells over time and to construct a time - lapse movie ( supplemental file s2 ) .
the percentage of closure of the scratch was calculated as : 100 [ ( surface area of the scratch at time 17 h / surface area of the scratch at time 0)100 ] . to account for differences in wound closure between experiments ,
the closure of the scratch relative to control ( relative closure ) was calculated by dividing the percentage of closure of the scratch with the average percentage of closure of the scratch under control conditions ( sfm)100% .
relative closure between 1 and 100% indicated inhibition of wound closure , with 0% indicating complete inhibition .
each treatment was performed in five different wells per experiment , and each experiment was completed on three separate occasions .
non - parametric tests were performed since not all assumptions for parametric testing were met .
differences in relative closure between the different mois of specific bacterial strains and control were tested for significance with the kruskal
whitney u tests were performed as post hoc tests , and was corrected for multiple testing . since three treatments were compared , and the number of pairwise comparisons was three , for the mann
differences between different mois of the same strain were tested with the wilcoxon signed rank test ( =0.05 ) .
spss version 21.0 ( ibm spss , chicago , il ) was used to perform the analyses .
figure 1 shows the percentage of closure of a scratch in oral epithelial cells challenged with a high concentration of p. gingivalis atcc 33277 ( moi 1,000 ) or with medium only ( control ) in time .
closure of the scratch under control conditions starts almost immediately , and closure in time follows a linear pattern .
a time - lapse movie of that experiment can be found in supplemental file s2 .
percentage closure of a scratch in oral epithelial cells challenged with p. gingivalis atcc 33277 and medium only .
the heat - inactivated p. gingivalis strains atcc 33277 , w83 , and w50 significantly inhibited the migration of oral epithelial cells at all tested mois compared to control in a dose - responsive manner , although the inhibition by the heat - inactivated w83 at moi 10 did not reach statistical significance ( fig .
2 ) . viable p. gingivalis atcc 33277 , w83 , and w50 significantly inhibited the migration of oral epithelial cells at all tested mois compared to control in a dose - responsive manner as well ( fig .
viable w83 and w50 inhibited cell migration more than their heat - inactivated variants ( not statistically significant for w50 at moi 10 ) ; however , no differences were found between heat - inactivated and viable p. gingivalis atcc 33277 ( p>0.05 ) .
mean relative closure ( sem ) from all biological replicates of scratches in oral epithelial cells challenged with heat - inactivated and viable p. gingivalis strains atcc 33277 , w83 , and w50 . a = significantly different from control ( p<0.0083 ) .
c = significantly different from moi 1,000 of the same strain ( p<0.0083 ) . * = significantly different from atcc 33277 of the same moi ( p<0.05 ) .
# = significantly different from heat - inactivated p. gingivalis of the same moi and strain ( p<0.05 ) . adding ultrapure lps from p. gingivalis to the oral epithelial cells
did not result in the inhibition of the migration of the epithelial cells ( p=0.39 ; fig .
when p. gingivalis lps was blocked by the addition of polymyxin b , it did not affect relative closure of the scratch in epithelial cells compared to p. gingivalis alone ( p=0.38 ; fig .
3a ) . mean relative closure ( sem ) from all biological replicates of scratches in oral epithelial cells challenged with lps from p. gingivalis and lps inactivated p. gingivalis by polymyxin b ( a ) and heat - inactivated p. gingivalis strains w83 and epsc mutant ( b ) .
gingivalis mutant epsc inhibited cell migration significantly more than the encapsulated wild - type strain w83 ( fig .
relative closure of the scratch in oral epithelial cells challenged with heat - inactivated p. gingivalis strains of different capsular serotypes is shown in table 2 .
not all capsular serotypes inhibited wound healing to the same extent ( all mois , p<0.002 ) . the non - encapsulated strains ( atcc 33277 and hg91 ) inhibited epithelial cell migration significantly more than the k1 and k6 capsular serotypes at all mois ( p<0.002 ) , except for moi 10 of strain hg91 versus atcc 33277 .
the relative closure of the k4 capsular serotype was close to that of the k strain atcc 33277 .
the k4 strain inhibited migration of oral epithelial cells significantly more than the k1 and k6 capsular serotypes at all mois ( p<0.001 ) .
relative closure ( meansem ) of scratch in oral epithelial cells challenged with heat - inactivated p. gingivalis strains with several capsular serotypes and a fimbriae - lacking mutant k , k1 , k4 , and k6 strains all significantly inhibited epithelial cell migration ( p=0.001 ) .
atcc 33277 and hg91 inhibited epithelial cell migration significantly more than k1 and k6 ( p<0.002 ) .
k4 inhibited epithelial cell migration significantly more than k1 and k6 ( p<0.001 ) , k4 inhibited epithelial cell migration significantly less than hg91 but not atcc 33277 .
the influence of protease activity was tested using gingipain mutants constructed from both the atcc 33277 and the w50 strains . for strain atcc 33277 , migration of oral epithelial cells
was significantly more inhibited by the wild - type strain than by the both gingipains mutants ( fig .
4a ) . there was no significant difference in relative closure between the kgp and rgpargpb mutants .
the viable gingipain mutants inhibited wound closure more strongly than the heat - inactivated variants at moi 1,000 ( fig .
, it was also found that the gingipain mutants , either viable or heat inactivated , inhibited the epithelial cell migration significantly less than the wild - type strain ( fig .
the viable gingipain mutants significantly inhibited wound closure more than the heat - inactivated variants at moi 1,000 and moi 100 ( fig .
3b ) . the viable rgpargpb mutant led to more inhibition of wound closure than the kgp mutant at moi 1,000 and moi 100 ( fig .
4b ) . adding the gingipain inhibitors kyt-1 or kyt-36 to viable p. gingivalis w50 led to significantly less inhibition of wound closure compared with the wild type ( fig .
wound closure was more inhibited by the rgpargpb gene compared with the kgp gene , as shown by the results from the mutant strain and the kyt inhibitors .
moreover , heat inactivation of the gingipain mutants resulted in a higher inhibition by the kgp mutant compared with the rgpargpb mutant .
mean relative closure ( sem ) from all biological replicates of scratches in oral epithelial cells challenged with heat inactivated and viable p. gingivalis wild type strain atcc 33277 , kgp , and rgpargpb mutants ( a ) and heat - inactivated and viable p. gingivalis wild - type strain w50 , kgp , and rgpargpb mutants and with addition of kyt-36 and kyt-1 peptides ( b ) . * = significantly different from wild - type strain of the same moi ( p<0.001 ) .
# = significantly different from heat - inactivated p. gingivalis of the same moi and strain ( p<0.05 ) . = significantly different from the kgp mutant ( p<0.05 ) .
viable fima mutants , lacking the major fimbriae , did not differ in inhibiting epithelial cell migration compared to the wild - type strain ( moi 100 : p=0.10 , moi 10 : p=0.627 ) .
the heat - inactivated fima mutant did inhibit epithelial cell migration significantly less than the wild type at high concentrations ( table 2 ; moi 1,000 : p<0.01 , moi 100 : p<0.05 , moi 10 : p=0.575 ) , although the difference was very small .
previous research described the effect of p. gingivalis on cell migration and wound healing , finding a significant inhibiting effect of p. gingivalis .
the responsible virulence factors , however , were not determined ( 4 , 5 , 16 , 17 ) . by comparing different naturally occurring variants and mutant strains of p. gingivalis , major virulence factors of p. gingivalis were studied in an in vitro wound - healing model to find out which of these affect wound healing . in the in vitro wound - healing model that was used ,
the migration of cells is studied , which is an important step in wound healing ( 18 , 19 ) .
epithelial cell migration under control conditions was a linear process , and the model used was successfully applied by oudhoff et al .
others also found that in vivo wound healing was a linear process ( 21 ) . measuring extra time points
could , however , give extra information about the relationship between bacteria and bacterial products and epithelial cell migration .
all of the p. gingivalis strains that we used in this study inhibited migration of oral epithelial cells .
the difference between viable and heat - inactivated p. gingivalis w83 and w50 suggests that there were heat - resistant and heat - sensitive factors of p. gingivalis that influenced migration of oral epithelial cells .
the heat - resistant virulence factors of p. gingivalis that were expected to account for this effect were lps , the capsular polysaccharide , and the fimbriae , whereas the heat - sensitive factors could be the gingipains ( 22 ) .
we found that the capsular polysaccharide and the kpg and rgpargpb genes influenced migration of oral epithelial cells significantly , whereas lps and the major fimbriae did not influence migration of oral epithelial cells at all .
until now , only the effect of the capsule on the immune system and on human cells has been described . both stimulating and inhibiting effects
were reported ( 11 , 23 , 24 ) . in the present experiments , the non - encapsulated bacteria inhibited wound closure more strongly than the encapsulated bacteria .
not only was the presence of the capsule important , but the thickness of the capsule as well .
the k4 serotype has a relatively thin capsule , while the k1 and the k6 serotypes have significantly thicker capsules ( 25 ) .
the k4 p. gingivalis strain had a relative closure rate that was in between that of the k and the k1k6 serotypes .
these findings suggest that a capsule may more or less depend on its thickness to hide cell - wall bound factors that might influence cell migration such as protease activity of the gingipains or other , yet unknown antigens on the cell wall of p. gingivalis .
gingipains are important for the adherence of p. gingivalis to epithelial cells ( 9 ) .
the absence of specific protease activity of the gingipain mutants used in this study was confirmed using the method described by kaman et al .
the protease activity of the gingipains of p. gingivalis was an important determinant of epithelial cell migration . knocking out and inhibiting gingipain activity led to improved wound closure , suggesting that gingipain activity inhibited cell migration .
the gingipains account for 85% of the protease activity of p. gingivalis
( 27 ) .
furthermore , in the rgpargpb mutant the kgp gene is still functional , and in the kgp mutant the rgpargpb gene is still functional .
thus , these viable gingipain mutants still possessed some protease activity , while the heat treatment inactivated almost all protease activities ( 22 ) .
furthermore , gingipains have a function in the modification of several cell wall components ( 28 ) .
therefore , p. gingivalis that lacks arginine or lysine gingipain activity may have a slightly different cell wall composition compared to the wild type , which may lead to differences in attachment to or recognition by the epithelial cells .
this effect would have been more pronounced in the experiments with the viable bacteria , which may contribute to the differences in relative closure between experiments with heat - inactivated and viable bacteria found in this study .
lps did not influence epithelial cell migration , and our results are in agreement with those of kraus et al .
however , many previous studies have shown that lps could stimulate cell migration through activation of the tlr4/nf-b signal pathway .
( 30 ) showed that lps from p. gingivalis was different from many other gram - negative bacteria .
moreover , lps from p. gingivalis is known to contain numerous lipid a species ( 31 , 32 ) .
the different lipid a structures could differentially modulate host innate immune responses in human gingival epithelia ( 32 ) and perhaps wound - healing processes .
it is worthwhile for studying further , even though lps did not appear to be a major virulence factor in this study .
the fimbriae are important for the adherence of p. gingivalis to epithelial cells ( 6 ) , and the inhibition of the fimbriae on wound healing was described by nakagawa et al .
( 33 ) . therefore , our hypothesis was that the absence of major fimbriae would lead to less inhibition of wound healing . in our experiments
, however , we proved that the fima gene was not involved in migration of oral epithelial cells because we found no difference in relative closure between the viable wild - type strain and the fima mutant .
when epithelial cells were challenged with heat - inactivated bacteria , we did find significantly less inhibition of wound healing of the fima mutant strain compared to the wild type .
we could not confirm the importance of fimbriae in the inhibition of migration of oral epithelial cells as described by nakagawa et al .
hela cells may react differently on a challenge with p. gingivalis than our oral epithelial cell line .
moreover , the amount of wound closure nakagawa et al . reported was generally lower . because there was an inhibition in migration of oral epithelial cells even in the presence of a capsule and the absence of either the arg- or lys- gingipains
, we hypothesize that there must be other , yet unknown , antigens on the cell wall that are responsible for the inhibiting effect of p. gingivalis .
this is supported by the finding that there is no difference in the percentage of wound closure between heat - inactivated and viable p. gingivalis atcc 33277 . in this strain
, it must be a heat - resistant factor that exceeds the influence of the gingipains .
another possibility is that a combination of virulence factors , the ones that were tested in this research or other yet unknown factors , have a synergistic effect in explaining the inhibiting effect on the migration of oral epithelial cells . in conclusion ,
the p. gingivalis strains atcc 33277 , w83 , and w50 strongly inhibited the migration of oral epithelial cells .
part of that effect was accounted for by the absence of a capsular polysaccharide exposing cell wall bound virulence factors and arg- and lys- gingipain activity .
lps and the major fimbriae did not influence migration of oral epithelial cells . because not all inhibition could be explained by the absence of capsular polysaccharide or the arg- and lys- gingipains ,
other yet unknown characteristics of p. gingivalis must have contributed to this effect , making further research necessary .
| background
porphyromonas gingivalis inhibits oral epithelial wound healing in vitro more strongly than other oral bacteria , but it is unknown why p. gingivalis is such a potent inhibitor of wound healing.objectivetherefore , the aim of this study was to investigate the influence of major virulence factors of p. gingivalis on wound healing in an in vitro wound - healing model .
the influence of the capsular polysaccharide , the arg- and lys- gingipains , the major fimbriae and lipopolysaccharide ( lps ) was investigated.designa standardized scratch was made in a confluent layer of human oral epithelial cells ho-1-n-1 .
the epithelial cells were then challenged with different concentrations of several p. gingivalis wild - type strains and knockout mutants .
closure of the scratch was determined after 17 h and compared to control conditions without bacteria.resultsthe p. gingivalis strains atcc 33277 , w83 , and w50 significantly inhibited wound healing .
the presence of a capsular polysaccharide lowered significantly the inhibition of epithelial cell migration , while gingipain activity significantly increased the inhibition of cell migration .
lps and the major fimbriae did not influence epithelial cell migration . none of the tested p. gingivalis strains completely prevented the inhibition of cell migration , suggesting that other characteristics of p. gingivalis also play a role in the inhibition of wound healing , and that further research is needed.conclusionsthe capsular polysaccharide and the arg- and lys- gingipains of p. gingivalis influenced the capacity of p. gingivalis to hinder wound healing , while lps and the major fimbriae had no effect . |
for more than 12 years , scientists have noticed that some individuals with autism also have a chromosomal change involving specific part of chromosome 15
.
trisomy or tetrasomy of the 15q11-q13 region has also been reported in some autistic patients with varying degrees of mental retardation [ 24 ] .
this report describes a male patient with autistic disorder , and a supernumerary marker
chromosome ( smc ) .
molecular cytogenetic investigation using fish
method allowed us to characterize the marker as an inv dup(15 )
including the prader willi / angelman locus .
this
observation reinforces the hypothesis that additional copies of
proximal chromosome 15 segment may be causally related to autism .
the patient , an 8-year - old male , is the sixth child born to
nonconsanguineous healthy parents .
the patient has developmental
delay , mental retardation , hyperactivity , epilepsy , antisocial
behavior , abnormal language skills and inability to maintain
social approaches .
he presents also stereotypies , including hand
flapping , hand - clapping over plane surfaces without growth delay
or microcephaly . at that time , the diagnosis of
autistic disorder ( dsmiv ) was established .
physical examination
revealed hyperlaxity of joints , hypotonia , and no evident facial
dysmorphism ( see figure 1 ) ; and the
electroencephalogram showed frequent bilateral diffuse fast
rhythms during sleep .
conventional cytogenetic analyses were performed in the department
of medical genetics at the national institute of health in
morocco .
cell culture , cytogenetic preparation gtg and rhg banding
were performed according to standard protocols .
briefly , this
included 72 hours culture of cells in rpmi 1640 medium
supplemented with 10% fetal bovine serum .
the smc was further
characterized by fluorescence in situ hybridization ( fish ) with a
whole chromosome 15 painting probe , wcp15 ( vysis , downers grove ,
ill , usa ) and the prader willi / angelman probe which contains three
locus / region - specific probes : d15z1 at the centromeric region
( spectrum green ) , d15s10 at the 15q11 - 12 pws / as critical region
( spectrum orange ) , and pml at 15q22 ( spectrum orange ) ( vysis ,
downers grove , ill , usa ) .
hybridizations were performed
following manufacturer 's instructions and 30 cells were counted
for each probe .
rhg and gtg banding analysis of the patient 's chromosomes revealed
an smc ( see figure 2 ) .
thus the karyotype is
47,xy,+mar [ 21 cells ] according to the iscn ( 1995 ) .
fish study
with chromosome 15 painting probe showed a complete hybridization
of the marker in 30 cells scored . to further characterize this
marker ,
fish analysis was carried out using the vysis prader
willi / angelman region probes .
the d15z1 probe showed two signals
for the centromeric region as the d15s10 probe and no signal for
the pml probe suggesting an inv dup(15 ) including the pws / as
region ( see figure 3 ) .
therefore patient 's karyotype was interpreted as
47,xy,+mar.ish idic(15)(wcp15 + , d15z1++ , d15s10++ , pml ) de novo .
cases with partial trisomy of a specific portion of chromosome 15
( 15q11-q13 ) have been reported in a considerable number of
individuals with autism or with pronounced autistic behavior
[ 1 , 5 ] .
supernumerary marker chromosomes occur at a frequency of 0.3 per
1000 live births and around 60% of all smcs are designated
smc(15)s were first described by van dyke et al .
and were consist in two inverted copies of
the short arm , centromere(s ) and proximal long arm of chromosome
15 . the majority of smc(15)s are dicentric , with one inactivated
centromere , and are also called
two
groups of smc(15)s are distinguished : the first one is small
smc(15)s which are metacentric chromosomes without euchromatic
materiel , the second is large smc(15)s which are acrocentric
chromosomes containing two copies of the 15q11-q13 region .
small
smc(15)s can be familial or de novo and are not directly
associated with an abnormal phenotype . in all cases , the smc(15)s
were maternally derived . in this article
we report a male child with tetrasomy of the
15q11-q13 chromosomal region and autistic disorder associated with
mental retardation , developmental problems and behavioral
disorders , epilepsy and no evident dysmorphy .
compiling classical
and molecular cytogenetics , the karyotype was
demonstrated as 47,xy,+mar.ish idic(15)(wcp15 + , d15z1++ ,
d15s10++ , pml ) de novo .
cytogenetic abnormalities found at the 15q11-q13
region are reported most frequently in patients with autism , up to
1% to 4% .
the chromosome region 15q11-q13 is known for its
instability and many rearrangements may occur in this imprinted
segment : deletion associated either with angelman syndrome ( as ) or
with prader - willi syndrome ( pws ) according to parental origin
translocations , inversions , and supernumerary marker chromosomes
formed by inverted duplication of proximal chromosome 15 .
inherited duplications are of maternal origin and seem to cause autism by creating an over abundance of product from the
nonimprinted maternally derived genes .
the inv dup(15 ) or idic(15 ) syndrome displays distinctive clinical findings
represented by hypotonia , developmental delay / mental retardation ,
difficult to control epilepsy , and autistic behavior .
several genes are incriminated in the unrests of the autistic
disorder bound at the region 15q11-q13 .
tetrasomy of these genes ,
as seen in inv dup(15 ) syndrome , may alter the gaba receptor
activity , upon which the major cns inhibitory mechanisms rely .
this alteration could represent the biological basis for some
clinical manifestations of the inv dup(15 ) , such as seizures ,
hyperactivity , aggressiveness , and autistic disorder . of interest ,
linkage disequilibrium between a marker in the g - aminobutiric
acida receptor subunit gene , gabrb3 155ca-2 , and autistic disorder
has been reported by battaglia .
ube3a is another candidate gene for autistic disorder , because of its position in this region and its known association with angelman syndrome , a genetic disorder which share some
symptoms in common with autistic disorder .
epigenetic studies in this region recently identified atp10c as another
imprinted gene that may contribute to autistic disorder . a suggested mechanism of formation of inv dup(15 ) involves
illegitimate recombination between homologous chromosomes followed
by nondisjunction and centromere inactivation .
proximal chromosome
15q is believed to be highly unstable as evidenced by its frequent
involvement in structural rearrangements .
the heterogeneous nature
of the breakpoints involved in these chromosomal abnormalities
suggests that either several low - copy repeat sequences are
involved in the genesis of these chromosomal abnormalities
or the same repeat sequence is located in many places
throughout proximal 15 . in conclusion , this observation approves the implication of the
region 15q11-q13 in the autistic disorder / behavior , and these
chromosomes 15 appear to hold particular promise in the search for
candidate genes . | we report the case of a moroccan boy with mental retardation , hyperactivity , epilepsy , developmental problems and behavioural disorders .
cytogenetic analysis showed the presence of a supernumerary marker chromosome .
molecular cytogenetics allowed us to determine the marker as an inverted duplication of chromosome 15 .
it is the first case of a moroccan patient with tetrasomy 15q in which fluorescence in situ hybridization ( fish ) enabled us to specify the diagnosis .
interestingly , this patient has an infantile autism with cytogenetic abnormalities on chromosomal region 15q11-q13 as reported in patients with autistic disorder . |
forward head posture ( fhp ) pushes the center of the head forward and upwards which , in
turn , increases the head weight supported by the neck , triggering relative compensatory
mechanisms , such as increased cervical lordosis and abnormal and sustained contractions in
the suboccipital muscle and muscles in the neck and shoulders .
as this posture causes
changes in the head and neck area1 , it is
thought to be one of the most well - known postures that result from poor posture . in this
posture , posterior cranial rotation occurs to adjust the eyes by rotating the head backwards
and in the end , there is an increase in the cervical extensor ; if sustained for long enough ,
there will be a loss of the normal cervical lordosis2 .
such increases in the lordosis and muscles in the suboccipital
region , neck , and shoulders in other words , such muscle contractions cause changes in the
head and neck area3 .
humans are greatly
influenced by physical , structural , and functional changes that result from posture .
therefore , it is highly likely that they experience a decline in learning performance ,
concentration , and memory with poor postures .
ultrasound therapy , therapeutic exercise ,
electrical stimulation therapy , and a combination of therapeutic interventions are often
used as treatments for fhp .
neurofeedback is a brain - training program that self - regulate
one s own brain waves by providing feedback on the status of brain waves induced using the
biofeedback technique4 .
the underlying
principle of neurofeedback is that it improves brain plasticity by enhancing the
self - regulation of brain homeostasis with brain waves . in other words , people self - train
their brains by looking , hearing , and feeling their own brains .
neurofeedback does not
merely involve increasing specific brain waves , but also maintaining and optimizing the
health of brain functions by bringing balance and harmony of brain waves .
because the brain
self - teaches and self - trains in accordance with the given information , a strong network of
nerve fibers that connect brain cells develops , leading to improvements in blood
circulation , which in turn increases the supply of oxygen and stimulates the delivery of
information , greatly improving the brain functions5 . as the self - training program of brain waves6 , neurofeedback training aims to normalize cerebral functions
by either decreasing or increasing specific brain waves when a patient receives visual or
auditory feedback on the activities in the cerebral cortex during a brain wave
measurement7 .
therfore , the training is
being used in various fields of study , such as sports training , that require
concentration8 .
since it is thought that cervical spine misalignment not only affects fhp , but also
concentration , the present study aims to investigate the effects of neurofeedback training
on the postural changes , range of motion ( rom ) , and neck disability index .
( ndi ) of the
cervical joints while tracking changes using x - ray imaging .
the subjects of the present study were students of university g in north gyeongsang
province whose cervical lordosis angle was below 21 degrees9 , whose anterior weight bearing ( awb ) of the head was above
15 mm10 , and whose cervical extension
rom was below 70 degrees ( neuman ) .
they were adequately informed of the purpose and methods
of the study and took part in the study after voluntarily signing the consent form .
the
subjects who regularly took medications or therapies to ease pain until recently and who had
neurological problems were excluded from the study .
table 1table 1.general characteristics of each group ( mean sd)nftg ( n=20)cg ( n=19)male / female9/119/10age ( years)20.9 3.322.8 4.4height ( cm)168.5 9.2167.6 8.7body weight ( kg)59.3 11.559.5 11.4nftg : neurofeedback training group , cg : control group shows the general characteristics of the study subjects ( table 1 ) . all parents of the enrolled participants provided
written informed consent prior to this experiment in accordance with the ethical standards
established in the declaration of helsinki .
nftg : neurofeedback training group , cg : control group the subjects in the present study who had signed the consent form were randomly divided
into a neurofeedback training group ( nftg ) and a control group ( cg ) and took x - ray images .
in accordance with their ndi measurements ,
each group underwent a four - week intervention
program of three sessions per week .
after four weeks , the subjects again took x - ray images
and measured their ndis .
the neurofeedback training took place in a
light - blocking and noise - cancelling space .
after a 10-minute break , the subjects wore
headbands while adjusting the midline electrode site , fpz to be over the center of the
forehead , which is the prefrontal region and connecting the reference electrode to the left
earlobe .
the training methods included pottery and archery games as part of the
concentration training .
first , for the pottery game , the subjects would score the most
points when they made most cups out of broken pieces of glass . when the stability of brain
waves met the satisfactory level of stability for training , a signal was sounded and a cup
was made , but when the brain waves became unstable , the training stopped .
second , for the archery game ( mind arrow )
where the subjects shot arrows with their brain waves , when the brain waves met the training
conditions , a signal was sounded .
if an arrow was shot when the signal was sounded , the
subjects had a high probability of hitting the bullseye .
the game ended when 15 arrows were
shot within two minutes , showing the brain training performance in terms of scores .
the absolute rotation angle ( ara)11 was measured to calculate a cervical lordosis angle , and
the anterior weight bearing ( awb)4 was
measured to assess fhp . and to find the neck flexion and extension angles , the range of
flexion and extension motion ( rfem)12 was
examined by x - ray imaging .
the ndi was comprised of a total of 10
questions to measure the neck pain and functional disorder of respondents .
the respondents
were asked to choose one option out of six ( 05 points ) in 10 survey items , namely neck
pain , daily activities , lifting objects , reading , headache , concentration , work , driving ,
sleep , and leisure activities . the higher the score , the greater the neck dysfunction .
the
score range was between zero and 50 , indicating 04 for no disability , 514 for mild
disability , 1524 for moderate disability , 2534 for severe disability , and above 35 for
complete disability13 . for the data
analysis , spss 14.0 for windows
was used to process the statistics , and independent t - tests
were carried out to compare differences within and between the groups .
there were no significant within - group differences in the awb , ara , cervical flexion and
extension angles , and rfem ( p>0.05 ) .
moreover , there was no significant within - group
difference in the ndi pre - intervention ( p>0.05 ) .
however , post - intervention , a
significant difference in the ndi was found within the nftg ( p<0.05 ) ( table 2table 2.comparison of the change of neck position and neck disability index ( ndi ) between
the neurofeedback training group and the control group ( mean sd)nftg cg pre - testpost - testpre - testpost - testawb22.5 5.421.9 4.922.2 4.723.7 6.5ara11.1 4.111.8 4.210.6 3.611.7 3.5flexion16.6 9.516.3 5.018.1 7.818.6 4.9extension50.6 7.251.7 5.451.3 7.950.8 6.8rfem67.2 12.768.0 7.269.4 9.869.3 7.5ndi7.2 1.56.7 1.7 * 7.5 1.87.7 1.9*significant difference from the pre - test at<0.05 .
awb : anterior weight bearing , ara : absolute rotation angle ,
rfem : range of flexion and extension motion , ndi : neck pain and disability index ) . *
awb : anterior weight bearing , ara : absolute rotation angle ,
rfem : range of flexion and extension motion , ndi : neck pain and disability index in addition , no significant between - group differences were found in the awb , ara , cervical
flexion and extension angles , and rfem , both pre- and post - intervention ( p>0.05 ) .
moreover , there was no between - group difference in the ndi pre - intervention ( p>0.05 )
.
however , post - intervention , a significant between - group difference in the ndi was found
( p<0.05 ) ( table 2 ) .
modern people suffer problems in the cervical spine due to continuous stress and poor
posture , as they overwork and overpressure their cervical spines by sitting at a computer
for a long period of time , using a smartphone , and lacking exercise .
such postural change in
the cervical spine causes dysfunction and imbalance in the head and neck and is accompanied
by limitations in the rom and pain1 .
neurofeedback training is a comprehensive training system that spurs the growth of and
change in brain cells as a part of biofeedback training13 .
biofeedback training involves a patient learning to control his or
her physical activities while effecting changes in his or her own physiological activities
as various physiological signals that occur in the human body are converted into
easy - to - understand visual signals by a computer14 .
brain waves , one of the physiological signals that occur in the
human body , reflect specific mental statuses in humans .
therefore , changes in mental status
can be observed with brain waves . in the present study ,
after college students with fhp
underwent the neurofeedback training , a comparative analysis of changes in x - ray images
( awb , ara , rfem ) and ndis was conducted to investigate whether there were any postural and
functional changes in the cervical joints .
no significant increases were found in the awb
and cervical flexion angle of the nftg and cg .
hence , to improve posture in the cervical
spine , physical therapy interventions , such as direct joint mobilization and direct
improvement in the muscle strength of stabilizers that maintain joint alignments , are
important15 .
however , such direct
interventions were excluded from the present study , and only neurofeedback training , an
internal biofeedback system of brain waves , was used , which is thought to have made it
difficult to directly cause postural change and increase the rom in the cervical spine .
such
research findings suggest that when joint mobilization , exercise therapy , and neurofeedback
training are applied in combination to people with poor posture of and pain in the cervical
spine , it will have a greater impact on improving their neck pain and functional recovery in
the future .
gong et al.12 reported that
after patients who had neck pain associated with acute whiplash performed exercise , there
was a reduction in the ndi in chronic neck pain patients . in the present study , a
significant increase was observed in the nftg .
this is thought to be the result of
neurofeedback training , which maintained stable and effective brain functions in the nftg .
moreover , with the improvement in the dynamic functions in the cervical spine , the group s
daily activity function improved partially . with the postural improvement in the neck ,
the
stress in the muscles around the neck was reduced , leading to a lower ndi , which in turn
improved concentration .
in addition , pain reduction in the ndi was found both in the
study15 of neurofeedback training on
patients with chronic pain and by ibric and dragomirescu16 , who applied neurofeedback training to patients who complained of
neck and temporomandibular pain as well as a sleep disorder .
teaching self - regulation to
either reduce or alleviate pain sensations by stabilizing the pain in the central nervous
system is the core of neurofeedback training . from the aforementioned results ,
it is
expected that the subjects in the nftg experienced pain reduction in the cervical spine .
moreover , the finding in which scores in the ndi indicating functional disorders improved is
in line with the research findings of yu yeon - ho8 who reported improved physical movement abilities , such as static
coordination ability , hand movement coordination ability , general movement coordination
ability , and the ability to perform simultaneous movement and single movement .
furthermore ,
yu saeng - yeol17 reported that
concentration training with biofeedback enhanced pitching performance and partially improved
attention , while chang jae - geun18 found
that concentration training with brain wave regulation had a positive impact on sprint
performance .
therefore , the findings of the present study can be seen in the same light . in the present study , although neurofeedback training could not be confirmed to directly
cause change in the postural alignment of patients with fhp , it is thought to have improved
their overall ability to carry out daily activities and their physical functions .
building
on the findings of the present study , it is hoped that there will be continuous research on
neurofeedback training to investigate its effects on improving concentration and direct
changes in brain waves . | [ purpose ] the purpose of the present study was to examine the effects of neurofeedback
training on postural changes in the cervical spine and changes in the range of motion of
the neck and in the neck disability index in adults with forward head posture .
[ subjects
and methods ] the subjects of the study were 40 college students with forward head posture ,
randomly divided into a neurofeedback training group ( nftg , n=20 ) and a control group ( cg ,
n=20 ) .
the neurofeedback training group received six sessions of pottery and archery
games , each for two minutes , three times per week for four weeks , using the neurofeedback
system .
[ results ] there were no significant effects within and between groups in terms of
the absolute rotation angle , anterior weight bearing , and range of extension and flexion
by x - ray imaging .
there were significant effects in the neurofeedback training group pre-
intervention and post - intervention in neck disability index .
there were significant
effects between groups in neck disability index .
[ conclusion ] it is thought that
neurofeedback training , a training approach to self - regulate brain waves , enhances
concentration and is therefore an effective intervention method to improve neck pain and
daily activities . |
cancer is one of the leading causes of death worldwide and the burden is increasing day by day .
though we have a number of anticancer agents now , adequate control of cancer is still lacking .
hence there is a persistent demand to develop newer and more effective anticancer drugs which can help tackle this problem .
the major groups of anticancer drugs such as vinca alkaloids , taxanes , camptothecins and epipodophyllotoxins which are currently a part of many standard anticancer regimens are derived from plants .
cleistanthin b is an arylnaphthalene lignan lactones glycosides , the major phytoconstituents responsible for most of the toxicological and pharmacological actions of cleistanthus collinus , a plant commonly found in the states of southern india .
cleistanthin b also present in dysosma versipellis ( berberidaceae ) , a chinese herb widely used to clear sputum , kill parasites and treat epidemic encephalitis b and epidemic parotitis , and hypoestes purpurea ( acanthaceae ) , a tropical herb eaten as spinach , used as a poultice for sore eyes.5 , 6 cleistanthins a and b were reported to possess hypotensive and diuretic properties .
while these compounds have no activity on motor function , they were demonstrated to exhibit significant antitumor activity.10 , 11 , 12 , 13 , 14 most of the studies about antitumor activity of these compounds are based on in vitro assays .
the gi50 ( 50% growth inhibition ) values are lower for tumor cells compared to normal cells.11 , 13 they exert cytotoxic activity on various cancer cell lines by interfering with cell cycle progression , causing dna damage and inducing apoptosis . while cleistanthin a has in vivo antitumor activity against dalton 's ascites lymphoma ( dal ) and s-180 sarcoma tumor bearing animals by prolonging the life span and reducing the tumor volume , there is no report of the in vivo antitumor activity of cleistanthin b so far .
hence the present study was focused to evaluate the in vivo antitumor activity of cleistanthin b in ehrlich 's ascites carcinoma ( eac ) and dal cell line induced malignant ascites and solid tumor in mice .
cleistanthin b was isolated from the leaves of c. collinus ( euphorbiaceae ) as described in our previous study .
taxonomically identified plant leaves were collected in the regions of pondicherry district and it was certified by botanical survey of india , coimbatore ( bsi / sc/5/23/08 - 09/tech.241 ) .
eac and dal cell lines ( amala cancer research center , thrissur , kerala ) were maintained by weekly intraperitoneal transplantation of respective tumor cells ( 2 10 cells per mouse ) in stock animals .
carboxymethyl cellulose sodium ( cmc ) ( lobachemie , mumbai ) , phosphate buffered saline ( pbs ) ( media labs , mumbai ) , diethyl ether ( thermo fisher scientific india pvt ltd , mumbai ) and 5-fluorouracil ( 5-fu ) ( ceon labs , andhra pradesh ) were used for the study . automated haematology analyzer (
sysmex xs-1000i ; sysmex corporation , kobe , japan ) was used for the complete blood cell counts .
female swiss albino mice were obtained from the central animal house of jipmer , pondicherry and they were maintained under standard laboratory conditions throughout the study .
the animals were fed with standard rodent pellet feed ( amrut feeds , sangli , india ) and water ad libitum .
the study protocol was approved by the institutional animal ethics committee ( no : jip / micro / jiaec/2012 ) and all the animal experiments were carried out in accordance with the guidelines of the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , india .
an exploratory study was designed to evaluate the in vivo antitumor activity of cleistanthin b using different mouse tumor models .
study i and ii were carried out with eac and dal cell lines induced malignant ascites mouse models , respectively .
25 , 50 and 100 mg / kg were chosen based on the results of a toxicity study done previously .
the animals were divided into five different groups as follows : group i : tumor controlgroup ii : 5-fu 20 mg / kggroup iii : cleistanthin b 25 mg / kggroup iv : cleistanthin b 50 mg / kggroup v : cleistanthin b 100 mg / kg group i : tumor control group ii : 5-fu 20 mg / kg group iii : cleistanthin b 25 mg / kg group iv : cleistanthin b 50 mg / kg group v : cleistanthin b 100 mg / kg study i and ii used 8 mice in each group while study iii used 6 mice per group .
group i served as a positive ( tumor ) control ( 0.5% cmc orally ) and group ii as a comparator control ( 5-fluorouracil 20
groups iii , iv and v were given cleistanthin b orally at the doses of 25 , 50 and 100 mg / kg respectively .
on day 1 , blood collection from retro - orbital plexus was carried out and the samples ( 0.3 ml ) in edta were used for the assessment of haematological parameters such as haemoglobin ( hb ) content , red blood cell ( rbc ) count , total white blood cell ( wbc ) count , neutrophils ( % ) , lymphocytes ( % ) and platelet count . on day 2
, tumor fluid was withdrawn from the stock animals for eac and dal cell lines and the tumor cell count was done using neubauer chamber under the light microscope .
the pbs was added to make a concentration of 1 10 cells in 0.1 ml . for tumor induction in study
i and ii , each experimental animal was injected with 2 10 dal / eac cells i.e. 0.2 ml intraperitoneally . in study iii ,
injection of 2 10 dal cells in 0.1 ml pbs on right hind limb of each experimental animal .
after 24 h of the tumor cells inoculation , the animals were treated according to their respective groups once daily for next 10 days . on day 15
, the retro - orbital blood collection was done again for haematological assessment , if the animal was alive .
the animals were followed till death or upto 35 days . the parameters for antitumor activity in study i and ii were recorded as follows : determination of the percentage increase in life span ( pils ) : it is calculated from the mean survival time ( mst ) values .
the mst for each group was calculated as , mst(days)=totalnumberofdayssurvivedbyallanimalsinthegroupnumberofanimalsinthegroup for each group , pils(%)=[(mstoftreatedgroup / mstofcontrolgroup)1]100 determination of the overall survival : it was based on the results of survival analysis . in study
determination of the solid tumor volume : the solid tumor volume was measured on the seventh day after tumor inoculation and then it was repeated every fifth day until the end of the study .
the tumor volume was calculated using the following formulatumorvolume(ml)=4/3r12r2(cm3)where , r1 and r2 are two perpendicular radii of the solid tumor measured using a vernier caliper .
the haematological parameters of all surviving animals such as haemoglobin , rbc , wbc , neutrophils , lymphocytes and platelets were assessed for all the three studies .
the tumor bearing animals alive at the end of the study were sacrificed by cervical dislocation .
the survival analysis was done by log - rank test using spss version 20 software package ( ibm spss , usa ) .
the mean survival time , haematological parameters and tumor volume comparison between groups were analyzed using instat version 3.06 ( graphpad , usa ) .
cleistanthin b 50 mg / kg and 5-fu significantly increased the pils . while 5-fu increased the life span of eac and dal animals by 40.6% and 65.1% respectively , cleistanthin b increased it by 25.5% and 26.2% respectively ( fig . 2 ) .
only cleistanthin b 50 mg / kg and 5-fu significantly improved the overall survival of eac and dal tumor bearing animals . while cleistanthin b 50 mg / kg and 5-fu were not significantly different from each other in improving the overall survival of eac animals , 5-fu was significantly more effective than all the three doses of cleistanthin b in improving the overall survival of dal animals ( fig . 3a and b ) .
the haematological parameters could not be analyzed in dal animals because only four out of 40 animals were alive on day 15 .
so mortality was less in 5-fu group compared to all the three doses of cleistanthin b. the hb and rbc count were lower in all the treatment groups compared to normal control group .
the wbc counts were significantly increased in tumor control , 5-fu and cleistanthin b 25 mg / kg group compared to normal control group . only cleistanthin b 50 mg / kg decreased the wbc counts to normal range .
the neutrophils were increased and lymphocytes were decreased significantly in all the treatment groups . the platelet count was significantly increased in tumor control and cleistanthin b 25 mg / kg group compared to normal group ( table 1 ) . all animals with solid tumor survived beyond 35 days .
the tumor volume was found to be significantly less only in 5-fu group compared to tumor control group .
none of the three doses of cleistanthin b significantly reduced the solid tumor volume ( fig .
there was no significant difference between the groups with regards to haematological parameters ( data not shown ) .
in the present study , cleistanthin b showed significant antitumor activity against eac and dal cell line induced malignant ascites tumor animals at 50 mg / kg dose while no significant antitumor activity was recorded against dal solid tumor animals with all the three doses of cleistanthin b. even the higher dose of cleistanthin b was found to be less effective than 5-fu which was used as a standard comparator .
the life span was prolonged by cleistanthin b more than 25% in both eac and dal tumor animals .
prolongation of life span is a valuable criterion for initial evaluation of antitumor activity of a new compound.20 , 21 if the compound increases the life span by 25% , it can be considered to have a significant antitumor activity .
the results of survival analysis should also be considered for assessing the antitumor activity and cleistanthin b has improved the overall survival of animals .
there is no previous report on the in vivo antitumor activity of cleistanthin b but in vitro studies with cleistanthins a and b have reported the same .
the antineoplastic activity was predicted in silico for both the compounds by pass bioinformatics tool and cleistanthin a which has been evaluated in vivo in dal tumor animals was reported to have significant antitumor activity .
since cleistanthins a and b are structurally similar , it is not surprising that cleistanthin b shares the same in vivo effect .
while cleistanthin b 50 mg / kg significantly decreased the wbc count in eac tumor animals , 5-fu could not do so .
the agents with antitumor activity tend to decrease the wbc count and it is a reliable criterion of their antineoplastic activity .
the increase in wbc count in tumor bearing animals was probably due to myeloid leukemoid reaction secondary to malignancy .
the antitumor effect of a compound will reduce the tumor cells and decrease the wbc count .
the reduction in leucocyte count probably reflected the decrease in tumor burden as well as the action of compound on the bone marrow precursors .
although 5-fu showed a marginal decrease in the wbc count , it was not significant while cleistanthin b maintained a near normal range .
so it can be said that 5-fu reduces the wbc count when it is raised due to malignancy and also when it is in normal range .
in contrast , cleistanthin b reduces wbc count only when it is raised due to malignancy and not when it is in normal state which is advantageous . the haemoglobin and rbc values were less in all treatment groups .
it is well known that development of anemia is a problem with most of the cancer chemotherapy agents . in the present study
also we found the same problem with 5-fu and cleistanthin b. the most common cause of anemia in cancer is chemotherapeutic agent induced myelosuppression .
the anemia in ascites carcinoma bearing mice can also occur due to iron deficiency or haemolytic conditions .
it is known that neutrophils and platelet counts increase while lymphocytes decrease in all the ascites tumor bearing animals .
reactive neutrophilic leucocytosis is known in malignancy and the agents with antitumor activity tend to reverse it .
both 5-fu and cleistanthin b reversed the deranged neutrophils ( % ) , lymphocytes ( % ) and platelet counts but not to the normal range .
it was reported that cleistanthin b has affinity towards the localized solid tumor and it may be helpful in the treatment of solid tumor .
but in the current study , we did not observe the beneficial effect of cleistanthin b in solid tumor animals .
cleistanthin b doses were chosen based on the results of a previously carried out toxicological study in animals.16 , 27 it is possible that the doses selected could have been inadequate against solid tumor .
this is evidenced by the fact that the tumor volume starts increasing rapidly after stopping the treatment .
so cleistanthin b given at a higher dose for longer duration might have been effective against solid tumor . out of all the three doses of cleistanthin
b only 50 mg / kg has shown a significant antitumor activity in malignant ascites tumor bearing animals .
while cleistanthin b 25 mg / kg dose may be ineffective because it did not significantly improve the survival of animals , cleistanthin b 100 mg / kg might have exerted toxic effect on the eac and dal tumor bearing animals because most of the animals in this group died early .
parameters such as viability testing of tumor cells and percentage cell growth inhibition were not evaluated in our study .
early death of ascites tumor animals did not allow us to collect blood on day 15 . reducing the initial load of tumor cells to 1 10 cells during inoculation
we have not carried out the tumor growth delay assay in solid tumor model which could be done by measuring the tumor volume every day .
these limitations have not affected the outcome of the study but eliminating them would have given more information .
from the present study it can be concluded that cleistanthin b at the dose of 50 mg / kg has significant antitumor activity against eac and dal ascites tumor bearing mice but it is not as effective as 5-fu .
cleistanthin b has a better effect on wbc count compared to 5-fu but it has no antitumor activity against solid tumor .
| to evaluate the in vivo antitumor activity of cleistanthin b in ehrlich 's ascites carcinoma ( eac ) and dalton 's ascites lymphoma ( dal ) cell lines induced malignant ascites mouse models and dal cell line induced solid tumor mouse model .
all animals were injected with 2 106 eac / dal cells i.p./s.c . to induce malignant ascites and solid tumor and treated with 5-fluorouracil ( 5-fu ) 20 mg / kg or cleistanthin b for 10 days .
cleistanthin b was given at three doses viz .
25 , 50 and 100 mg / kg . the percentage increase in life span and the overall survival in malignant ascites animals and the tumor volume in solid tumor animals were measured .
the haematological parameters were assessed in all animals before and 2 weeks after the treatment .
cleistanthin b 50 mg / kg and 5-fu significantly prolonged the life span ( > 25% ) of malignant ascites tumor bearing animals .
the overall survival was significantly improved by both . only cleistanthin b 50 mg / kg significantly reduced the elevated wbc counts in eac tumor bearing animals .
both 5-fu and cleistanthin b 50 mg / kg reversed the malignancy induced increase in neutrophils and platelet counts and decrease in lymphocyte counts but not to the normal range .
only 5-fu significantly reduced the solid tumor volume .
none of the three doses of cleistanthin b was effective against the solid tumor .
cleistanthin b has antitumor activity against eac and dal tumor mice but it is not as effective as 5-fu . at 50 mg / kg
dose cleistanthin b exerts significant antitumor activity compared to 25 and 100 mg / kg dose .
its effect on wbc count is higher and advantageous when compared to 5-fu .
but cleistanthin b in the doses used is not effective against solid tumor . |
hypertrophic olivary degeneration ( hod ) is an unusual neuronal degenerative disease involving inferior olivary nuclei with increased t2-weighted signal intensity on magnetic resonance imaging ( mri ) .
oppenheim first described hod in 1887.1,2 however , guillain and mollaret widely described and studied the hod with more detailed experiments in 1931 and first reported the theory of the dento - rubro - olivary pathway , which is referred to as the triangle of guillain and mollaret.3,4 researchers have reported that hod is a transsynaptic disease of degeneration and that the inferior olivary nucleus is hypertrophic , but not atrophic .
it has also been reported that many factors can affect the dento - rubro - olivary pathway , such as surgical operation , hemorrhage , tumor , trauma , inflammation , demyelination , degeneration , and radiation damage.58 in a recent study , we can see the etiology , clinical manifestation , mri presentation , and prognosis of hod clarified in detail.9 although the theory of the triangle of guillain and mollaret has been described , diagnosing hod following resection of cavernomas of the brain stem by using diffusion tensor imaging ( dti ) and diffusion tensor tractography ( dtt ) has not been reported .
therefore , we report a case concerning delayed hod after resection of cavernomas of the brain stem and demonstrate the value and significance of dti and dtt in diagnosis of hod
. this article will present new knowledge of hod by means of a case report and review of the literature .
a 62-year - old woman was hospitalized in the department of neurosurgery in qi lu hospital ( jinan , people s republic of china ) .
the chief complaint was sudden - onset dizziness and superficial sensory decrease in the right limbs for 1 week .
the computed tomography scan results showed that there was a high - density change in the posterior cranial fossa , which was considered to be a hemorrhage of the pons .
there was soft high signal intensity on t1-weighted imaging and high signal intensity on t2-weighted imaging ( t2wi ) , with a low signal surrounding the lesion in the mri scan .
then , we performed a cavernoma resection via posterior fossa craniotomy and confirmed the disease histologically .
the patient recovered well and was discharged 10 days after operation without apparent abnormal signal on the primary lesion area on mri ( figure 2 ) .
one year later , the patient was admitted to qi lu hospital again , with diplopia , restricted motion of the left eye , involuntary movement of the uvula , and inaccuracy of bilateral finger - to - nose test and heel knee
mri scan displayed an enlarged mass in the left anterolateral part of the medulla , with a high signal on t2wi and no obvious increased signal intensity on enhancement scan ( figure 3 ) .
the dti and dtt showed the nerve fiber of the left was decreased compared to the right and the other part of the interrupted areas ( figure 4 ) .
we made the diagnosis of hod and did follow - up visits at regular intervals .
we continue to perform clinical and radiologic ( t2wi ) follow - up , and the patient has shown hod - associated symptoms on radiologic imaging at each time of follow - up . therefore , we can confirm that our diagnosis of hod is correct .
this study has also been approved by the ethics committee of shandong university , jinan , people s republic of china .
hod was first described by oppenheim in 1887,1,2 and hod was chacterized by cytoplasmic vacuolation and the pathological changes of the neural cell body enlargement.10 ogawa et al11 confirmed the clinical pathology by postmortem examination . according to a previous report about hod , there were no significant differences in morbidity according to age , sex , and single or multiple lesions , but there were for position .
that is to say , the morbidity of hod was higher when the primary disease was located in the mesencephalon.12 clinical manifestations of hod include dentatorubral tremor , ocular myoclonus , diplopia , and palatal myoclonus .
the high signal intensity on t2wi and absence of contrast enhancement could help us to distinguish hod from a tumor lesion or an infection.1315 a lesion - damaged dento - rubro - olivary pathway could result in hod , according to guillain and mollaret s report in 1931.3,4 anatomically , the dento - rubro - olivary pathway , or triangle of guillain and mollaret , is composed of three structures , including the ipsilateral red nucleus in the midbrain , ipsilateral inferior olivary nucleus in the medulla , and contralateral dentate nucleus in the cerebellum .
the efferent nerve tract travels from the dentate nucleus , ascends to the contralateral red nucleus via the superior cerebellar peduncle , and forms the decussation .
then , the tract descends through the central tegmental tract to the ipsilateral inferior olivary nucleus .
finally , the afferent pathway originates from the inferior olivary nucleus to the contralateral cerebellum bypassing the inferior cerebellar peduncle .
the above clarifies the pathway of the triangle of guillain and mollaret.16 if a lesion damages the red nucleus or central tegmental tract , degeneration occurs in the ipsilateral inferior olivary nucleus . on the contrary , if the dentate nucleus or superior cerebellar peduncle is insulted , degeneration would happen in the contralateral corresponding area .
only if the pathological changes damage both central tegmental tracts and the bilateral superior cerebellar peduncle or the decussation of the superior cerebellar peduncle would bilateral hod result.17 hod often happens when the dentate nucleus
red nucleus pathway or central tegmental tract pathway is damaged , rather than the pathway from the inferior olivary nucleus to the contralateral cerebellum.18 the clinical presentation of hod includes oscillopsia diplopia and palatal myoclonus.19 to our knowledge , the inferior olivary nucleus could regulate the cerebellum neural activity by generating an inhibitory substance , such as gamma - aminobutyric acid ( gaba).17,20 when some pathological changes may have damaged the triangle of guillain and mollaret and clinical symptoms ( such as palatal myoclonus ) occur , the diagnosis of hod should be strongly suspected .
the time interval between pathological changes and mri manifestation of hod can vary to some extent .
pathological changes of the inferior olivary nucleus can be classified into six stages : stage 1 : no apparent changes of the inferior olivary nucleus in both macropathology and microscopic pathology ; stage 2 : degeneration of the olivary amiculum and inferior olivary nucleus neuron pathological hypertrophy with no glial proliferation under the microscope after approximately 3 weeks ; stage 3 : soft hypertrophy of the inferior olivary nucleus because of neuron and astrocyte proliferation after approximately 6 months ; stage 4 : significant hypertrophy and enlargement of the inferior olivary nucleus ; stage 5 : olivary pseudohypertrophy , which persists for 34 years ; and stage 6 : inferior olivary nucleus atrophy due to neuronal disappearance.12,20,21 in addition , researchers have recently assessed the mri presentation and propose three phases of hod : phase 1 : increased signal begins on t2wi in about the third week after lesion and without olivary changes within 6 months ; phase 2 : both olivary hypertrophy and high signal on t2wi , persisting for approximately 34 years ; and phase 3 : disappearance of olivary hypertrophy and persistence of increased signal on t2wi for a long time.2023 the present case of hod belongs to the second phase and corresponded to pathological changes and mri manifestation .
dti is a method which can analyze the diffusion movement of water molecules in human tissue in three - dimensional space on the basis of fractional anisotropy ( fa ) of water hydrone distribution.24 the parameter is described by fa value , which is related to the compactness and completeness of the neural fiber tract . by fa imaging and directionally encoded color imaging , the white matter fiber could be visualized by a special method to evaluate the completeness of the organic structure.25,26 the bilateral central tegmental tracts are distributed symmetrically and uniformly on dtt at the anatomical level , as is the decussation of the superior cerebellar peduncle . when a primary lesion damages the area of the triangle of guillain and mollaret , the dtt will be abnormal . from the present case of hod , in figure 4 , we can see the value of the apparent diffusion coefficient increased significantly on the left diseased region compared to the right diseased region . similarly , the value of fa decreased relative to the contralateral area .
the dti and dtt showed that the nerve fiber tract of left was decreased compared to the right and the other part of that interrupted .
however , there was no apparent change in the decussation of the superior cerebellar peduncle , which could illustrate that the lesion mainly damaged the left central tegmental tract and the left superior cerebellar peduncle rather than the decussation . according to the theory of the triangle of guillain and mollaret , damage to the ipsilateral central tegmental tract
the present case was a patient with hod after resection of cavernomas of the left dorsomedial pons , which can insult the left central tegmental tract and lead to hod .
the diagnosis of this case was made according to the presentation of dtt and dti . in clinical studies ,
hod was mainly caused by two kinds of treatment , including surgical resection and gamma knife radiosurgery of brain stem cavernomas.27,28 the frequency of hod caused by surgical resection was equal to that caused by gamma knife radiosurgery.27,28 yun et al21 reported that the time interval from resection to hod diagnosis was a mean of 7.7 months ( range : 112 months ) in surgical resection and 9.5 months ( range : 315 months ) in the gamma knife radiosurgery .
therefore , the interval between resection and hod occurrence is random , and not limited within a certain period range .
hod , as a rare disease of transsynaptic degeneration , has been the focus of an increasing amount of research in recent years .
the development of imageology promotes and improves the level of hod diagnosis and differential diagnosis .
unfortunately , knowledge of hod is still so limited and many mechanics of this disease are still so unclear that the therapeutic outlook is fairly pessimistic . more research and effort need to be put into practice . | hypertrophic olivary degeneration is a transsynaptic form of degeneration , which is also a result of primary or secondary lesion and can damage the dento - rubro - olivary pathway .
the dento - rubro - olivary pathway was first described by guillain and mollaret and is referred to as the triangle of guillain and mollaret .
multiple factors can destroy the dento - rubro - olivary pathway , such as surgical operation , hemorrhage , tumor , trauma , inflammation , demyelination , degeneration , and radiation damage .
all of the above factors can result in delayed hypertrophic olivary degeneration .
articles related to this disease cover etiology , clinical presentation , pathology changes , etc .
however , to our knowledge , there has been no literature reporting the use of diffusion tensor imaging and diffusion tensor tractography to improve the diagnosis of hypertrophic olivary degeneration following resection of cavernomas in the brain stem .
herein , we report a case who was diagnosed with hypertrophic olivary degeneration following resection of cavernomas of the brain stem , verify the significance of diffusion tensor imaging and diffusion tensor tractography , and review previous literature .
the development of imageology promotes and improves hypertrophic olivary degeneration diagnosis and differential diagnosis . |
the energy required for the cell survival is produced through the energy metabolism
process , during which oxygen and nutrients react .
therefore , the lungs that supply oxygen
are important for maintaining and improving health1 .
in particular , movement of the thorax causes movement of the pulmonary function
because the lungs are attached to the thorax . when the thorax expands , the intrapulmonary
pressure becomes lower than the atmospheric pressure , leading to inhalation ; when the thorax
contracted , the intrapulmonary pressure becomes higher than the atmospheric pressure ,
leading to exhalation2 .
contraction of the
diaphragm has the greatest effect on the reduction of intrapulmonary pressure , but the
expansion and contraction of the thorax also influence pulmonary function .
therefore , to
enhance pulmonary function , functions of the major respiratory muscles that directly expand
the thorax and the respiratory support muscles that help thoracic expansion by lifting the
thorax need to be improved .
major respiratory muscles include the diaphragm , intercostal
muscles , and abdominal muscles3 that are
involved in resting respiration4 .
respiratory support muscles include the sternocleidomastoid ( scm ) , scalene , trapezius ,
pectoralis major , pectoralis minor , and anterior serratus5 , which are involved in deep , forced breathing4 .
therefore , if neck muscles are not easily controllable or
the joint range of motion is limited , not only the neck movement but also the thoracic
movement become limited , which weaken respiratory function6 . in relation to this , legrand et al.7 conducted an experiment on dogs and verified that the movement of
scm and scalene , which are respiratory support muscles , lowered intrapulmonary pressure . in
particular
, they reported that the movement of the scalene had greater action compared to
scm .
the scm appears to have greater effect on thoracic movement because it is attached to
the clavicle and sternum , whereas the scalene muscles are attached to the first and second
ribs8 . in agreement with this ,
han et
al.9 also reported that stretching
exercises for the neck muscles partially improve pulmonary function .
furthermore , han et
al.10 reported improved pulmonary
function as a result of stretching exercise of the scm and scalene and strengthening
exercise for the neck muscles in patients with allergic rhinitis . likewise , cho et al.11 also reported improved pulmonary function
with neck muscle stretching and cervical joint motion exercises in stroke patients implanted
with a tracheotomy tube .
however , as reported in the study by de troyer and kelly12 , the scm and scalene are not involved in
resting respiration ; on the other hand , legrand et al.7 reported that the scalene muscles could be involved in resting
respiration .
the
anterior scalene is attached to the two - third of the medial point of the first rib and the
middle scalene is attached to the superior surface of the first rib .
the posterior scalene is attached to the dorsal
surface of the second rib ; thus , it lifts the second rib8 .
therefore , if the length of the scalene becomes shorter , the extent
of the lift of the thorax becomes smaller during inhalation , which can decrease the extent
of reduction of intrapulmonary pressure , thereby decreasing inhalation volume .
furthermore ,
during exhalation , the lowering of the thorax can be hindered , which decreases the extent of
exhalation , thereby reducing the total ventilation volume .
nevertheless , studies
investigating the correlation between the scalene muscles and pulmonary function are very
rare .
in particular , no study has been conducted on the correlation between scalene muscles
and resting vital capacity .
therefore , in this study , the possibility that the relaxation of
the scalene muscles can improve slow vital capacity ( svc ) was investigated .
the subjects of this study were 20 female students in their 20s who were attending
none of them had a past medical history of diseases of the nervous
system , musculoskeletal system , or cardiovascular system that could have affected the
results of this experiment .
the subjects were randomized into an experimental group of 10
subjects and a control group of 10 subjects who received no treatment .
all subjects were
informed of the purpose and methods of the study beforehand and voluntarily agreed to
participate .
this study complied with the ethical standards of the declaration of helsinki ,
and written informed consent was obtained from all subjects . before applying stretching exercise to the scalene muscles , svc of every subject was
measured using a digital spirometer ( pony fx , cosmed inc . ,
they were instructed to straighten their waist and shoulder , spread
their feet at shoulder width , and at right angle to the ground .
then , they were instructed
to block their nose with a nose clamp and slightly bite into a mouth piece of the measuring
instrument .
when ready , they were instructed to inhale slowly to the maximum when the start
message appeared on the monitor while breathing as usual and then exhale slowly to the
maximum , and to breathe as usual .
three measurements were taken , and the average values were
used for analysis . after pulmonary function was measured , stretching exercises for the scalene were performed ,
in the order of the middle , anterior , and posterior scalene . while manual stretching
exercises of the middle scalene , in the starting position , subjects were instructed to bend
their neck laterally in the opposite direction . then , the practitioner standing behind the
subject pressed the shoulder with one hand while touching one side of the head and applied a
lateral load with the other hand . for the manual stretching exercise of the anterior
scalene , in the same position
, the subject was instructed to bend the neck laterally in the
opposite direction , direct the eyes upwards , and slightly bend the neck posteriorly . in this
position ,
the practitioner standing behind the subject pressed the shoulder with one hand
while touching one side of the head and applied a load with the other hand laterally and
posteriorly . for manual stretching exercises of the posterior scalene , in the starting
position
, the subject was instructed to bend the neck forward and then laterally in the
opposite side , and to look to the side and downward .
the practitioner standing behind the
subject pressed and fixed the shoulder with one hand while touching one side of the head and
applied a lateral load with the other hand . for this manual stretching exercise , a
hold - relax technique that involved holding for 6 s , followed by relaxing for 4 s
the total duration of manual stretching exercise was about 15 min . in the
experimental group ,
the svc was measured again after scalene muscle stretching . in the
control group ,
the svc was measured again after resting in the sitting position on a chair
for 15 min , without treatment .
first , a test of normality was performed with the
shapiro - wilk test .
as the normality condition was satisfied , parametric statistical analysis
was performed in this study . to examine homogeneity between the experimental and control
groups before the experiment ,
then , a paired t - test was
performed to examine changes in svc between the experimental and control groups .
differences in the general characteristics and svcs of the experimental and control groups
before the intervention are listed in table
1table 1.differences in general characteristics and slow vital capacities between the
experimental and control groups before interventionvariablesexperimentalcontrolage ( years)21.3 0.721.6 0.5height ( cm)163.0 5.8162.6 3.7weight ( kg)60.2
( l)1.3 0.31.2 0.3minute ventilation ( l)15.6 6.614.3 6.4tidal volume ( l)0.7 0.20.6 0.2 . regarding general characteristics , the ages of subjects in the experimental
and control groups were 21.3 and 21.6 years ; the heights were 163.0 cm and 162.6 cm ; and the
weights were 60.2 kg and 57.3 kg , respectively .
for svc , the expiratory vital capacities
( evcs ) of the experimental and control groups were 3.1 l and 2.9 l ; expiratory reserve
volumes ( ervs ) were 1.2 l and 1.2 l ; inspiratory reserve volumes ( irvs ) were 1.3 l and 1.2
l ; ve was 15.6
l and 14.3 l ; and tidal volume ( vt ) were 0.7 l and 0.6 l , respectively .
the effects of stretching on svc are shown in table
2table 2.differences in slow vital capacities between the experimental and control groups
before interventionvariablesgrouppre - stretchingpost - stretchingexpiratory vital capacity ( l)experimental*3.1 0.33.3 0.3control2.9 0.32.9 0.3expiratory reserve volume ( l)experimental1.2 0.21.3 0.3control1.2 0.21.2 0.3inspiratory reserve volume ( l)experimental1.3 0.31.2 0.3control1.2 0.31.1 0.4minute ventilation ( l)experimental15.6 6.617.2 7.1control14.3 6.416.5 7.5tidal volume ( l)experimental*0.7 0.20.8 0.4control0.6 0.20.6 0.3*p<0.05 . in the experimental group ,
evc ( p<0.05 ) and vt ( p<0.05 ) showed a
significant increase after stretching . on the other hand , erv and
in the control group ,
ve and vt increased , erv and irv decreased , and evc did not change .
inspiratory and expiratory volumes are influenced by the degree of contraction and
relaxation of the diaphragm2 .
however , the
roles of respiratory support muscles that lift the thorax can also influence ventilation
volume .
de troyer and kelly12
electrically stimulated the respiratory support muscles originating from the neck of dogs ,
examined changes of the thorax and found that the volume of the thorax increased .
muza et
al.13 also verified that lung volume
increases when electric stimuli are applied to the scm of dogs .
these results clearly show
that the scm and scalene which are respiratory support muscles are involved in pulmonary
function . in relation to this ,
wilson and de troyer14 , 15 assumed that the muscles
involved in the respiratory system could be involved in the expansion of the chest wall . in
particular , because the contracting power of the muscles involved in inhalation is the
driving force behind the opening of the respiratory tract , muscle mass , the maximal active
muscle tension per unit cross - sectional area , and fractional changes in muscle length per
unit volume increase of the relaxed chest wall influence the expansion of the respiratory
tract16 .
in other words , if the
inspiratory main and support muscles have many muscular fibers , a large muscular contraction
force per unit area , and a large change in muscle length per unit time , the pressure
expanding the respiratory tract and the inhalation volume increase .
therefore , if the scm
and scalene have a large volume , a large muscular contraction force per unit area , and a
large change in muscle length per unit time , the thorax is lifted to a greater extent and
the inhalation volume increases . in this
regard , han et al.17 also noted that forward head posture could reduce vital capacity ,
possibly because of weakness of the accessory respiratory muscles .
this means that
respiratory function is related to the functions of the scm and scalene muscle . to verify
this , legrand et
al.7 examined changes in
the length of the scm and scalene while manually injecting external air into dogs to expand
their lungs . as a result
, they found that the scm and scalene both became short , and the
scalene in particular became shorter .
furthermore , when muscular contraction was induced by
stimulating the two muscles , the rib cage significantly moved toward the head and the
alveolar pressure decreased .
consequently , the more the lengths of the scm and scalene
changed per unit volume , the more the alveolar pressure lowered .
this result experimentally
confirmed that the ventilation volume through thoracic expansion increases when the scm and
scalene maintain reasonable lengths and the contraction and relaxation are normal . in line
with this , the results of the present study show that the vt and evc increased in the svc .
the vt increased
because the stretching of the scalene increased its length , which increased changes in the
contracting muscle length per unit time and accelerated the lifting of the thorax , and the
intrathoracic pressure became lower as a result .
likewise , the evc increased because the
scalene was relaxed and the descent of the thorax during exhalation became larger , further
increasing intrathoracic pressure .
in addition , han et al.17 also mentioned that forward head posture could reduce vital
capacity , possibly because of weakness of the accessory respiratory muscles .
the results of this study verified that stretching of the scalene improves vt and evc , and
this has positive effects on increased svc .
hence , stretching of the scalene should be
included in the programs for improvement of respiratory functions . | [ purpose ] the purpose of this study was to examine whether stretching of the scalene
muscles would improve slow vital capacity ( svc ) . [ subjects and methods ]
the subjects of
this study were 20 healthy female students to whom the study s methods and purpose were
explained and their agreement for participation was obtained .
the svc was measured using
spirometry ( pony fx , cosmed inc . ,
italy ) .
the intervention used was stretching of the
scalene muscles . stretching was carried out for 15 min , 10 times at per each portion of
scalene muscles : the anterior , middle , and posterior parts .
[ results ] expiratory vital
capacity ( evc ) and tidal volume ( vt ) noticeably increased after stretching .
however , there
were no changes in any of the svc items in the control group .
[ conclusion ] this study
demonstrated that stretching of the scalene muscles can effectively improve svc . in
particular , we confirmed that stretching of the scalene muscles was effective in
increasing evc and vt , which are items of svc . |
as molecular genetic testing and electronic healthcare records become increasingly common features of modern medical practice , there is a need to integrate information in genetic databases to establish a detailed understanding of how genome sequence differences impact on human health . to date
, there are several depositories that fall under the banner of mutation databases , which can be divided into three main categories : central ( or core ) databases , such as online mendelian inheritance in man [ omim , ; ( 1 ) ] , the human gene mutation database [ ; ( 2 ) ] , locus - specific databases ( lsdbs ) , a large group with over 570 members [ ; reviewed in ref . ( 3 ) ] and national / ethnic mutation databases [ nemdbs , ( 4 ) ] .
the recent emergence of national and ethnic mutation databases ( nemdbs ) is justified by the fact that the spectrum of mutations observed for any gene or disease will oftendiffer not only between population groups across the planet but also between distinct ethnic groups within a geographical region .
nemdbs provide data that can be used to e.g. stratify national molecular diagnostic services , study human demographic history , admixture patterns and gene / mutation flow ( 5,6 ) .
we have previously described the development of specialized software , namely ethnos ( 7 ) , which has facilitated the construction of the hellenic , cypriot , iranian , lebanese and serbian nemdbs ( 79 ) . however
, this software could not handle advanced data querying and storing of large datasets . here , we report the construction of frequency of inherited disorders database ( findbase ) , a relational database based on an upgraded version of the ethnos software , capable to accommodate large datasets pertaining to frequencies of causative mutations , leading to inherited disorders in various populations and ethnic groups worldwide .
the initial information data sources in findbase were acquired from the european union fp5 cystic fibrosis thematic network consortium data ( ) , also reported by the world health organization (; the molecular epidemiology of cystic fibrosis ) and partially published elsewhere ( 10 ) , and population - specific mutation frequency data derived from two lsdbs , namely the hbvardatabase of human hemoglobin variants and thalassemia mutations [ ; ( 1114 ) ] and the pahdb phenylalanine hydroxylase locus knowledgebase [ ; ( 15,16 ) ] . in addition , original and review articles retrieved by manual searching of all articles listed in the pubmed literature database ( ) , international conference proceedings and personal communications by research groups also consist part of findbase data source .
the information source ( publications , conference proceedings , etc . ) is displayed by selecting the
submissions option , located in the overview section . in case of multiple articles on a single genetic disorder in a certain population group , calculation of mutation frequencies
is based on a single and most representative study , involving sufficient numbers of patients and controls .
estimation of mutation frequencies based on multiple reports has the inherent danger of including redundant cases , which can alter the calculated frequencies . by only including number of chromosomes and not sensitive personal data of their carriers , the important issue of anonymity
findbase is the richest nemdb among those currently available content - wise ( 4 ) with a total of 3 379 records and contains information on 31 different inherited disorders studied in 83 population groups , resulting from 1 223 causative mutations in 24 genomic regions ( october 2006 release ) .
automated summary listings on findbase contents can be generated by selecting the respective options ( records ,
mutations and genes respectively ) in the menu , located at the left side of the screen ( see also supplementary figure ) .
the initial information data sources in findbase were acquired from the european union fp5 cystic fibrosis thematic network consortium data ( ) , also reported by the world health organization (; the molecular epidemiology of cystic fibrosis ) and partially published elsewhere ( 10 ) , and population - specific mutation frequency data derived from two lsdbs , namely the hbvardatabase of human hemoglobin variants and thalassemia mutations [ ; ( 1114 ) ] and the pahdb phenylalanine hydroxylase locus knowledgebase [ ; ( 15,16 ) ] . in addition , original and review articles retrieved by manual searching of all articles listed in the pubmed literature database ( ) , international conference proceedings and personal communications by research groups also consist part of findbase data source .
the information source ( publications , conference proceedings , etc . ) is displayed by selecting the
submissions option , located in the overview section . in case of multiple articles on a single genetic disorder in a certain population group , calculation of mutation frequencies
is based on a single and most representative study , involving sufficient numbers of patients and controls .
estimation of mutation frequencies based on multiple reports has the inherent danger of including redundant cases , which can alter the calculated frequencies . by only including number of chromosomes and not sensitive personal data of their carriers , the important issue of anonymity
findbase is the richest nemdb among those currently available content - wise ( 4 ) with a total of 3 379 records and contains information on 31 different inherited disorders studied in 83 population groups , resulting from 1 223 causative mutations in 24 genomic regions ( october 2006 release ) .
automated summary listings on findbase contents can be generated by selecting the respective options ( records ,
mutations and genes respectively ) in the menu , located at the left side of the screen ( see also supplementary figure ) .
findbase was developed to facilitate easy creation and maintenance of fully web - based population - specific databases , is platform - independent and uses php and mysql ( , mysql ab , uppsala , sweden ) , open source software . at present , data are stored in a single data table , although a future upgrade includes splitting the data in multiple tables , corresponding to each population group documented in findbase ( see below ) . in other words , findbase will ultimately be a collection of many nemdbs , operating under the same software .
database design follows all content criteria and human genome variation society ( hgvs ; ) recommendations .
finally , findbase design follows certain database guidelines in order to conform to quality ( 17 ) .
findbase is a freely available online resource , which can be accessed on the world wide web at the url address : .
detailed instructions for both using and querying the database are also available from the same site ( user guide ) .
there has been no claim of ownership of the information stored in this database by anyone involved in this initiative .
however , this compilation and representation of it are subject to copyright and usage principles to ensure that findbase and its contents remains freely available to all interested individuals .
there are three levels of registered users , as outlined below in hierarchical order :
administrators have full access rights to all database 's functionalities and contents.national coordinators have data entry and modification rights , registration and account activation for advisors / curators and data re - allocation to another advisor / curator .
national coordinators are responsible for managing the overall construction and maintenance of a nemdb , contributing to findbase .
their role is also to promote the usage of their nemdb in their population and to promote investigations on those genetic disorders , which mutation spectrum is not yet known in their countries.advisors and curators have data entry and modification rights only for those data entered by him / herself and under no circumstances can alter data entered by another advisor / curator .
advisors and curators are responsible for data entry in the nemdb , for which they are registered . if an advisor / curator wish to end his involvement with findbase , their data are allocated to another curator who will then be responsible for their curation .
national coordinators have data entry and modification rights , registration and account activation for advisors / curators and data re - allocation to another advisor / curator .
national coordinators are responsible for managing the overall construction and maintenance of a nemdb , contributing to findbase .
their role is also to promote the usage of their nemdb in their population and to promote investigations on those genetic disorders , which mutation spectrum is not yet known in their countries .
advisors and curators have data entry and modification rights only for those data entered by him / herself and under no circumstances can alter data entered by another advisor / curator .
advisors and curators are responsible for data entry in the nemdb , for which they are registered . if an advisor / curator wish to end his involvement with findbase , their data are allocated to another curator who will then be responsible for their curation .
national coordinator or advisor / curator accounts can be requested in the corresponding ( register ) section .
the request is then automatically sent to the administrator and national coordinator of that particular nemdb respectively .
the account is subject to eligibility criteria , such as active involvement in the area of human genetics , and thorough knowledge of the genetic basis of inherited disorders in a particular population .
the data entry page can be found in the curators section . once logged in
, the registered user connects with publication data editor , for further guidance through the data entry procedure .
each entry contains empty fields in a table format , where the registered user enters the genetic disorder , the gene , accompanied by the respective omim i d , the mutation in the correct ( official ) nomenclature , the number of chromosomes or mutation frequencies .
population name is automatically set , depending on the population / nemdb for which the curator is registered , and , where available , the entry of the ethnic group or geographical region is required .
findbase provides a browsing / filtering interface to the underlying data allowing one to formulate queries in order to better explore the variety and depth of information recorded therein .
such queries can be useful in a clinical setting to reach a diagnosis or to better coordinate mutation screening .
stored data can be queried via the search page in two different ways , namely automated summary listings , sorted by disorder , gene or mutation and user - defined queries , by population or mutation .
first of all , a user can query for all the different inherited disorders found in a particular population , such as all the hungarian population . in this case
, the user needs either to click on hungary 's capital from the world map ( figure 1a ) in the home page or to select for the
hungarian population from the population list in the home ( supplementary figure ) or search page ( figure 1b ) .
query results are then provided in a summary list , displaying the disorder , the gene found mutated and the mutations found in the population in question . if a genetic disorder is further specified for the selected population , the query returns a detailed list of all relevant mutations together with their frequencies and data source in a table of graphical format . in the latter case ,
a chart is automatically created with the corresponding mutation frequencies shown in the bars ( figure 1c and d ) , while mutation frequencies from different ethnic groups or geographical regions are displayed in different colors ( figure 1d ) .
the disorder and gene names and omim i d are hyperlinked to further information in the omim central database .
the search page also allows the user to insert the desired frequency range ( figures 1d and 2a ) . querying findbase for frequencies of mutations leading to inherited disorders in a certain population .
( a ) the world map in the home page , from where the user can quickly select the desired population to query upon by clicking on a country 's capital ( in this case , hungary ) .
a communication box automatically appears containing the corresponding national flag and the number of disorders currently documented in findbase .
find all inherited disorders in the hungarian population. only parts of the search page are shown .
the user needs either to click on hungary 's capital from the world map ( as previously described ) or to select for the hungarian population from the population list , located either in the home page [ next to the world map ( supplementary figure ) ] or at the left part of the search page .
query results are in a table format , indicating the disorder , the gene found mutated and the mutations in their official nomenclature .
( c ) construction of the query find all mutations leading to -thalassemia in the hungarian population. by selecting
hungarian and beta - thalassemia from the population and disorder 's list respectively , the query returns a list of all -thalassemia mutations found in the hungarian population together with their frequencies and data source in a table ( by selecting the show results as table button ) or graphical format ( by selecting the show results as chart button ) .
the disorder and gene names and omim i d are hyperlinked to further information in the omim central database .
( d ) sample query find all mutations leading to cystic fibrosis in the austrian population with a frequency range of 1% and above. by selecting austrian and
cystic fibrosis from the population and disorder 's list respectively and specifying the desired frequency range in the corresponding boxes ( indicated with an arrow ) , the query returns a list of all cftr mutations with a frequency range of 1% and above found in the austrian population together with their data source in a table or graphical format . in the latter case ,
the different cftr mutation frequencies found in different geographical regions are differentially displayed , with the corresponding mutation frequencies shown in the bars . querying findbase for frequencies of mutations leading to a certain inherited disorder in different populations .
( a ) construction of the query find frequent ( between 2 and 100% ) familial hypercholesterolemia mutations in all populations. ( b ) the user needs to select for the
familiar hypercholesterolemia disorder from the disorder 's menu , located either in the home page [ next to the world map ( supplementary figure ) ] or at the right part of the search page .
query results are in a table format , indicating the populations and ethnic groups ( if applicable ) where ldlr mutations are found .
( c ) construction of the query find the frequency of the p.s188f mutation leading to glucose 6p - dehydrogenase ( g6pd ) deficiency in all populations. by selecting
glucose 6p - dehydrogenase deficiency from the disorder 's list and p.s188f from the mutation 's list , the query returns a list of populations in which the p.s188f mutation is found together with the corresponding frequencies .
query output can be in a graphical ( d ) or table format ( e ) , always accompanied by the data source .
findbase data can be also queried by disorder and refined by mutation . in this case
, the user needs to select from the disorder 's menu , located either in the home page [ next to the world map ( supplementary figure ) ] or at the right part of the search page ( figure 2a ) and refine the search by specifying the mutation name ( figure 2c ) .
query results are again in a table ( figure 2b and e ) or graphical format ( figure 2d ) , indicating the populations and ethnic groups ( if applicable ) where this disorder is found .
national and ethnic - specific mutant alleles , identified from diagnostic laboratories , should be ideally stored in such databases to become freely accessible to all individuals , researchers and laboratories involved in molecular genetic testing in an either national or even international scale . combining relevant information from many population groups and with numerous links for the documented genetic disorders , genes and lsdbs
, findbase has the potential to expand significantly to become a reference source of information on the incidence of inherited disorders in various populations .
findbase is being upgraded : ( i ) to enrich the existing data collection , ( ii ) to upgrade ethnos software , ( iii ) to optimize database content and quality management .
upgrade is estimated to last 2 years , while funding for maintenance and upgrade is secured for a 5-year period from ec projects and private sources .
the latter is encouraging since opportunities for funding database projects are difficult to secure ( 18 ) .
we are currently in the process of collecting population - specific data from the published literature by manual or automated means , i.e. data mining software . also , being part of two european commission consortia increases not only the numbers of findbase end - users but also the number of potential data contributors . in june 2006
, we have started dispatching invitation letters to consortia members to contribute population - specific mutation frequency data in those cases where existing data were either not representative , e.g. based on a small population size , or really outdated .
so far , we have seen a very satisfactory response and we hope that more users will positively react to our call .
we also plan to provide database pages in a language other than english , a feature , currently not available in any nemdb ( 4 ) , which will appeal the database to non - specialist users , such as patients and family members .
as far as further upgrading of the ethnos software is concerned , we are currently working on establishing separate nemdbs per population ( source databases ) , hosted either in the main findbase server or in a remote location ( see also database design , implementation and access ) , which will intercommunicate with findbase [ central ( warehouse ) database ] bi - directionally .
the latter is facilitated by the fact that both the source and central databases operate under the same software , which not only contributes towards nemdb uniformity ( 4 ) but also allows for maintaining updated versions of the linked nemdbs using a very simple server task tool .
this will also allow local ( national ) curators to maintain a curator 's page that will differ from nemdb to nemdb and could redirect the end - user to other websites , containing information that may not appear in findbase itself , urls from local human genetic societies , conferences of interest , etc .
our preliminary data show that such operation is feasible and we anticipate that the existing nemdbs , currently based on the previous ethnos ( version1.0 ) flat - file database format [ ; ( 79 ) ] will migrate to the sql - based ethnos software in the beginning of 2007 .
last , but not least , database content and quality management is one of the key elements for success of database projects .
so far , findbase control is maintained locally , but as data influx will continue to rise , it would seem logical for its control and decision making to pass to a multinational consortium of experts from the field of human molecular genetics to form findbase steering committee ( 4 ) . also , in order to provide incentives to potential contributors and researchers to submit their data to findbase and at the same time to prevent population frequency data from being lost or kept unpublished , findbase would act as a specialized database - journal , exclusively dedicated to document the molecular basis of inherited disorders in different populations .
the main advantage of such an approach is that wherein all data gets immediately submitted and , once accepted , deposited into an internet - accessible structured depository , like findbase , all of which will be interconnected [ see also ref . ( 18 ) ] .
findbase steering committee can serve as the editorial board of such database - journal , evaluating the quality of the data submitted , i.e. conforming to a number of pre - determined requirements ( e.g. minimum population size tested , etc ) , while each submission , once entered into findbase , would be registered under a unique pubmed i d , providing authors with a certain degree of credit for their contribution . such approach has been previously proposed by the hgvs for lsdbs ( 19 ) .
we believe that this innovative approach , if adopted , could lead the way and can , thereby , produce a non - profit model for sustainable funding for such an entity .
findbase would be also linked to other online repositories , such as the waystation ( ) as part of the human variome project ( 20 ) and to complement efforts with genetests - geneclinics [ ; ( 21 ) ] and orphanet ( ) , which are useful online resources for the various molecular genetic laboratories and tests provided in the united states and europe respectively .
finally , it is known from our previous experience that the user input is fundamental for improving the overall database quality and data accuracy .
database users frequently contact the administrators in order to report missing information and pinpoint inconsistencies and/or erroneous entries .
therefore , we urge findbase users to frequently communicate their opinions and notify the administrators and national coordinators for errors or for incomplete information .
this will certainly contribute towards keeping the database as complete and up - to - date as possible .
| frequency of inherited disorders database ( findbase ) ( ) is a relational database , derived from the ethnos software , recording frequencies of causative mutations leading to inherited disorders worldwide .
database records include the population and ethnic group , the disorder name and the related gene , accompanied by links to any corresponding locus - specific mutation database , to the respective online mendelian inheritance in man entries and the mutation together with its frequency in that population .
the initial information is derived from the published literature , locus - specific databases and genetic disease consortia .
findbase offers a user - friendly query interface , providing instant access to the list and frequencies of the different mutations .
query outputs can be either in a table or graphical format , accompanied by reference(s ) on the data source .
registered users from three different groups , namely administrator , national coordinator and curator , are responsible for database curation and/or data entry / correction online via a password - protected interface .
databaseaccess is free of charge and there are no registration requirements for data querying .
findbase provides a simple , web - based system for population - based mutation data collection and retrieval and can serve not only as a valuable online tool for molecular genetic testing of inherited disorders but also as a non - profit model for sustainable database funding , in the form of a database - journal. |
multiple sclerosis ( ms ) is an immune - mediated , neurodegenerative disease that affects an estimated 2.5 million adults worldwide . in isfahan
/ iran , an increase in prevalence and incidence figures ( 73.3 ; 9.1 : 2004 - 2005 vs. 43.8 ; 3.64 : 2003 - 2010/1,00,000 ) was reported .
ms typically has its onset in early adulthood , and affects more women than men .
this disease manifests in a wide range of symptoms including muscle weakness , extreme fatigue , imbalance , visual , speech disturbances , and cognitive impairment .
such symptoms often lead to poor health - related quality of life ( hrqol ) , neurologic disability , and high health care costs .
ms poses multiple challenges for both physical and psychological well - being ( pwb ) people with ms experience unpleasant and unpredictable symptoms , difficult treatment regimes , and drug side effects , and increasing levels of physical disability .
they also face psychosocial consequences including disruptions to life goals , employment , income , relationships , leisure activities , and daily living activities .
psychological difficulties are extremely common in ms compared to both healthy populations and other chronic diseases .
the empirical literature attests to elevated rates of depression and distress , increased anxiety , low subjective well - being , quality of life ( qol ) , social role and relationship difficulties .
nonetheless , a substantial proportion of people with ms manage to adapt well to living with the illness .
illness factors such as the extent of neurological disability , symptom severity , remission status , and length of illness can influence levels of psychological adjustment in ms . however , these factors are inconsistently associated with adjustment , and are often only modest predictors .
research demonstrates that psychological factors are often better predictors of individual differences in adjustment than illness factors .
psychological factors , unlike illness factors , are potentially modifiable through psychological interventions . studies on emotional distress and hrqol broadened the traditional biomedical focus in ms research , but little attention was paid to total well - being indicators . although research on these topics has broadened the traditional biomedical focus to include subjective evaluations of living conditions , the limitations of such approach are increasingly evident . first , in ms literature qol is substantially related to health issues , while other relevant life domains are neglected .
second , research and intervention primarily target physical and emotional symptoms , namely the negative aspects of illness .
such focus implicitly equates health with the absence or reduction of disease / infirmity , and not with a state of complete physical , mental , and social well - being , as defined by the who . in this respect , psychologists recently called for a shift in attention from human shortcomings and deficits to personal resources and potentials , showing through empirical studies that well - being is not the opposite of ill - being ; rather , it comprises unique dimensions .
the hedonic one focuses on emotions and operationalizes well - being as life satisfaction and prevalence of positive over negative affect ( hedonic balance ) .
one of the eudaimonic constructs is pwb , purport by ryff that well - being is a multi - dimensional construct made up of life attitudes necessary for positive functioning .
this conception of qol has been termed by ryff as pwb and has received extensive empirical support .
ryff suggests a model representative of six dimensions of well - being :
self - acceptance ( sa ) ; the ability to feel good about oneself while being aware of one 's limitations , environmental mastery ( em ) ; the attempt to shape one 's environment to meet personal needs and wishes , positive relations with others ( pr ) ; having affirming relationships with others , personal growth ( pg ) ; making the most of one 's capability and skills , purpose in life ( pl ) ; ability to find meaning in one 's struggles or hardships , andautonomy ( a ) ; seeking a sense of personal authority and independence .
self - acceptance ( sa ) ; the ability to feel good about oneself while being aware of one 's limitations , environmental mastery ( em ) ; the attempt to shape one 's environment to meet personal needs and wishes , positive relations with others ( pr ) ; having affirming relationships with others , personal growth ( pg ) ; making the most of one 's capability and skills , purpose in life ( pl ) ; ability to find meaning in one 's struggles or hardships , and autonomy ( a ) ; seeking a sense of personal authority and independence .
the experience of having ms has the potential to impact seriously each one of these aspects of well - being , but little research has examined these constructs in ms population .
research has shown that conditions such as chronic disease are not necessarily perceived as stressful threats .
they can also be interpreted as challenges and opportunities for growth , thus not hampering well - being , especially its eudaimonic components such as meaning - making , interpersonal relations , and engagement in daily activities .
accordingly , in the few studies targeting hedonic well - being in ms research , people with ms were more dissatisfied with their lives than healthy individuals .
as for eudaimonic well - being , only a few studies were carried out : it showed that people with ms and healthy controls reported similar levels of pg .
well - being ( both hedonic and eudaimonic aspects ) decreased with a higher disability .
hart et al . found that treating depression was significantly associated with improvements in 4 of the 6 scales of pwb included em , positive relations , pl , and self acceptance . because there is no published research on pwb in the iranian population
, we decided to examine pwb in people with ms in comparing to normal individuals .
the aims of the present study were to compare pwb in people with ms with normal people in isfahan , iran , and to determine whether there are relationships between demographic variables and pwb in ms population .
fifty - five male and female people with ms and 83 people with no neurological diseases aged 18 - 60 years were included in this case - control study , which was conducted in july 2012 .
the people with ms randomly were selected from ms clinic ( kashani hospital , isfahan neurosciences research centre ) .
criteria for the participants with ms were :
diagnosed by a neurologist based on systemic examination and laboratory findings such as magnetic resonance imaging andno other neurological and psychiatric disorders .
the normal sample also was selected through multistage sampling from 15 municipal zones in isfahan .
diagnosed by a neurologist based on systemic examination and laboratory findings such as magnetic resonance imaging and no other neurological and psychiatric disorders .
the normal sample also was selected through multistage sampling from 15 municipal zones in isfahan .
ryff 's measure defines well - being as a composition of six different psychological constructs include a : independence and self - determination , sa : positive attitude towards oneself and one 's past life , em : the ability to manage one 's life , pr : having satisfying high quality relationships , pg : being open to new experiences , pl : believing that one 's life is meaningful .
the participants were asked to grade their agreement to the sentences in seven categories , from strong disagreement to a strong agreement .
the responses were coded from 0 to 6 and summed across the three items of each dimension .
some items were reversed , and all items randomly distributed in an attempt to keep participants from seeing a pattern .
the instrument , in this study showed varied internal consistency reliability for the six subscales :
a ( cronbach 's = 0.34),em ( cronbach 's = 0.56),sa ( cronbach 's = 0.78),pl ( cronbach 's = 0.42),pg ( cronbach 's = 0.58 ) , andpr ( cronbach 's = 0.34 ) .
a ( cronbach 's = 0.34 ) , em ( cronbach 's = 0.56 ) , sa ( cronbach 's = 0.78 ) , pl ( cronbach 's = 0.42 ) , pg ( cronbach 's = 0.58 ) , and pr ( cronbach 's = 0.34 ) . a total psychological well - being ( tpwb ) score was calculated by adding all six constructs ( cronbach 's = .7 ) . the same results seen in reported in other studies .
t - test was performed to examine the differences in pwb scales in two normal and ms samples .
anovas were conducted to examine differences in pwb scales in ms group with regard to gender , married status , and level of education .
this study was approved by the local ethics committee and all patients in this study , and all subjects gave their written consent .
demograghic characteristics of two samples are presented in table 1 . as shown in table 2 , people with ms reported lower level pwb in all subscales than the normal group .
demographic characteristics of two samples a comparison of the mean scores for the psychological well - being scales in patients with ms according to the table 3 there is no significant difference in tpwb in people with ms regard to gender and married status , but individuals with a higher level of education reported higher tpwb . for exploring relationship between the level of education and pwb in details ,
the results showed that patients with higher level of education reported a higher level of pr and pl .
total psychological well - being regard to gender , married status and level of education in ms sample
our results indicate a reduction in all aspects of pwb of people with ms in comparison to the normal population and only the level of education has significantly positive relationship to pwb in ms group .
reduction in pwb could be attributed directly and indirectly to the symptoms and outcomes of ms disease .
people with ms are faced with uncertainty about the future , unpleasant and unpredictable symptoms , difficult treatment regimes , and drug side effects .
ms can have profound consequences , including disruption of life goals , employment , income , relationships , social , leisure activities , and activities of daily living .
this may be particularly significant because , for the majority of people with ms , the disease begins in young adulthood , a period that is often important for career development and starting families .
individuals with low positive well - being were 7.16 times more likely to be depressed 10-years later .
historically the management of ms has been predominantly about limiting disability by the symptomatic management of acute relapses and attempting to influence the long - term course . even though this type of management is important , we suggest this is accompanied by an equal effort at improving participation pwb . because the previous studies showed that both physical and psychological aspects are important and interact with each other .
it is necessary to continue the research on pwb in people with ms to evaluate the impact of factors such as the type of pharmacological treatment and rehabilitation . regarding the correlation between pwb and level of education
, we believe that firstly it is assumed that ms symptoms are to be diagnosed sooner in people with higher education , thus treatment affairs sooner be started and consequence impairment in pwb would be minimized . in the other hand , there is more purpose for people with higher education especially who enter to universities in life , and they have a broader range of relationship options . the limitations of this study were the lack of a control group .
furthermore , patients who participated in this research in terms of disability were mild to moderate .
srd contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work as first author .
fh contributed in the conception of the work , conducting the study , data analysis and agreed for all aspects of the work.fa contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work.vsh contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .
all authors have read and approved the content of the manuscript and are accountable for all aspects of the work . | background : to date , few results on well - being in individuals with neurological disease have been published , while several studies in other groups have indicated that well - being may not be the only absence of psychological distress , but also positive psychological function .
the aim of the present study was to compare the psychological well - being ( pwb ) between the people with multiple sclerosis ( ms ) and normal individuals and identify correlated demographic factors to pwb in people with ms disorder.materials and methods : a case - control study was performed in july 2012 on 55 people with ms who were referred to ms clinic ( located at the kashani hospital ) , isfahan neurosciences research centre and 83 normal individuals with matched mean of age , level of education , and gender .
the participants filled up the 18-item ryff 's pwb and demographic profile .
the data were analyzed by spss software based on the independent t - test , and anova.results:there is significant different in all pwb dimensions between people with ms and normal groups .
there were no significant differences in pwb in people with ms in relation to gender and marital status , but individuals with higher level of education scored higher in total pwb , positive relationship with others and purpose in life.conclusion:people with ms are at risk of lower level of pwb .
interventional programs for improving pwb are strongly recommended . |
it is a paradox that at the same time these drugs are amongst the top 5 drugs all categories causing most serious and fatal events .
the major reason is the difficulty to tailor the dose according to the individual dose requirement which varies widely between patients .
few other drugs than warfarin have a broader therapeutic dose window , 2030-fold , possibly with exception for narcotic analgesics .
historically , it has been known for long that a vitamin k containing diet has an influence on the efficacy of warfarin and other coumarins .
it has also been known for a long time that increasing age reduces the dose needed to achieve a therapeutic anticoagulant effect through inhibition of coagulation factor synthesis .
other factors that are known to influence dose requirement include concurrent medication , and body surface area [ 24 ] .
we embarked upon a prospective study of patients in need of anticoagulation because of atrial fibrillation , deep venous thrombosis , pulmonary emboli , and so forth . in our prospective study ,
warfarin nave patients consented to donate a blood sample for genotyping at the start of warfarin treatment .
all of them were monitored according to local clinical routines at 40 different study sites in sweden .
warfarin bleedings defined according to who definition of severe adverse drug reaction as well as other adverse reactions were reported to the study centre where the blood samples were stored in a biobank sample collection . in this cohort of patients starting treatment with warfarin , the incidence of first - time severe haemorrhage was 2.3 per 100 patient years ( 95% confidence interval 1.43.1 ) . during a mean followup time of 10 months ,
1.8% of the included patients died and the overall incidence of severe bleeding or death ( all - cause ) was 4.3 per 100 patient years ( 95% c.i .
our assessment of the incidence was lower than has been reported earlier [ 79 ] , probably because of the more strict criteria stipulated by who and applied by us .
although the incidence of bleeding was lower than that seen in many older studies , it is evident that haemorrhagic complications remain a major clinical problem in anticoagulation with vitamin k antagonists .
when investigating potential predictors of severe bleeding in a multivariable model ( table 1 ) , we identified two factors significantly associated with this adverse outcome male gender ( hr = 2.8 , 95% c.i .
1.17.3 ) and use of drugs potentially interacting pharmacologically with warfarin ( hr = 2.3 , 95% c.i .
the vastly increased risk in men was an unexpected finding and previous studies have oppositely showed an increased risk in female patients [ 8 , 10 , 11 ] . although the association between the use of interacting drugs and bleeding may have been anticipated , the evidence of an overall effect of the full spectrum of interacting drugs has been scarce prior to our findings .
the association remained even after several years of treatment ( figure 2 ) indicating prevailing difficulties in managing the effects of drug interactions in warfarin - treated patients .
with the development of pharmacogenetics , the interest was focused on enzymes catalysing the metabolism of anticoagulants to see if there was a relation between dose requirement and genetic polymorphisms in these genes .
early studies [ 12 , 13 ] demonstrated an important role of cyp2c9 in the metabolism of warfarin , in particular the 7-hydroxylation of the more active s - enantiomer .
they studied hospitalised patients and found that low - dose requirement was associated with cyp2c9 * 2 and cyp2c9 * 3 alleles .
these findings have subsequently been confirmed in numerous clinical or experimental study settings [ 4 , 1518 ] .
the common cyp2c9 * 2 and * 3 allelic functional variants have a compromised ability to catalyze the oxidation of s - warfarin , with in vitro activities of only 12 and 5% , respectively , for the homozygous mutant forms [ 19 , 20 ] .
other enzyme members of the cyp2c family have some catalyzing capacity albeit very little and of minor importance for the clearance of s - warfarin .
the identification and localisation of the vkorc1 gene [ 21 , 22 ] paved the way to studies that demonstrated a profound dependence of warfarin dose on the polymorphic vkorc1 gene [ 2326 ] .
since warfarin targets the vitamin k epoxide reductase , it is conceivable that any functional mutations in this gene will perturb the balance between the influence of warfarin and vitamin k on the enzyme activity .
one of these snps is located in the promoter region and usually included in studies of the vkorc1 genetics and warfarin dose requirements , namely 1639 g > a ( rs 9923231 ) .
the a allele is associated with the need of lower doses than the g allele .
the association is such that a homozygous carrier of the a allele requires a warfarin dose approximately 50% of that of an individual that is homozygous for the g allele .
similar effects have been ascribed to the vkorc 1173c > t ( rs9934438 ) polymorphism . however , these two snps are in pronounced linkage disequilibrium and are interchangeably used as tag snps for differentiation between a low - dose haplotype a ( 1639a and 1173 t ) and a high - dose haplotype b ( 1639 g and 1173c ) .
other snps have been independently associated with altered warfarin dose requirements , but their contribution to the interindividual variability is small compared to the 1639 g > a and 1173c >
candidate gene work with univariate regression analysis of small samples has limited power to detect warfarin dose association with allelic variants with weak influence .
thus , both cyp2c9 * 3 and * 2 were statistically significant predictors of warfarin dose when data were corrected for nongenetic and other dose predictors .
genome - wide association studies ( gwass ) are increasingly applied within pharmacogenomics and in studies of multiple gene factors behind certain diseases .
this approach was used by us in 1053 of 1523 patients of the warg cohort and was sufficiently powered to detect a genome - wide significance of p < 1.5 10 for polymorphisms with modest influence on therapeutic dose need .
univariate regression analysis of each snp analysed ( n = 325,997 ) disclosed just two important loci / snp regions .
this snp is located in a gene encoding a cytochrome p450 enzyme protein , cyp4f2 , a putative catalyst of vitamin k1 oxidation .
the pharmacogenetic knowledge about warfarin pharmacokinetics and pharmacodynamics has to be translated into useful dosing guidelines in order to be accepted in the clinical care of patients in need of warfarin .
several algorithms have been suggested on the basis of patient information with or without genetic data on the vkorc1 and cyp2c9 polymorphisms . by now
, there is unequivocal evidence that combined clinical and genetic information generates more accurate dose prediction than non - genetic information alone [ 33 , 34 ] .
considering the allele frequencies of cyp2c9 and vkorc1 , it is obvious that about 3/4 of the caucasian population carry at least one allelic variant in these genes with lower warfarin dose requirement ( figure 3 ) .
table 2 demonstrates the fractional distribution of different haplotypes based on the allele frequencies given for each of the genes .
when based on a multiple regression model that was developed in the warg material , the algorithm for estimation of warfarin dose ( table 3 ) predicts a 12-fold difference between the extreme haplotypes in the upper left and lower right corners of table 2 . according to our algorithm ( table 3 ) ,
this difference increases to 14-fold if the patients are treated with three interacting drugs , demonstrating the complexity of interactions between all predictors of the warfarin dose .
this is because the dose requirement decreases much less ( 5% ) for the cyp2c9 * 1/*1 , vkorc1b / b combination than for the cyp2c9 * 3/*3 , vkorc1a / a genotype ( 18% ) .
figure 4 demonstrates the effect of genotype , sex , interacting drugs , and age , alone or in different combinations , on warfarin dose requirement .
it is obvious from figure 4 that any genetic variation in the two genes compared to cyp2c9 *
1/*1 , vkorc1b / b , all other parameters being equal , causes more than 1520% decrease in dose requirement .
this feature corresponds to about 3/4 of the population . the conspicuous effect on dose requirement of common patient characteristics is also shown , for example , 80 versus 50 years of age , three interacting drugs versus none , and so forth . in the clinical setting , it is common that warfarin treatment has to be commenced without genetic information about the patient .
such information , if requested , is usually not provided until the patient has already received 510 warfarin doses .
the value of genetic information is expected to decrease the later it is available and after one week the information is considered irrelevant by some investigators .
however , data generated in a broad warfarin consortium have demonstrated an additive informative value of genetic data on vkorc1 and cyp2c9 even when it is obtained 79 days after the start of treatment .
previous and ongoing studies have found that after 46 , 79 , and 14 days of inr - guided therapy , genotyping still improves the prediction of maintenance dose by 415% , 214% , and 4% , respectively [ 3539 ] .
individual warfarin dose predictions based on vkorc1 and cyp2c9 genotype and clinical factors ( with or without inr data ) are available , for example , at the nonprofit website http://www.warfarindosing.org/.
other coumarin derivates such as phenprocoumon ( pc ) and acenocoumarol ( ac ) are widely prescribed in continental european countries and latin america , whereas warfarin is the drug of choice in us , uk , and the scandinavian countries .
thus , acenocoumarol has a much shorter half - life ( 28 hours ) as compared to warfarin ( 3045 hours ) or phenprocoumon ( 156172 hours ) . the genetic contribution to variation in kinetics and dose requirement
is more pronounced for warfarin than for acenocoumarol or phenprocoumon . on the other hand , adjustment of warfarin dose is easier than that of phenprocoumon because of its shorter half - life and short time to achieve steady state .
cyp3a4 and cyp2c9 are the major catalysts of the phenprocoumon 7-hydroxylation , and therefore the polymorphic cyp2c9 enzyme contributes less to the metabolism of phenprocoumon compared to warfarin [ 40 , 41 ] .
in addition , a part of the drug is excreted directly via the kidneys or the bile .
because of this difference , phenprocoumon is sometimes preferred as an alternative in patients harboring the cyp2c9 * 2 or cyp2c9 * 3 alleles which significantly compromise the metabolism of warfarin , and to a high degree also acenocoumarol [ 42 , 43 ] . identification of the vitamin k epoxide reductase c1 gene paved the way to the identification of functional mutations in vkorc1 gene , both those conferring warfarin resistance and those causing an increase in warfarin dose requirement . a recent publication compared the relation between on one hand the vkorc1 and cyp2c9 genotype , and on the other , the mean weekly dose requirement for acenocoumarol and phenprocoumon .
the difference in weekly average dose for individuals carrying vkorc1 aa and bb genotypes was approximately two - fold ( as for warfarin ) for each of the drugs , with the highest doses in the bb group .
the average reduction in acenocoumarol and phenprocoumon dose requirements in patients with mutant variants of cyp2c9 included in their study was at most 1/3 of the average dose in wild type individuals .
these results clearly demonstrate the difference in dependence on the cyp2c9 genetic polymorphisms of these drugs as compared to warfarin .
other genes of relevance for warfarin disposition or vitamin k action were not found to have any impact on the dose requirement of acenocoumarol and phenprocoumon .
although phenprocoumon is less dependent on cyp2c9 for its elimination compared to acenocoumarol , polymorphisms in cyp2c9 have been associated with an increased risk of overanticoagulation and bleedings for both drugs [ 4548 ] .
there is still limited information regarding the potential impact of vkorc1 polymorphisms on the risk of adverse outcomes in patients treated with acenocoumarol or phenprocoumon .
nevertheless , the vkorc1 a haplotype has been associated with an increased risk of overanticoagulation ( inr > 4 ) in patients treated with acenocoumarol and with major bleeding in phenprocoumon - treated individuals [ 49 , 50 ] . in summary ,
the risk of severe adverse reactions including fatal outcome is among the highest known in drug therapy .
it has been manageable by virtue of inr monitoring but needs to be further reduced .
the new knowledge about genes important for the metabolism and effect of these drugs suggest genotyping to be applied in the clinic since the future development of anticoagulant therapy is uncertain even if new therapeutic principles are on the way to be introduced .
ongoing large clinical trials such as coag ( clarification of optimal anticoagulation through genetics ) and eu - pact ( the european pharmacogenetics of anticoagulant therapy trial ) will further elucidate these important issues and it remains to be seen if their results will modify our current opinion [ 51 , 52 ] . | warfarin , acenocoumarol , and phenprocoumon are among the major anticoagulant drugs worldwide . because of their low therapeutic index and serious adverse reactions ( adrs ) , their wide use , and their varying kinetics and pharmacogenetic dependence , it is of great importance to explore further possibilities to forecast the dose beyond conventional inr measurements . here
, we describe particulars of the relative pharmacogenetic influence on the kinetics of these agents , the population distribution of genetics risk groups , and novel data on clinical features with influence on dose requirement and adr risk . the usefulness of genetic information prior to and soon after start of therapy is also discussed . the current renewed focus on these issues
is caused not only because of new genetic knowledge and genotyping facilities but also because of the high rate of serious adrs .
application of these measures in the care of patients with anticoagulant therapy is important awaiting new therapeutic principles to be introduced , which may take long time still . |
phaeohyphomycosis is a group of mycotic infections caused by dematiaceous fungi ( pigmented ) that contain melanin in their cell walls .
melanin is a known virulence factor in these fungi , and it acts by scavenging free radicals produced by phagocytic cells in the oxidative process , which would kill most organisms , and also prevents their action on the plasma membrane by binding to hydrolytic enzymes . the formation of fungal appressorium , which helps in fungal penetration into the host cell is also influenced by the melanin .
all these actions explain the pathogenic potential of dematiaceous fungi even in an immunocompetent host .
the fungus is present in host tissues as brownish hyphae , pseudohyphae , yeast cells , or combination of these forms .
these organisms are widespread in the environment , being found in soil , wood , and decomposing plant debris .
the major etiologic agents of phaeohyphomycosis are species of bipolaris , exophiala , curvularia , chaetomium , phoma , exserohilum , and wangiella .
it is a rare infection mostly involving the skin and subcutis , and rarely involving the paranasal sinuses , eyes , central nervous system , lymph nodes , and bone .
the subcutaneous infections typically occur on the exposed areas of the body , especially the extremities , fingers , wrists , knees , or ankles , resulting from traumatic inoculation .
we report this case for its uncommon clinical presentation with multiple cystic swellings on both the feet and right hand in an immunocompetent individual .
a 35-year - old male agriculturist presented with multiple asymptomatic swellings on the dorsum of right hand , both feet , and ankle region of 6 months duration .
there was no history of trauma , fever or intake of medication , and any other underlying systemic disease .
cutaneous examination revealed multiple skin - colored , nontender , firm - to - soft , cystic swellings approximately 3 cm in diameter present on the ankle region and feet .
multiple swellings were coalesced to form a large fluctuant mass measuring 810 cm , present on the dorsum of right hand [ figure 1 ] .
a differential diagnosis of multiple abscesses , calcinosis cutis , and deep fungal infection were considered .
8 10 cm confluent nodular swelling over the dorsum of the hand routine investigations were normal .
while performing an excisional biopsy , there was purulent discharge from the biopsy site , and hence all the lesions were incised [ figure 2 ] .
cyst material was sent for hpe gram stain was negative for bacteria and on culture with sabouraud 's dextrose agar , pigmented fungi were identified and lactophenol cotton blue showed exophiala jeanselmi species [ figure 3 ] .
cyst material for hpe showed multiple granulomas and grocott 's methanamine silver ( gms ) stain showed broad brownish filaments [ figures 4 and 5 ] .
multiple cystic swellings over the feet pigmented fungi on sabouraud 's dextrose agar ( a ) ( h and e , 10 ) granuloma in the dermis composed of epithelioid cells , lymphocytes , and fibroblasts .
( b ) ( h and e , 100 ) closer view ( a ) granulomas with pigmented fungal growth ( scanner view gms ) ( b ) pigmented fungal filaments ( high power view gms ) the patient was treated by surgical excision of the lesions without any antifungal therapy .
the infections caused by dematiaceous ( melanized ) fungi are grouped into three classes that include phaeohyphomycosis , chromoblastomycois , and eumycotic mycetoma .
phaeohyphomycosis represents a broad range of primary and opportunistic mycoses classified into four clinical forms , which includes superficial , cutaneous , subcutaneous , systemic , and disseminated forms ( brain , eye , central nervous system , peritoneum , bones ) .
subcutaneous infections mostly occur on the limbs , fingers , wrist , knees , or ankles .
the immune status of the host plays a major role in the clinical presentation which could range from papulonodules , verrucous , hyperkeratotic , or ulcerated plaques , cysts , abscesses , pyogranuloma , nonhealing ulcers , or sinuses .
subcutaneous phaeohyphomycosis is a rare infection , however , the number of cases appears to be increasing in recent years as the number of immunocompromised patients also have increased .
however , our case showed that immunocompromised state is not a necessary prerequisite for phaeohyphomycosis .
infection usually occurs through traumatic inoculation of the skin and subcutaneous tissue with contaminated matter , with majority of lesions occurring on the feet and legs of outdoor workers , as observed in this patient .
the most common etiological agents of subcutaneous phaeohyphomycosis are e. jeanselmi followed by e. dermatitidis .
the genus exophiala is widely distributed in the environment and may cause infections in both immunocompromised ( human immunodeficiency virus patients , transplant recipients , debilitating chronic diseases , diabetes , and immunosuppressive therapy ) and rarely , in immunocompetent persons .
e. jeanselmi usually causes mild cutaneous and subcutaneous infections which are often localized and solitary ( phaeohypomycotic cyst ) . even in severely immunosuppressed patients
common clinical presentation is solitary subcutaneous cyst or abscess , firm to fluctuant , usually sparing the overlying skin .
the common site of involvement is upper and lower limbs over the fingers , wrist , knees , or ankles , and less frequently on the face , neck , and scalp .
all dematiaceous fungi are similar in morphology and can be differentiated only by culture . according to sharma et al . who reviewed the literature on phaeohyphomycosis ,
23 patients with subcutaneous phaeohyphomycosis have been reported from india predominantly involving the extremities and very few cases with dissemination . in a study by murayama
, 54 cases of phaeohyphomycosis due to e. jeanselmi were reviewed , however , they failed to identify any underlying disease in 31 cases .
our patient also showed that there was no underlying disease predisposing to phaeohyphomycosis , as reported by murayama et al .
our case report highlights the fact that any subcutaneous soft swellings must also be considered under differential diagnosis for fungal infections as they may be mistaken for lipoma , fibroma , epidermal cyst , or foreign body reaction .
surgical excision of the lesion with or without antifungal agents ( itraconazole , ketaconazole , amphotericin b ) has been widely used in treatment of subcutaneous phaeohyphomycosis . the present case presented with multiple lesions , and all the lesions
were excised . surgical excision of lesions showed no relapse even after 6 months without any antifungal therapy .
| phaeohyphomycosis is a rare mycotic infection caused by various heterogenous groups of phaeoid ( dematiaceous ) fungi involving the skin and subcutaneous tissue .
common clinical manifestations are subcutaneous abscesses or cystic swellings . here
, we report a case of subcutaneous phaeohyphomycosis presenting as multiple asymptomatic cystic swellings over the hands and feet without any predisposing factors .
histopathology showed granulomatous inflammation and special stain with grocott 's methanamine silver stain revealed broad pigmented hyphae .
culture showed black - colored colonies identified as exophiala jeanselmi .
the patient was treated with surgical excision of the lesions . |
a simple , precise , specific , accurate high performance thin layer chromatography method was developed for simultaneous estimation of aloe emodin and emodin from medicinal plants like rheum emodi ( rhubarb ) , barbados aloes ( dried juice of aloe barbadensis leaf ) and cassia alata ( candle bush ) .
thin layer chromatographic aluminum plates pre - coated with silica gel 60 f254 was used as the stationary phase for chromatographic separation of the drugs .
toluene : ethyl acetate : formic acid ( 10:2:1 v / v / v ) was selected as mobile phase and analysis was carried out in absorbance mode at iso - absorptive wavelength of 263 nm .
this method shows good resolution for both drugs with retention factor 0.370.03 and 0.550.03 for aloe emodin and emodin , respectively .
the regression analysis data indicated good linear relationship for the calibration plots for aloe emodin and emodin in the range of 300 - 800 ng / spot and 150 - 400 ng / spot and regression coefficient was 0.9993 and 0.9994 , respectively .
validation of the method was performed according to international conference on harmonisation guidelines for following parameters : accuracy , precision , limit of detection , linearity , limit of quantification , robustness and specificity . in conclusion ,
the developed method was found to be rapid , simple , reliable and specific for the identification and quantitation of these anthraquinones in medicinal plants and marketed formulations . | |
data were acquired with the prototype ( 4 detectors ) and a second - generation instrument ( nanosims 50l , cameca : 7 detectors ) .
( n - thymidine , c - thymidine ) and sigma - aldrich , inc .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
| mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells .
we developed multi - isotope imaging mass spectrometry ( mims ) that allowed us to view and measure stable isotope incorporation with sub - micron resolution3,4 . here
we apply mims to diverse organisms , including drosophila , mice , and humans .
we test the immortal strand hypothesis , which predicts that during asymmetric stem cell division chromosomes containing older template dna are segregated to the daughter destined to remain a stem cell , thus insuring lifetime genetic stability . after labeling mice with 15n - thymidine from gestation through post - natal week 8
, we find no 15n label retention by dividing small intestinal crypt cells after 4wk chase . in adult mice administered 15n - thymidine pulse - chase , we find that proliferating crypt cells dilute label consistent with random strand segregation .
we demonstrate the broad utility of mims with proof - of - principle studies of lipid turnover in drosophila and translation to the human hematopoietic system .
these studies show that mims provides high - resolution quantitation of stable isotope labels that can not be obtained using other techniques and that is broadly applicable to biological and medical research . |
cortical venous thrombosis ( cvt ) is any thrombosis that occurs in intracranial veins or sinuses .
cerebral vein and sinus thrombosis is rare compared to arterial stroke and often occurs in young individuals .
worldwide estimated annual incidence is 34 cases/1 million population , about 75% of the adult patients are women . with reference to india , there are no definitive data as there are no multicenter hospital - based studies .
seizures occur in about 7%15% of cases during presentation and in about 40% during the course of illness , and they may be focal or generalized in almost equal proportion .
patients may also present with long - lasting focal neurological deficits or transient focal neurological deficits mimicking transient ischemic attacks .
etiological factors vary depending on geographical area and also according to anatomical site involved . primary or idiopathic cvt is mainly caused by hypercoagulable state .
secondary thrombosis results from a variety of causes that include injury , infection , hematological disturbances , and dehydration . in india , cvt is very common in the early puerperal period ( postpartum period till 6 weeks ) , 1012 times higher than in the west and as a whole puerperal cvt may account for about 15%20% of stroke victims of younger age .
a meta - analysis of the most recent prospectively collected series , in which the weight of the international study on cerebral veins and dural sinus thrombosis cohort is very important , showed an overall 15% case - fatality or dependency rate . with the advent of imaging modalities such as computed tomography ( ct )
scan and magnetic resonance imaging ( mri ) and mr venography ( mrv ) , the diagnosis and respective management has improved significantly .
hence , we started our study with following objectives to study clinical profile and assess the outcome and prognostic factors of cvt patients .
a case series study was done for 2 years in a tertiary care hospital in north india . during study period , patients aged more than 18 years which are confirmed as cvt from clinical features and investigations such as ct scan of the brain with contrast or mri with mrv were considered .
cases where ct scan of the brain showing hemorrhage or infarct in the distribution of arterial territory , neoplastic and granulomatous disease of the brain , and eclampsia or preeclampsia cases were excluded from the study .
all patients were assessed at the time of discharge for physical and mental status to look for outcome .
data were entered in excel sheet , and appropriate descriptive statistics were done to describe demographic and clinical features .
accordingly , modified rankin scale was used to assess outcome , scores between 0 and 2 were considered as good and 36 as poor outcome , respectively . to analyze data , spss version 20 ( spss version 20 , ibm , new york , usa ) was used .
a p < 0.05 was fixed as significant and p < 0.01 as highly significant .
a total of 81 patients formed as our study participants , more than half of the patients were in the age group of < 35 years ( 55.6% ) , and majority of patients were females ( 79% ) .
the most common symptom of presentation of patients was with headache ( 82.7% ) , seizure ( 63% ) , and focal neurological deficit ( 63% ) and least common symptom was fever ( 14.8% ) [ table 1 ] .
clinicodemographic and radiological profile of patients according to radiological findings , the most common involved sinus was superior sagittal sinus ( 74.1% ) and then right transverse sinus ( 42% ) . nearly half of the patients were in puerperal period ( 44.1% ) followed by other risk factors such as central nervous system infection ( 14.8% ) , pregnancy ( 13.6% ) , and idiopathic ( 12.4% ) .
pie chart showing distribution of patients according to risk factors more than half of them had good scores , 45 ( 55.6% ) with score 0 ( 8.6% ) , score 1 ( 27.2% ) , score 2 ( 19.8% ) , whereas 36 ( 44.4% ) had poor outcome [ table 2 ] .
distribution of patients according to outcome at the time of discharge ( modified rankin score ) patients aged more than 35 years had poor outcome ( 72.2% ) compared to patients aged < 35 years ( 22.2% ) and was statistically significant ( or : 9.1 , ci : 4.46319.750 ) .
patients who had symptoms such as fever ( or : 3.442 , ci : 1.08812.140 ) , impaired conciousness ( or : 5.467 , ci : 2.06415.330 ) , coma ( or : 23.99 , ci : 3.844544.1 ) , papilledema ( or : 25.15 , ci : 7.565101.5 ) , and presenting with focal neurological deficit ( or : 9.366 , ci : 2.6933.41 ) had statistically significant poor outcome [ table 3 ] .
a total of 81 patients were included during study period with age range from 18 to 70 years ( mean standard deviation : 30.2 9.15 ) .
more than half of our patients were in the age group < 35 years ( 55.6% ) .
this is in conformity with most of the earlier studies by ameri and bousser ( 61% ) , daif et al . , deschiens et al .
the present study showed a female preponderance with ratio of male to female patients being 1:3.76 because of influence of pregnancy , puerperium , and oral contraceptive pills , whereas ratio was 1:1.29 in study done by ameri and bousser and 1:1 by daif et al . , and studies done in india showed higher number of female patients such as 1:1.38 by patil et al .
patients presented with headache , foal deficit , seizure , papilledema , impaired consciousness , and coma in the order of decreasing frequency . in the present study , most of the patients ( 82.7% ) presented with headache .
( 95% ) . around ( 63% ) of patients presented with focal deficit such as hemiparesis , monoparesis , and paraparesis with cranial nerve palsy ( 18.5% with 7 nerve , 4.9% with 3 nerve , and 2.5% with 4 nerve palsy ) .
( 56.9% ) , whereas in a study done by halesh et al . , only 48% presented with focal deficits , this may be due difference in number of patients .
et al . ( 48% ) and in barinagarrementeria et al . ( 60% ) , patients presented with seizures which were comparable to our study .
accordingly , it varied from ( 27% ) einhupl et al . to ( 45% ) by bousser et al . in the present study ,
54% of patients presented with altered sensorium , out of which 38% were having glasgow coma scale ( gcs ) between 9% , 13% and 16% were having gcs < 9 , i.e. , comatose . in a study conducted by barinagarrementeria et al .
other symptom was fever seen in 16% of cases which may be secondary to infection such as otitis media , meningitis , and septic thrombotic process .
female patient contributed majority , among females , more than half were in puerperal period , i.e. , 36 ( 56.25% ) and only 6 ( 9.37% ) were on oral contraceptive pills .
a very low proportion of < 15% postpartum cases was noted from studies done by ferro et al . ,
de bruijn et al . , and deschiens et al . the reasons may be because of postpartum hypercoagulable state combined with culturally practiced water restriction and high fatty diet .
all patients were suffering from meningitis secondary to otitis media which was comparable to bousser et al .
other risk factors for cvt in decreasing order of frequency were pregnancy ( 13.6% ) , hyperhomocysteinemia ( 3.7% ) , dehydration ( 2.5% ) , and neurosarcoidosis ( 1.2% ) . in remaining 12.4% of cases , causes could not identify and named as idiopathic .
the superior sagittal sinus was most common sinus involved in 74.1% of the cases , either alone or with other sinuses . in studies by ameri and bousser ( 72% ) and daif et al .
( 85% ) , superior sagittal sinus was involved which was comparable to our study .
other sinuses involved in decreasing order of frequency were right transverse sinus ( 42% ) , left transverse sinus ( 38.3% ) , sigmoid sinus ( 34.6% ) , and straight sinus ( 22.2% ) , respectively . at the time of discharge , 44.3% of patients had poor outcome with 27.3% of patients having dependent morbidity and 16% of mortality .
patients aged more than 35 years had poor outcome compared to younger age group ( or : 9.1 , ci : 4.46319.750 ) .
similar results were observed in studies done by stolz et al . , ferro et al . , and de bruijn et al .
the present study showed that males had a poor outcome compared to females but was statistically not significant ( or : 2.822 , ci : 0.9279.219 ) .
however , there was not much difference in outcome in a study done by narayan et al .
patients who had history of fever ( or : 3.442 , ci : 1.08812.140 ) and impaired consciousness ( or : 5.467 , ci : 2.06415.330 ) were associated with poor outcome .
the study showed that patients who had coma ( or : 23.99 , ci , 3.844544.1 ) , papilledema ( or : 25.15 , ci : 7.565101.5 ) , and presenting with focal neurological deficit ( or : 9.366 , ci : 2.6933.41 ) were also associated with poor outcome which were statistically significant .
similar results were observed by ferro et al . with respect to coma and hemiparesis or any deficit at the time of diagnosis .
patients who had seizure also had a poor outcome but was statistically not significant ( or : 2.061 , ci : 0.8095.441 ) , whereas in a study by stolz et al . who studied 79 patients , more than two seizures despite antiepileptic treatment
was associated with poor outcome , and patients with history of headache had a comparatively better outcome but that was also statistically not significant ( or : 1.542 , ci : 0.4655.566 ) .
however , involvement of only straight sinus was associated with poor outcome but was statistically not significant ( or : 1.766 , ci : 0.60465.283 ) , and similar findings were seen in de bruijn et al . as compared to earlier studies , the present study shows higher number of dependency and death rate .
this discrepancy in the studies from western countries and our study may be attributed to the fact that our institution being a referral center receiving patients with comparatively poor prognosis .
in addition , patients in this setup seek alternative interventions leading to delay in medical intervention .
| background : cortical venous thrombosis ( cvt ) is a rare condition , compared to arterial stroke and often occurs in young individuals presenting with varying clinical features.aim:the aim is to study clinical profile and assess the outcome and prognostic factors of cvt patients.methodology:a case series study was done for 2 years .
cvt cases confirmed by magnetic resonance imaging were included in this study . clinical presentation and risk factors
were noted then patients were assessed at the time of discharge for their physical and mental status .
modified rankin scale was used to group patients , accordingly scores 02 were considered as good and 36 as poor outcome , respectively .
data were analyzed using chi - square test to know the association between prognostic factors and outcome.results:out of 81 patients , more than half of the patients were in the age group of < 35 years ( 55.6% ) , and majority of patients were females ( 79% ) .
most common symptom was headache ( 82.7% ) and least was fever ( 14.8% ) .
superior sagittal sinus was most commonly involved ( 74.1% ) . nearly half of the patients were in puerperal period ( 44.1% ) .
patients aged more than 35 years ( odds ratio [ or ] : 9.1 , confidence interval [ ci ] : 4.46319.750 ) presenting with symptoms such as fever ( or : 3.442 , ci : 1.08812.140 ) , impaired consciousness ( or : 5.467 , ci : 2.06415.330 ) and having clinical signs such as coma ( or : 23.99 , ci , 3.844544.1 ) , papilledema ( or : 25.15 , ci : 7.565101.5 ) , and with focal neurological deficit ( or : 9.366 , ci : 2.6933.41 ) had statistically significant poor outcome.conclusion:females formed a major bulk of patients .
higher number patients showed poor outcome .
study showed association between age , headache , impaired consciousness , coma , papilledema , and neurological deficit to poor outcome . |
periodontal disease is a chronic , non - resolving inflammatory lesion that may result in the destruction of the tooth supporting structures .
although bacteria are thought to initiate the inflammatory response , the tissue destruction is believed to be largely mediated by the host response .
the host response involves a complex interplay of several cell types of the innate and adaptive immune responses .
t cells are believed to play a critical role in the etiopathogenesis of periodontal disease by regulation of both tissue homeostasis and inflammatory/ immune responses.[2 - 3 ] t cell differentiation and activation is a two - step process .
the initial step involves antigen recognition by antigen presenting cells ( apcs ) and subsequent presentation to the t cell receptor .
this is followed by ligation of costimulatory molecules belonging to the b7 family on apcs to their receptors on t cells : cd28 and ctla4 .
costimulation is essential for optimal differentiation and prevention of t cell apoptosis / anergy .
the traditional costimulatory molecules such as cd80 ( b7 - 1 ) and cd86 ( b7 - 2 ) were thought to be primarily stimulatory in function .
t cell activation or inactivation was thought primarily to be the function of the receptors such as cd28 or ctla4 to which these costimulatory molecules adhere . in recent years
evidence for this gained strength with the expansion of the family of costimulatory molecules to include newer members such as inducible costimulator ligand ( icos - l ) , programmed death-1 ligand ( pd - l1 ) , programmed death- 2 ligand ( pd - l2 ) , b7-h3 , and b7-h4 .
of these costimulatory molecules , there is extensive literature regarding b7 - 1 and b7 - 2 in gingival tissue.[6 - 8 ] these molecules have been reported to promote th1 and th2 responses respectively , along with other factors .
however , the role of newer members of the b7 family is not yet well recognized .
recently , increased expression of pd-1 has been noted in gingival tissues of patients with chronic periodontitis and is reportedly involved in regulation of t cell responses .
b7-h3 is a new member of the b7 family , whose expression can be induced in activated dendritic cells , lymphocytes , and synovial - like fibroblast.10 this costimulatory molecule has been proposed to play a role in the etiopathogenesis of non - linear inflammatory diseases such as rheumatoid arthritis and asthma.[10 - 11 ] the role of b7-h3 in t cell function is yet to be fully understood .
though an earlier study demonstrated a stimulatory function , subsequent studies have shown that b7-h3 mediated an inhibitory as well as a stimulatory effect on t cells.[12 - 15 ] the expression of b7-h3 in periodontal tissue and its role , if any , in periodontal disease pathogenesis have not yet been documented .
thus , the aim of the present study was to
investigate its expression in healthy and diseased gingival tissue samples .
thirty subjects selected from the patient pool attending the outpatient clinic , department of periodontics , ragas dental college and hospital , were enrolled in the study between january and july of 2011 .
subjects exhibiting good general health with no history of smoking or periodontal treatment or antimicrobial therapy for the past 6 months were chosen for the study .
group a consisted of 15 subjects ( 5 males and 10 females ; age : 20 to 50 years ; mean 35.6 years ) with clinically healthy gingiva ( probing depth [ pd ] 3 mm ; no clinical attachment loss [ cal ] ;
no bleeding on probing and minimum 20 teeth ) .
group b consisted of 15 subjects ( 7 males and 8 females ; age : 20 to 65 years ; mean 45.7years ) with chronic generalized periodontitis
( pd 5 mm , cal 3 mm in at least six sites and minimum 20 teeth ) .
the study protocol was approved by the institutional review board of ragas dental
college and hospital , and a written informed consent was obtained from each subject in accordance with the principals of good clinical practice and the declaration of helsinki .
gingival samples were collected from subjects in group a during the crown lengthening procedure . in the group b subjects ,
gingival samples were taken during periodontal surgery after completion of phase i therapy , from sites with the deepest pd.[17 - 18 ] the patients were evaluated for persistent disease activity based on the clinical parameters of pd 5 mm and cal 3 mm after completion of phase i therapy .
the samples were taken after a thorough phase i therapy to reduce the tissue friability during their collection and processing .
the tissues obtained from the subjects were fixed with 10% neutral buffered formalin and paraffinized .
sections that were 4 microns thick were made from the specimens and mounted on apes ( 3-aminopropyltriethoxysilane ) pretreated slides to carry out immunohistochemistry .
sections were also made from same paraffin blocks for hematoxylin and eosin stained slides to confirm the gingival specimens .
the sections for immunochemistry were deparaffinized using xylene following which rehydrated in descending series of isopropanol ( 100% , 70% and 50% ) .
antigen retrieval was carried out using edta ( ethylene diamino tetraacetic acid ) , by autoclaving at a temperature of 121c and pressure of 20 psi for 15 minutes .
the slides were cooled to room temperature and treated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity , then put in one change of pbs ( phosphate buffer saline ) and treated with avidin blocking agent for 15 minutes .
avidin and biotin blocking agents were used to suppress endogenous avidin binding activity ( eaba ) .
the primary antibody , polyclonal goat antihuman b7-h3 ( r&d systems , usa ) was added to the tissue and the slides were incubated overnight at 2 - 8. the slides were then washed in three changes of cold pbs for 5 minutes to remove the excess antibody . from the secondary antibody kit ( r&d systems , usa ) ,
a drop of biotinylated secondary antibody was added onto the sections and the slides were incubated for 1 hour after which they were washed in three changes of cold pbs for 15 minutes each and wiped carefully without touching the tissue section to remove excess pbs .
then a drop of hss- hrp ( high sensitive streptavidin- horse radish peroxidase conjugate ) from the kit was added onto the sections and incubated for 30 minutes .
the sections were then washed in three changes of cold pbs for 2 minutes each and wiped carefully to remove excess pbs .
a drop of freshly prepared dab ( diamino benzidine- a substrate chromogen ) was added onto the sections and incubated for 15 minutes .
slides were then washed in three changes of pbs for 10 minutes each and then rinsed in distilled water and counterstained with harris hematoxylin .
the tissue sections were washed with ascending series of isopropanol ( 50% , 100% ) and xylene and mounted with dpx ( di - n - butyl phthalate in xylene ) for microscopic examination .
negative controls were used by incubating the sections without a primary antibody prior to the addition of appropriate secondary antibodies was also used .
all of the slides were observed under the microscope at magnifications of 10 x and 40 x.
the slides were observed by two independent examiners .
the staining on the epithelium was evaluated based on the intensity of staining , scored as negative ( - ) , mildly positive ( + ) , moderately positive ( + + ) , or highly positive ( + + + ) , respectively .
positively stained cells in the connective tissue were evaluated by counting the number of cells that were stained .
the percentage of positive cells was the ratio of the number of cells in the connective tissue that showed intense staining divided by the total number of cells counted .
thus , labeling index ( li)= ( number of positive cells/1 , 000 ) x 100 .
data entry and descriptive analysis was performed using a statistical software program ( spss version 10.0.5 ) .
independent t test , was used to compare the groups ( healthy and periodontitis ) with regard to the b7-h3 expression in the connective tissue .
the intensity of staining was determined by two examiners and the inter - examiner variation was determined by kappa analysis .
thirty subjects selected from the patient pool attending the outpatient clinic , department of periodontics , ragas dental college and hospital , were enrolled in the study between january and july of 2011 .
subjects exhibiting good general health with no history of smoking or periodontal treatment or antimicrobial therapy for the past 6 months were chosen for the study .
group a consisted of 15 subjects ( 5 males and 10 females ; age : 20 to 50 years ; mean 35.6 years ) with clinically healthy gingiva ( probing depth [ pd ] 3 mm ; no clinical attachment loss [ cal ] ;
no bleeding on probing and minimum 20 teeth ) .
group b consisted of 15 subjects ( 7 males and 8 females ; age : 20 to 65 years ; mean 45.7years ) with chronic generalized periodontitis
( pd 5 mm , cal 3 mm in at least six sites and minimum 20 teeth ) .
the study protocol was approved by the institutional review board of ragas dental
college and hospital , and a written informed consent was obtained from each subject in accordance with the principals of good clinical practice and the declaration of helsinki .
gingival samples were collected from subjects in group a during the crown lengthening procedure . in the group b subjects ,
gingival samples were taken during periodontal surgery after completion of phase i therapy , from sites with the deepest pd.[17 - 18 ] the patients were evaluated for persistent disease activity based on the clinical parameters of pd 5 mm and cal 3 mm after completion of phase i therapy .
the samples were taken after a thorough phase i therapy to reduce the tissue friability during their collection and processing .
the tissues obtained from the subjects were fixed with 10% neutral buffered formalin and paraffinized .
sections that were 4 microns thick were made from the specimens and mounted on apes ( 3-aminopropyltriethoxysilane ) pretreated slides to carry out immunohistochemistry .
sections were also made from same paraffin blocks for hematoxylin and eosin stained slides to confirm the gingival specimens .
the sections for immunochemistry were deparaffinized using xylene following which rehydrated in descending series of isopropanol ( 100% , 70% and 50% ) .
antigen retrieval was carried out using edta ( ethylene diamino tetraacetic acid ) , by autoclaving at a temperature of 121c and pressure of 20 psi for 15 minutes .
the slides were cooled to room temperature and treated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity , then put in one change of pbs ( phosphate buffer saline ) and treated with avidin blocking agent for 15 minutes . slides were rinsed in pbs and treated with biotin blocking agent for 15 minutes .
avidin and biotin blocking agents were used to suppress endogenous avidin binding activity ( eaba ) .
the primary antibody , polyclonal goat antihuman b7-h3 ( r&d systems , usa ) was added to the tissue and the slides were incubated overnight at 2 - 8. the slides were then washed in three changes of cold pbs for 5 minutes to remove the excess antibody . from the secondary antibody kit ( r&d systems , usa ) , a drop of biotinylated secondary antibody
was added onto the sections and the slides were incubated for 1 hour after which they were washed in three changes of cold pbs for 15 minutes each and wiped carefully without touching the tissue section to remove excess pbs .
then a drop of hss- hrp ( high sensitive streptavidin- horse radish peroxidase conjugate ) from the kit was added onto the sections and incubated for 30 minutes .
the sections were then washed in three changes of cold pbs for 2 minutes each and wiped carefully to remove excess pbs .
a drop of freshly prepared dab ( diamino benzidine- a substrate chromogen ) was added onto the sections and incubated for 15 minutes .
slides were then washed in three changes of pbs for 10 minutes each and then rinsed in distilled water and counterstained with harris hematoxylin .
the tissue sections were washed with ascending series of isopropanol ( 50% , 100% ) and xylene and mounted with dpx ( di - n - butyl phthalate in xylene ) for microscopic examination .
negative controls were used by incubating the sections without a primary antibody prior to the addition of appropriate secondary antibodies was also used .
all of the slides were observed under the microscope at magnifications of 10 x and 40 x.
was evaluated based on the intensity of staining , scored as negative ( - ) , mildly positive ( + ) , moderately positive ( + + ) , or highly positive ( + + + ) , respectively .
positively stained cells in the connective tissue were evaluated by counting the number of cells that were stained .
the percentage of positive cells was the ratio of the number of cells in the connective tissue that showed intense staining divided by the total number of cells counted .
thus , labeling index ( li)= ( number of positive cells/1 , 000 ) x 100 .
data entry and descriptive analysis was performed using a statistical software program ( spss version 10.0.5 ) .
independent t test , was used to compare the groups ( healthy and periodontitis ) with regard to the b7-h3 expression in the connective tissue .
the intensity of staining was determined by two examiners and the inter - examiner variation was determined by kappa analysis .
a good inter - examiner agreement was observed between the two examiners ( health-0.718 ; disease - 0.797 ) .
table 1 summarizes the results of the present study . in the periodontal health group ( group a )
the gingival epithelium demonstrated intense staining in 5 specimens , moderate staining in 7 specimens and pale staining in 3 specimens .
buccal , l- lingual , p- palatal , cl- crown lengthening , mwf- modified widman flap , -ve- negative immunohistochemistry of membranous staining b7-h3 in gingival epithelia and connective tissue of healthy ( group a ) samples ( original magnification 10x ) with high intensity staining .
in the periodontitis group ( group b )
, the gingival epithelium demonstrated intense staining in 4 specimens , moderate staining in 8 specimens and pale staining in 1 specimen .
the difference in intensity of epithelial staining between the groups was statistically insignificant ( p > 0.713 )
while in the connective tissue , the mean li demonstrated a statistically insignificant decrease in chronic periodontitis group ( group b ) group ( 41.3725.29 ) when compared to healthy ( group a ) group ( 50.28 26.09 )
with p value = 0.415 as described in table 2 .
comparison of intensity scoring and mean labeling index between health and disease
( p value < 0.001-statistically significant ; p value > 0.001- statistically insignificant )
the progression of periodontal disease is characterized by short bursts of disease activity and long periods of quiescence .
though the factors that have been proposed to play a role are not yet well defined ,
it has been postulated to be related to a change in quantity or quality of periodontopathogenic bacteria and/or host response.[19 - 20 ] this study sought to identify b7-h3 in the gingival tissues as it may play a role in regulating t cell responses by either co - stimulation/ co - inhibition .
t cell activation may in turn regulate the host response thereby
significantly affecting disease progression .
the role of the newer b7 family members in periodontal disease is not yet fully understood . to the best of our knowledge ,
the present study is the first to identify the
presence of b7-h3 in gingival tissues .
in the healthy samples ,
b7-h3 was present predominately in the basal layer of gingival epithelium and in the connective tissue just beneath the basal lamina .
the role of the gingival epithelium in providing innate immune functions is well documented .
in addition to functioning as a mechanical barrier , immune cells such as
dendritic cells and neutrophils help in antigen presentation and clearance .
the results of our study pro vides evidence that the epithelium may , in addition to influencing
innate immune mechanisms , activate or influence t cell behavior through expression of costimulatory molecules .
our results suggest that b7-h3 was expressed constitutively
in the epithelium most likely as a result of exposure to commensal bacteria present in the oral cavity .
in the connective tissue , b7-h3 was present just beneath the basal lamina and was not detected in the deeper layers of the connective tissue .
( figure 2a , 2b ) although , it is difficult to assign
a role or suggest a function for these molecules based on this study , we speculate that connective tissue infiltration is part of the basal inflammatory tone that is crucial to mounting an effective defense against the periodontopathogens .
[ 22 - 23 ]
a : immunohistochemistry of membranous staining b7-h3 in gingival epithelia and connective tissue of healthy ( group a ) samples ( original magnification 40x ) with high intensity staining .
b : immunohistochemistry of membranous staining b7-h3 in gingival epithelia and connective tissue of periodontitis ( group b ) samples ( original magnification 40x ) with high intensity staining
in the present study , periodontally diseased samples demonstrated a statistically insignificant decrease in the expression of b7-h3 when compared to healthy samples .
it is beyond the scope of this study to accurately identify the role of b7-h3 in t cell activation .
the exact mechanism of b7-h3 mediated t cell activation has not yet been elucidated at its receptor , and downstream signaling remains as yet unknown .
investigators have examined the role of b7-h3 in inflammatory bone disorders and chronically inflamed tissues .
b7-h3 has been identified in fibroblast- like synovicytes obtained from patients
with rheumatoid arthritis , and air way epithelium.[10 - 11 ] their role in the development of inflammation in these systems by regulating t - helper cell function has been proposed .
luo et al .
concluded from their study that b7-h3 differentially regulates t - helper subsets .
it has been found to play a costimulatory role on th-1 and th-17 subsets
and a co - inhibitory role on th-2 subsets .
it is proposed to be the reason for the differential expression pattern of b7-h3 in chronic inflammatory diseases . since this study clearly proves the presence of b7-h3 in gingival tissues ,
future
studies are needed to clearly outline the role of b7-h3 in the pathogenesis of periodontal disease
.
the limitations of this study include the small sample size and the lack of staging of periodontal disease at time of biopsy .
hence , it is possible that this may not truly represent the picture during active periodontal disease .
other costimulatory signal molecules such pd-1 has been demonstrated in periodontal tissues previously in a similar manner .
finally , since polyclonal b7h3 antibodies were used this could have further affected the results .
finally , since polyclonal b7h3 antibodies were used this could have further affected the results .
in conclusion , in the light of the present study it may be hypothesized that the costimulatory molecules play a more complex role than originally believed in the progression of periodontal disease . targeted therapeutic strategies aimed at controlling periodontal disease and periodontal regeneration | statement of the problem : t cells have been shown to play a role in the etiopathogenesis of periodontal disease .
b7-h3 , a costimulatory molecule , is found to be associated with regulation of t cell function in some tumoral tissues , as well as autoimmune and inflammatory diseases .
purpose : the aim of this study was to investigate the expression of b7-h3 molecule in healthy and diseased gingival tissue samples .
materials and method : gingival samples were taken from 2 groups ( a and b ) representing periodontal health and periodontal disease , respectively .
these were paraffinized and processed to carry out immunostaining to identify b7-h3 expression .
the slides were then examined under light microscope to assess the positive staining in epithelium and connective tissue .
the intensity of positive staining in epithelium and the number of positive cells in the connective tissue were evaluated .
statistical analysis was done using kappa analysis and independent t - test .
results : kappa analysis revealed good inter - examiner agreement for both the groups ( group a : 0.718 ; group b : 0.797 ) .
intensity of staining in the epithelium ranged from
intense to moderate for both the groups . in the connective tissue
, there was a statistically insignificant decrease ( p value= 0.415 ) in the number of positive cells from group a ( mean labeling index : 50.2826.09 ) to group b ( mean labeling index= 41.3725.29 ) .
conclusion : b7-h3 molecule has been found to be expressed in gingival tissue samples ; however , it showed a statistically insignificant decrease in periodontal disease group compared to healthy group . |
the pancreas is an important organ in both the digestive system and the endocrine system .
severe acute pancreatitis ( sap ) has a high mortality rate due to sepsis , necrosis , and multiple organ failure caused by pancreatic or peripancreatic infection .
approximately 80% of deaths caused by sap are related to sepsis , necrosis , and multiple organ failure . in the first several hours after onset of ap , the local inflammatory response of injured pancreatic glands
consists of increased release of chemokines , cytokines , neutrophils , and other inflammatory mediators . due to the local inflammatory response
, the gut barrier is also damaged , with significantly increased intestinal permeability within 72 h . through the damaged gut barrier ,
the bacterial flora in the intestine gain access to systemic circulation , leading to exacerbated systemic inflammatory response and possible infected pancreatic necrosis and sepsis at the early stage of the disease .
thus , maintaining the normal structure and function of the gut barrier is critical to reducing the possibility of organ failure and infected necrosis in ap .
early enteral nutrition ( een ) was introduced for sap patients due to its benefits in modifying the lactulose / mannitol ratio , reducing inflammatory response , maintaining the gut barrier , and lowering bacterial translocation .
previous meta - analyses confirmed that een is a relatively safer route to feed patients with sap or predicted sap ( psap ) compared to total parental nutrition ( tpn ) .
however , the appropriate time at which to start een administration it is still controversial .
some scholars thought 72 h was best because the onset of the disease should be the cut - off time , while others thought en provided within 48 h of admission had more definite advantages .
one recent meta - analysis even indicated that een given within 24 h was associated with more benefits than between 24 h and 48 h .
however , that meta - analysis missed several highly randomized controlled studies and only extracted 1 arm s data of the included studies to make their assessment . for better understanding of the appropriate start time of een , the present study tried to pool all relevant studies to assess the complications of een by stratifying relevant rcts into < 24 h and 2472 h groups .
relevant studies were searched for in pubmed , embase , medline , the cochrane library , and clinicaltrials.com from jan 1990 to may 2014 by 2 authors independently ( xl and fm ) . the following terms and strategy were applied for searching in the databases : ( enteral nutrition or
nasojejunal or nasogastric ) and ( acute pancreatitis ) and ( randomized controlled trial or rct or
no language restriction was set during searching . to avoid missing qualified trials , manual searching with backward snowballing method
was used to check the reference lists of included studies , relevant meta - analyses , and reviews .
studies included in this meta - analysis had to fulfill the following criteria : ( 1 ) randomized comparative trials ( rcts ) ; ( 2 ) consecutive patients with acute pancreatitis ; ( 3 ) patients were randomized assigned to experimental een group initiated within 72 h of admission or control group with tpn or den ( beyond 72 h ) .
the following information was extracted from the included rcts : first author , year of publication , start time and route of en administration , intervention of the control group , and number of participants in experimental and control groups . to compare the clinical outcomes in the 2 groups , cases of pancreatic infection , mortality , hyperglycemia , organ failure , and catheter - related septic complications were extracted .
stratified analysis was performed by the starting time of en after admission ( within 24 h or 2472 h ) .
quality of the included rcts was assessed according to methodological criteria of the cochrane handbook for systematic reviews of interventions .
publication bias was assessed by visually inspecting of funnel plots of rr of organ failure , which was reported in most of the studies included .
data of discontinuous outcomes extracted from original rcts were pooled to estimate risk ratios ( rr ) and corresponding 95% confidence intervals ( cis ) .
subgroup analysis was performed by stratifying the studies into < 24 h and 2472 h subgroups . between - studies
heterogeneity was measured with chi - square - based q test and i. p<0.1 or i>50% indicate significant heterogeneity . to identify the level of heterogeneity , a primary analysis was performed with a fixed - effects model .
if no significant heterogeneity observed , a fixed - effects model was used to make estimates , otherwise a random - effects model was applied . for the pooled estimates ,
relevant studies were searched for in pubmed , embase , medline , the cochrane library , and clinicaltrials.com from jan 1990 to may 2014 by 2 authors independently ( xl and fm ) . the following terms and strategy were applied for searching in the databases : ( enteral nutrition or
nasojejunal or nasogastric ) and ( acute pancreatitis ) and ( randomized controlled trial or rct or
no language restriction was set during searching . to avoid missing qualified trials , manual searching with backward snowballing method
was used to check the reference lists of included studies , relevant meta - analyses , and reviews .
studies included in this meta - analysis had to fulfill the following criteria : ( 1 ) randomized comparative trials ( rcts ) ; ( 2 ) consecutive patients with acute pancreatitis ; ( 3 ) patients were randomized assigned to experimental een group initiated within 72 h of admission or control group with tpn or den ( beyond 72 h ) .
the following information was extracted from the included rcts : first author , year of publication , start time and route of en administration , intervention of the control group , and number of participants in experimental and control groups . to compare the clinical outcomes in the 2 groups ,
cases of pancreatic infection , mortality , hyperglycemia , organ failure , and catheter - related septic complications were extracted .
stratified analysis was performed by the starting time of en after admission ( within 24 h or 2472 h ) .
quality of the included rcts was assessed according to methodological criteria of the cochrane handbook for systematic reviews of interventions .
publication bias was assessed by visually inspecting of funnel plots of rr of organ failure , which was reported in most of the studies included .
data of discontinuous outcomes extracted from original rcts were pooled to estimate risk ratios ( rr ) and corresponding 95% confidence intervals ( cis ) .
subgroup analysis was performed by stratifying the studies into < 24 h and 2472 h subgroups . between - studies
heterogeneity was measured with chi - square - based q test and i. p<0.1 or i>50% indicate significant heterogeneity . to identify the level of heterogeneity
if no significant heterogeneity observed , a fixed - effects model was used to make estimates , otherwise a random - effects model was applied . for the pooled estimates ,
through searching databases , 12 studies were included in the final meta - analysis [ 12,1525 ] .
the searching and screening of eligible studies is summarized in a prisma flow diagram in figure 1 .
the key characteristics of included studies are summarized in table 1 . among the 12 studies
included , 4 provided een to patients within 24 h after admission [ 1518 ] , while 8 studies provided een to patients at 2472 h after admission [ 12,1925 ] . except for eckerwall s study , which used
en delivered through the nasogastric - feeding route , all other studies used the nasojejunal feeding route . only 2 studies had den in the control group , while the remaining studies all used tpn in the control group .
a total of 625 participants were included in the 12 studies , with 301 patients in the een group and 324 in the control group .
the methodological quality of the rcts was generally high , but only 2 studies had blind design for intervention .
were both single - blind ; this design deficiency is largely due to the nature of the intervention .
therefore , nearly all patients randomized in the rcts completed the study and thus provided complete data of composite endpoints .
eight studies reported the outcome of pancreatic infection 4 in the < 24 h subgroup and 4 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of pancreatic infection than tpn or den ( 21/186 vs. 53/206 ) ( rr : 0.44 , 95% ci : 0.280.69 , p=0.0004 , i=0% ) .
subgroup analysis confirmed a similar trend in both < 24 h ( 11/86 vs. 24/97 ) ( rr : 0.51 , 95% ci 0.270.94 , p=0.03 , i=0% ) and 2472 h subgroup ( 10/100 vs. 29/109 ) ( rr : 0.39 , 95% ci : 0.200.74 , p=0.004 , i=0% ) . although the een in the 2472 h subgroup was associated with lower rr than in the < 24 h group , the difference was not significant ( p=0.56 , i=0% ) ( figure 2a ) .
the pooled analysis showed that een was generally associated with lower mortality rate than tpn or den ( 16/300 vs. 36/323 ) ( rr : 0.51 , 95% ci : 0.300.86 , p=0.01 , i=34% ) ( figure 2b ) .
subgroup analysis observed significantly decreased risk of mortality in the < 24 h subgroup ( 3/76 vs. 15/86 ) ( rr : 0.27 , 95% ci : 0.100.78 , p=0.02 , i=31% ) , but not in the 2472 h subgroup ( 13/224 vs. 21/237 ) ( rr : 0.67 , 95% ci : 0.361.25 , p=0.29 , i=19% ) .
ten studies reported the outcome of organ failure , 4 in the < 24 h subgroup and 6 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of organ failure than tpn or den ( 51/297 vs. 90/317 ) ( rr : 0.59 , 95% ci : 0.440.79 , p=0.0003 , i=10% ) ( figure 3a ) .
subgroup analysis observed significantly decreased risk of organ failure in both the < 24 h subgroup ( 12/107 vs. 30/116 ) ( rr : 0.42 , 95% ci : 0.240.77 , p=0.004 , i=21% ) and the 2472 h subgroup ( 39/190 vs. 60/201 ) ( rr : 0.67 , 95% ci : 0.490.94 , p=0.02 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.18 , i=44.7% ) ( figure 3a ) .
five studies reported the outcome of hyperglycemia , 2 in the < 24 h subgroup and 3 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of hyperglycemia than tpn or den ( 18/116 vs. 51/120 ) ( rr : 0.38 , 95% ci : 0.240.59 , p<0.0001 , i=0% ) ( figure 3b ) .
subgroup analysis observed significantly decreased risk of hyperglycemia in both the < 24 h subgroup ( 8/58 vs. 26/60 ) ( rr : 0.34 , 95% ci : 0.180.64 , p=0.0008 , i=0% ) and the 2472 h subgroup ( 10/58 vs. 25/60 ) ( rr : 0.42 , 95% ci : 0.220.79 , p=0.008 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.64 , i=0% ) ( figure 3b ) .
five studies reported the outcome of catheter - related septic complications , 2 in the < 24 h subgroup and 3 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of catheter - related septic complications than tpn or den ( 5/113 vs. 23/117 ) ( rr : 0.29 , 95% ci : 0.130.64 , p=0.002 , i=0% ) ( figure 3c ) .
subgroup analysis revealed significantly decreased risk of hyperglycemia in both the < 24 h subgroup ( 0/43 vs. 6/43 ) ( rr : 0.15 , 95% ci : 0.021.11 , p=0.06 , i=0% ) and the 2472 h subgroup ( 5/70 vs. 17/74 ) ( rr : 0.35 , 95% ci : 0.150.82 , p=0.02 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.43 , i=0% ) ( figure 3c ) . because most of the included studies reported outcome of organ failure , funnel plots of or of organ failure
the plots demonstrate asymmetric distribution at the bottom of the funnel , suggesting potential of publication bias ( figure 3d )
. however , since the number of studies involved in this meta - analysis was relatively small , it is difficult to estimate the publication bias accurately .
through searching databases , 12 studies were included in the final meta - analysis [ 12,1525 ] .
the searching and screening of eligible studies is summarized in a prisma flow diagram in figure 1 .
the key characteristics of included studies are summarized in table 1 . among the 12 studies
included , 4 provided een to patients within 24 h after admission [ 1518 ] , while 8 studies provided een to patients at 2472 h after admission [ 12,1925 ] . except for eckerwall s study , which used
en delivered through the nasogastric - feeding route , all other studies used the nasojejunal feeding route . only 2 studies had den in the control group , while the remaining studies all used tpn in the control group .
a total of 625 participants were included in the 12 studies , with 301 patients in the een group and 324 in the control group .
the methodological quality of the rcts was generally high , but only 2 studies had blind design for intervention .
were both single - blind ; this design deficiency is largely due to the nature of the intervention .
therefore , nearly all patients randomized in the rcts completed the study and thus provided complete data of composite endpoints .
eight studies reported the outcome of pancreatic infection 4 in the < 24 h subgroup and 4 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of pancreatic infection than tpn or den ( 21/186 vs. 53/206 ) ( rr : 0.44 , 95% ci : 0.280.69 , p=0.0004 , i=0% ) .
subgroup analysis confirmed a similar trend in both < 24 h ( 11/86 vs. 24/97 ) ( rr : 0.51 , 95% ci 0.270.94 , p=0.03 , i=0% ) and 2472 h subgroup ( 10/100 vs. 29/109 ) ( rr : 0.39 , 95% ci : 0.200.74 , p=0.004 , i=0% ) . although the een in the 2472 h subgroup was associated with lower rr than in the < 24 h group , the difference was not significant ( p=0.56 , i=0% ) ( figure 2a ) .
the pooled analysis showed that een was generally associated with lower mortality rate than tpn or den ( 16/300 vs. 36/323 ) ( rr : 0.51 , 95% ci : 0.300.86 , p=0.01 , i=34% ) ( figure 2b ) .
subgroup analysis observed significantly decreased risk of mortality in the < 24 h subgroup ( 3/76 vs. 15/86 ) ( rr : 0.27 , 95% ci : 0.100.78 , p=0.02 , i=31% ) , but not in the 2472 h subgroup ( 13/224 vs. 21/237 ) ( rr : 0.67 , 95% ci : 0.361.25 , p=0.29 , i=19% ) .
ten studies reported the outcome of organ failure , 4 in the < 24 h subgroup and 6 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of organ failure than tpn or den ( 51/297 vs. 90/317 ) ( rr : 0.59 , 95% ci : 0.440.79 , p=0.0003 , i=10% ) ( figure 3a ) .
subgroup analysis observed significantly decreased risk of organ failure in both the < 24 h subgroup ( 12/107 vs. 30/116 ) ( rr : 0.42 , 95% ci : 0.240.77 , p=0.004 , i=21% ) and the 2472 h subgroup ( 39/190 vs. 60/201 ) ( rr : 0.67 , 95% ci : 0.490.94 , p=0.02 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.18 , i=44.7% ) ( figure 3a ) .
five studies reported the outcome of hyperglycemia , 2 in the < 24 h subgroup and 3 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of hyperglycemia than tpn or den ( 18/116 vs. 51/120 ) ( rr : 0.38 , 95% ci : 0.240.59 , p<0.0001 , i=0% ) ( figure 3b ) .
subgroup analysis observed significantly decreased risk of hyperglycemia in both the < 24 h subgroup ( 8/58 vs. 26/60 ) ( rr : 0.34 , 95% ci : 0.180.64 , p=0.0008 , i=0% ) and the 2472 h subgroup ( 10/58 vs. 25/60 ) ( rr : 0.42 , 95% ci : 0.220.79 , p=0.008 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.64 , i=0% ) ( figure 3b ) .
five studies reported the outcome of catheter - related septic complications , 2 in the < 24 h subgroup and 3 in the 2472 h subgroup .
the pooled analysis showed that een was generally associated with lower risk of catheter - related septic complications than tpn or den ( 5/113 vs. 23/117 ) ( rr : 0.29 , 95% ci : 0.130.64 , p=0.002 , i=0% ) ( figure 3c ) .
subgroup analysis revealed significantly decreased risk of hyperglycemia in both the < 24 h subgroup ( 0/43 vs. 6/43 ) ( rr : 0.15 , 95% ci : 0.021.11 , p=0.06 , i=0% ) and the 2472 h subgroup ( 5/70 vs. 17/74 ) ( rr : 0.35 , 95% ci : 0.150.82 , p=0.02 , i=0% ) .
no significant heterogeneity was observed between the results of these 2 subgroups ( p=0.43 , i=0% ) ( figure 3c ) .
because most of the included studies reported outcome of organ failure , funnel plots of or of organ failure were used to assess publication bias .
the plots demonstrate asymmetric distribution at the bottom of the funnel , suggesting potential of publication bias ( figure 3d )
. however , since the number of studies involved in this meta - analysis was relatively small , it is difficult to estimate the publication bias accurately .
this meta - analysis based on 12 rcts showed that een is generally associated with reduced risk of pancreatic infection , mortality , organ failure , hyperglycemia , and catheter - related septic complications compared to tpn or den .
een within 24 h of admission presented significantly better outcome in reducing morality than een administered at 2472 h. however , no significant subgroup difference was observed in the risk of pancreatic infection , organ failure , hyperglycemia , or catheter - related septic complications between the 2 subgroups .
patients included in the rcts had high levels of compliance to the een since only 1 patient withdrew from the study .
one large retrospective study based on 197 patients with psap showed that een started within 48 h after admission contributed to reduced infected pancreatic necrosis , respiratory failure , intensive care unit admission , and mortality .
therefore , for patients with sap or psap , nutrition support should be initiated as soon as possible .
generally , the cut - off time points for en after onset of symptoms is 24 h according to the espen ( european society for parenteral and enteral nutrition ) and 48 h according to the aspen ( american society for parenteral and enteral nutrition ) guidelines .
previous studies proposed that een could help reduce infection complications through 2 aspects : firstly , to maintain the integrity of the intestinal mucosa barrier and thus to lower the possibility of bacteria translocation from small intestine to systemic circulation at the early phase of the disease ; and secondly , to reduce tpn - associated high rates of catheter - related infection . in this meta - analysis ,
pooled evidence showed that en could simultaneously reduce the risk of pancreatic infection and catheter - related infection better than tpn or den .
higher intestinal permeability is not only associated with increased bacteria translocation , but is also associated with increased serum endotoxin level and cytokine level . therefore , systemic inflammatory response syndrome ( sirs ) might involve remote organs or even cause remote organ failure .
in fact , the integral gut mucosa acts as a barrier against bacteria translocation and spreading of toxins and inflammation factors .
since increased permeability of the gut barrier was observed during the first 24 h of sap , the importance of maintaining the gut barrier integrity was already emphasized in previous studies .
een initiated within 24 h may reasonably be supposed to more strongly protect the gut barrier .
however , this study also failed to demonstrate significantly greater benefits of een within 24 h in reducing the risk of pancreatic infection , hyperglycemia , and catheter - related septic complications than at 2472 h. although the < 24 h subgroup had slightly lower rr of organ failure than the 2472 h subgroup , the difference was not significant .
interestingly , the mortality rate of the < 24 h subgroup was significantly lower than that in the 2472 h subgroup , suggesting that een within 24 h might have some unidentified benefits .
firstly , there is still no consensus about the definition of early en .
therefore , in this study , rcts were arbitrarily divided into < 24 h and 2472 h subgroup according to previous clinical guidelines .
secondly , some of the rcts included did not clearly report the interval between onset of disease symptoms and patient admission , which might be a factor confounding accurate classification of een .
however , overall and subgroup analysis all had small heterogeneity , suggesting the influence of this limitation was not large .
two rcts had den in the control group , while tpn was applied in controls in other studies .
fourthly , only 5 studies reported the outcome of hyperglycemia and catheter - related septic complications . due to the small sample size of these 2 outcomes ,
in addition , since various studies might be based on a small sample of patients and were not reported , the possible reporting bias should not be ignored .
this study showed that een is associated with reduced risk of pancreatic infection , mortality , organ failure , hyperglycemia , and catheter - related septic complications compared to tpn or den .
een within 24 h of admission presented significantly better effect in reducing morality than een at 2472 h. however , no significant difference was observed in the risk of pancreatic infection , organ failure , hyperglycemia , and catheter - related septic complications between the 2 subgroups .
if a patient may reasonably expected to have good compliance with en therapy , it should be considered as early as possible . | backgroundalthough ( een ) is a relatively safer route by which to feed patients with severe acute pancreatitis ( sap ) or predicted sap ( psap ) compared to total parental nutrition ( tpn ) , the appropriate starting time for een administration after admission is still controversial .
this study pooled all relevant studies to assess the complications associated with een by stratifying relevant rcts into subgroups according to the starting time ( < 24 h or between 24 and 72 h after admission).material / methodsrelevant studies were searched for among 5 databases .
the association between intervention and complications , including pancreatic infection , mortality , hyperglycemia , organ failure , and catheter - related septic complications , were assessed by using pooled risk ratio ( rr ) and the corresponding 95% confidential interval ( ci).resultstwelve rcts were identified through our literature search .
pooled analysis showed that een , but not tpn or delayed enteral nutrition ( den ) , is associated with reduced risk of pancreatic infection , mortality , organ failure , hyperglycemia , and catheter - related septic complications .
een within 24 h of admission presented significantly better outcome in morality than een between 24 and 72 h. however , no significant heterogeneity was observed in the risk of pancreatic infection , organ failure , hyperglycemia , and catheter - related septic complications between the 2 subgroups.conclusionsif the patients are reasonably expected to have high compliance to en therapy , it could be considered as early as possible . |
oral contraceptives increase the risk of venous phenomenon due to the hypercoagulable state as a result of their effect on increasing the levels of the circulating coagulation factors . since their introduction , hormonal contraceptives have always been associated with an increased risk of stroke ( cerebrovascular accidents ) . thus , thrombotic or ischemic strokes have frequently been associated with the use of oral contraceptives where the risk is directly proportional to the amount of estrogen present in the pill . as the expected risk is presumed to be higher in women who take more than 50 mcg of estrogen per day ,
thus , the ideal combined oral contraceptive ( coc ) is the one that contains the lowest amount of estrogen , which in combination with progesterone is effective in preventing pregnancy although minimizing the adverse effects .
hence , all the available coc in the market contain lower doses of estrogen and progesterone than they did in the past , and the current recommendations are to restrict the use of coc to younger women who do not have the associated risk factors for cardiovascular disease .
a common cause of ischemic strokes is lacunar cerebral infarction ( laci ) that results from the occlusion of any one of the cerebral penetrating arteries , which accounts for nearly 25% of all ischemic strokes , with an annual incidence of approximately 15/100,000 people .
laci are particularly commonly associated with hypertension and diabetes due to the associated hypercoagulable state .
there occur small infarcts either in the basal ganglia , thalamus , and white matter or even in the brain stem .
common symptoms and complications of laci include anemia , high blood pressure , cerebral infarction , pneumonia , dizziness , cerebral hemorrhage .
laci has usually been seen in uncontrolled hypertension , osteoporosis , arthritis , diabetes and depression .
it is also a common adverse effect of certain drugs such as , prednisolone , alendronate , atorvastatin , levothyroxine sodium , etc .
sukhi is a type of an oral combined contraceptive pill for women that are available in bangladesh only .
out of the 28 pills in a strip , seven are iron supplements which are brown in colour and the rest of the pills are coc containing lynestrenol 2.5 mg along with 50 mcg of ethinyl estradiol per tablet .
although the current recommendation suggests the use of lower doses ( 30 to 35 g ) of estrogen in oral combined contraceptive pills , sukhi is the only coc contraceptive that contains high amounts of estrogen .
further , gullible consumers have a wrong notion that sukhi is a homeopathic contraceptive since the composition of the pill is written in bengali .
for this reason and because of its easy availability as an over the counter drug ( otc ) it is also being popularly used in india . though literature search revealed no documented adverse drug reaction with this coc we report a case of laci in a 35 year old woman who was on sukhi for contraception for the last 3 years . to our knowledge , this is the first ever documented case , for there are no such available evidences suggesting such side effects with the otherwise popular combined pill sukhi .
a 35-year - old previously healthy female patient on sukhi for contraception for the last 3 years presented to the emergency department of s.c.b .
medical college and hospital , cuttack in november 2011 , with complaints of sudden onset ( 45 min earlier ) of slight paralysis of the right side of her body and the inability to express herself by speech , writing , or signs .
she was afebrile , alert , oriented with blood pressure of 122/98 mm hg , pulse rate 100/min .
she was a non - smoker , not a known diabetic or hypertensive , but there was history of repeated abortions in the past .
fundoscopic findings were normal although neurological examination revealed right sided hemiparesis with upward right babinski reflex . on investigations the parameters such as hemogram , esr , random blood sugar , kidney function tests , liver function tests , electroencephalogram , electrocardiogram , serum cholesterol , serum electrolytes , urine analysis and x - ray chest were within normal limits .
plasma amino acid , vascular profile , and metabolic profile ( including levels of homocysteine , ammonia , and lactate ) were also within normal limits .
additional tests such as prothrombin time , activated partial thromboplastin time , antinuclear antibody test , anticardiolipin antibody immunoglobulin levels , protein c level and antithrombin iii level were within normal limits . however a non - contrast enhanced computed tomography of brain showed a laci [ figure 1 ] which was confirmed on a magnetic resonance imaging scan .
the patient was then hospitalized and treated with intravenous unfractionated heparin , which was then switched on to subcutaneous enoxaparin .
after 7 days , her condition completely improved and she was discharged with oral warfarin .
an ischemic stroke results from inadequate blood supply due to occlusion of an artery usually due to thrombus or atherosclerotic plaques .
oral contraceptives , particularly that containing high estrogen dose can increase the risk of thrombus formation . though studies have shown an increased risk of acute myocardial infarction and hemorrhagic stroke among users of oral contraceptive agents this type of laci reported in our patient within a span of 3yrs of usage with an oral contraceptive has not been reported so far .
it is well documented that oral contraceptive use increases the risk of venous thromboembolic disease as well as arterial stroke via a mechanism of paradoxical embolism due to a resultant hypercoagulable state .
certain studies have suggested an observed increase in the levels of serum procoagulants such as factor i , vii , viii , ix , x , xii , and xiii along with a decrease in anticoagulant factors such as protein s and antithrombin iii in persons on combined pills .
in addition to the above mentioned mechanisms underlying risk of oral contraceptive - associated venous thromboembolism , there is also an evidence for a genetic susceptibility for oral contraceptive associated stroke .
the most susceptible are the women who carry the alleles of the methylene tetrahydrofolate reductase c677 t and the factor v leiden ( fv g1691a ) genes .
persons with polymorphic forms of the angiotensin - converting enzyme and the angiotensinogen gene may also have increased risk of stroke among women using oral contraceptives .
elevated levels of the factor vii , lipoprotein - associated phospholipase a2 has also been shown to double the risk of oral contraceptive associated stroke .
though oral contraceptive drugs are one of the most effective forms of birth control , concern still exists about the short - term and long - term risks associated with these agents .
since the amount of estrogen in oral - contraceptive preparations has been thought to be chiefly responsible for these risks , in recent years newer , low - estrogen oral contraceptives ( containing less than 50 g ) have been developed and are widely being used nowadays . furthermore estrogen - containing hormonal contraception
are better avoided among women with a history of cardiovascular disease , including stroke where progestin - only hormonal contraceptives can be used in the setting of stroke .
hence , where oral contraceptives containing the least possible estrogen are recommended and are usually prescribed coc like sukhi are also irrationally prescribed .
thus , clinicians must always be extra cautious even when prescribing contraceptives for many issues must be taken into account such as the amount of the hormones in them , the genetic pattern of persons thought to be at risk and the rationality of the otc available coc . | combined oral contraceptives are one of the risk factor for stroke in women .
we report a case of an arterial ischemic stroke due to lacunar infarction in a 35-year - old previously healthy female patient induced after 3 years on sukhi an oral contraceptive after two times artificial abortions .
a brain mri finding was suggestive of lacunar infarction .
her symptoms improved after stopping the oral contraceptive and putting her on i.v heparin therapy . |
in the last 30 years the spanish nhs has evolved from a tightly centralized structure to seventeen autonomous regional health systems . as a result a number of benefits have been obtained but also important side - effects such as unjustified variation in clinical practice and a substantial loss of equity .
to deal with these unwanted developments the spanish inter - territorial board of the nhs has recently approved and launched several health strategies on priority areas : diabetes , coronary ischemia , palliative care , cancer and mental health .
the strategies are based on scientific evidence or consensus and they have been agreed on by all the regions
the gaps from research to policy and from policy to practice in the strategy for mental health are identified and potential solutions are discussed . | introductionin the last 30 years the spanish nhs has evolved from a tightly centralized structure to seventeen autonomous regional health systems . as a result a number of benefits have been obtained but also important side - effects such as unjustified variation in clinical practice and a substantial loss of equity.descriptionto deal with these unwanted developments the spanish inter - territorial board of the nhs has recently approved and launched several health strategies on priority areas : diabetes , coronary ischemia , palliative care , cancer and mental health .
the strategies are based on scientific evidence or consensus and they have been agreed on by all the regions
. however , the degree of implementation is not as good as expected.conclusionthe gaps from research to policy and from policy to practice in the strategy for mental health are identified and potential solutions are discussed . |
talon cusp is a prominent accessory cusp - like structure projecting from the cingulum area or the cementoenamel junction ( cej ) of the maxillary or mandibular teeth in both primary and permanent dentition .
this accessory cusp , which can vary in size , is formed by enamel and dentin , and may or may not have a projection of the pulp tissue .
it was thereafter named a talon cusp by mellor and ripa due to its resemblance to an eagle 's talon .
there are limited data available on the prevalence of talon cusps , which ranges from 0.06% to 7.7% .
the maxillary lateral incisors are the most commonly affected ( 67% ) followed by the central incisors ( 24% ) and canines ( 9% ) .
. only few cases of talon cusps in facial surfaces of teeth were reported in the literature.[35814 ]
a 25-year - old female reported to the dental clinic with complaint of decayed tooth in the left back region of the lower jaw . on examination
an elevation on the labial aspect of crown of the maxillary right permanent central incisor was noticed .
the elevation was broader at the cervical area and gradually tapered toward the incisal edge to give a ridge - like appearance .
intraoral periapical radiograph of the area revealed v - shaped radiopacity in relation to the coronal aspect of maxillary right central incisor , extending 1 mm below the incisal edge to the cementoenamel junction .
no changes in the periapical area were noticed [ figure 2 ] . based on these clinical and radiological features diagnosis of facial talon cusp was made .
as the patient had no complaints with the abnormal tooth ( with facial talon ) she was kept on a yearly periodic recall .
photograph showing elevated area on the crown of the right central incisor extending from the cervical area to the incisal margin intraoral periapical radiograph showing v - shaped radiopaque area overlapping the crown of right maxillary central incisor
genetic and environmental factors , aberrant hyperactivity of dental lamina , outward folding of the inner enamel epithelium , altered endocrine functions , and a transient focal hyperplasia of the mesenchymal dental papilla might be responsible .
the shape , size , and location of talon cusp may vary from a slight tubercle - like projection to a well - delineated prominent cusp extending from the cementoenamel junction to the incisal edge . in our case , the tubercle - like projection extending 1 mm below the incisal edge to the cementoenamel junction was observed . they may be found on the palatal / lingual or labial faces of the tooth , in primary or permanent dentition .
talon cusp occurs more frequently in permanent than primary dentitions and shows a predilection for the maxilla over the mandible .
our case was of a 25-year - old female with talon cusp on permanent dentition in the maxillary arch .
in vast majority of cases the talon cusp originates from the lingual surface of the tooth , on very rare occasions from the facial aspect of the tooth.[3814 ] as a matter of fact only seven case reports of facial talon in permanent maxillary teeth have been found in the literature till date.[381214 ] so far nine cases of faciolingual talon cusps have been reported.[141722 ] few archeological studies have reported of labial talon cusp in ancient skeletal remains .
graded the lingual talon cusp from 1 ( the most extreme form ) to 3 ( slightest form ) .
where as a classification for the labial talon cusp was suggested after examining the dentition of 301 skeletons from a pre - european contact american indian population as follows . the slightest form , consists of a slightly raised triangle on the labial surface of an incisor extending the length of the crown , but not reaching the cementoenamel junction or the incisal edge . the moderate form , is a raised triangle on the labial surface of an incisor that extends the length of the crown , does not reach the cementoenamel junction , but does reach the incisal edge .
the most extreme form , is a free - form cusp extending from the cementoenamel junction to the incisal edge on the labial surface of an incisor .
radiographically , it may appear typically as a v - shaped radiopaque structure , as in true talon or semitalon , or be tubercle - like , as in trace talon , originating from the cervical third of the root .
the radiopaque v - shaped structure is superimposed over the normal image of the crown of the tooth .
this appearance varies with the shape and size of the cusp , and the angle at which the radiograph is taken .
the site of the junction of a cusp with the dental surface frequently allows the buildup of plaque , caries lesions , and periodontal or pulpal inflammation .
other sequelae attributed to talon cusp are dental movement , irritation of soft tissues , occlusal trauma , speech or mastication problems , attrition , cusp fracture , and unfavorable esthetic aspects .
discoloration of the cervicofacial aspect of crown due to extrinsic staining was observed in the our case due to abnormal morphology .
caries susceptibility due to abnormal groove morphology has been observed in few other cases . irritation and interference to tongue and buccal mucosa when occurring on the lingual and buccal side , respectively , have been reported .
where there are deep developmental grooves , simple procedure such as pit and fissure sealants can be considered . in the case of occlusal interference reduction of bulk of the cusp and fluoride application
can be carried out gradually over a period of 6 - 8 weeks . in the case of a fully matured tooth root canal therapy
is advisable where as in immature teeth calcium hydroxide pulpotomy is the treatment of choice . orthodontic correction may become necessary when there is tooth displacement or malalignment of affected or opposing teeth . since there was no risk of pulpal exposure , caries formation or esthetic concern in our patient oral prophylaxis and periodic review was considered .
the slightest form , consists of a slightly raised triangle on the labial surface of an incisor extending the length of the crown , but not reaching the cementoenamel junction or the incisal edge .
the moderate form , is a raised triangle on the labial surface of an incisor that extends the length of the crown , does not reach the cementoenamel junction , but does reach the incisal edge .
the most extreme form , is a free - form cusp extending from the cementoenamel junction to the incisal edge on the labial surface of an incisor .
radiographically , it may appear typically as a v - shaped radiopaque structure , as in true talon or semitalon , or be tubercle - like , as in trace talon , originating from the cervical third of the root .
the radiopaque v - shaped structure is superimposed over the normal image of the crown of the tooth .
this appearance varies with the shape and size of the cusp , and the angle at which the radiograph is taken .
the site of the junction of a cusp with the dental surface frequently allows the buildup of plaque , caries lesions , and periodontal or pulpal inflammation .
other sequelae attributed to talon cusp are dental movement , irritation of soft tissues , occlusal trauma , speech or mastication problems , attrition , cusp fracture , and unfavorable esthetic aspects .
discoloration of the cervicofacial aspect of crown due to extrinsic staining was observed in the our case due to abnormal morphology .
irritation and interference to tongue and buccal mucosa when occurring on the lingual and buccal side , respectively , have been reported .
where there are deep developmental grooves , simple procedure such as pit and fissure sealants can be considered . in the case of occlusal interference reduction of bulk of the cusp and fluoride application
can be carried out gradually over a period of 6 - 8 weeks . in the case of a fully matured tooth root canal therapy
is advisable where as in immature teeth calcium hydroxide pulpotomy is the treatment of choice . orthodontic correction may become necessary when there is tooth displacement or malalignment of affected or opposing teeth . since there was no risk of pulpal exposure , caries formation or esthetic concern in our patient oral prophylaxis and periodic review was considered .
the slightest form , consists of a slightly raised triangle on the labial surface of an incisor extending the length of the crown , but not reaching the cementoenamel junction or the incisal edge .
the moderate form , is a raised triangle on the labial surface of an incisor that extends the length of the crown , does not reach the cementoenamel junction , but does reach the incisal edge .
the most extreme form , is a free - form cusp extending from the cementoenamel junction to the incisal edge on the labial surface of an incisor .
radiographically , it may appear typically as a v - shaped radiopaque structure , as in true talon or semitalon , or be tubercle - like , as in trace talon , originating from the cervical third of the root .
the radiopaque v - shaped structure is superimposed over the normal image of the crown of the tooth .
this appearance varies with the shape and size of the cusp , and the angle at which the radiograph is taken .
the site of the junction of a cusp with the dental surface frequently allows the buildup of plaque , caries lesions , and periodontal or pulpal inflammation .
other sequelae attributed to talon cusp are dental movement , irritation of soft tissues , occlusal trauma , speech or mastication problems , attrition , cusp fracture , and unfavorable esthetic aspects .
discoloration of the cervicofacial aspect of crown due to extrinsic staining was observed in the our case due to abnormal morphology .
irritation and interference to tongue and buccal mucosa when occurring on the lingual and buccal side , respectively , have been reported .
where there are deep developmental grooves , simple procedure such as pit and fissure sealants can be considered . in the case of occlusal interference reduction of bulk of the cusp and fluoride application
can be carried out gradually over a period of 6 - 8 weeks . in the case of a fully matured tooth root canal therapy
is advisable where as in immature teeth calcium hydroxide pulpotomy is the treatment of choice . orthodontic correction may become necessary when there is tooth displacement or malalignment of affected or opposing teeth . since there was no risk of pulpal exposure , caries formation or esthetic concern in our patient oral prophylaxis and periodic review was considered .
the aim of this report was to describe the clinical and radiological feature of an extremely rare anomaly of facial talon cusp .
the information in this report would help the clinicians to identify such anomalies more efficiently and prevent further complications and inconvenience to patient . | talon cusp is a rare developmental anomaly that occurs most commonly on the lingual side of the incisors .
occurrence of the labial talon cusp is rare in the dental literature . till date
only seven cases of isolated nonsyndromic labial talon cusps have been reported in the maxillary permanent dentition . however , a few cases of syndromic labial talon cusps and faciolingual talon cusps have been reported .
the aim of our report is to highlight clinical and radiological features of this rare anomaly . |
recently , neuroscientists and cognitive psychologists have promoted the idea of the existence of a mirror system in our mind and brain .
the mirror system simulates the actions we observe by mapping an observed action onto a motoric representation of the same action in the observer ( jeannerod , 2003 ) .
this modern motor theory of action and perception could be explained by the ideomotor model .
the model proposes that actions are represented not only in terms of body movements but also in terms of the distal perceptual effects they aim to generate .
in this way , action and perception are commensurate and are coded in a common representational medium ( prinz , 1997 ) . performing a movement
creates an association between the motor pattern it has been generated by and the sensory effects it leads to .
this association can also be used in a reverse direction to induce a movement by perceiving its sensory effects ( hommel et al . , 2001 ) .
from this viewpoint , the potential motor and social roles of mirror mechanisms are stressed . mirroring actions might help a person not only understand what another person is doing , but also explore how his or self - awareness is developing ( schtz - bosbach and prinz , 2007 ) .
these potential functions of action mirroring form a possible part of the mirror mechanisms from the perspective of the perceiver ( fig .
a growing body of research suggests that the mirror neuron system activates when an action is observed , imitated , and empathically understood ( rizzolatti et al . , 2001 ; rizzolatti and craighero , 2004 ) .
the aim of this paper is to draw attention to basic neurophysiological findings about the mirror neuron system .
these effects have so far been largely neglected in modern motor theories of neuro and social cognition .
finally , i will examine how basic concepts of the mirror neuron system could have clinical applications in exercise rehabilitation .
when observing other people s performance , human brains simulate the performance of the observed action .
this simulation process could reinforce sophisticated mental functions such as observational learning ( rizzolatti and arbib , 1998 ) .
researchers have reported that within the human premotor and parietal cortex , mirror neurons
discharge both when the person performs an action and when the person observes another person performing the same action .
the mere observation of a performance induces a selective increase in motor - evoked potentials ( meps ) from the muscles that would be active if the observed actions were performed ( rizzolatti , 2005 ) .
rizzolatti and colleagues ( 2001 ) showed that the mirror system integrates observed actions of others with the motor repertoires of the individual .
mirror neurons respond to the execution of an action , and to the observation of a conspecific carrying out that same action ( rizzolatti and craighero , 2004 ) .
activation of mirror neurons is greater when observing familiar rather than unfamiliar movement .
neuroscience studies show that motor expertise modulates the activation of the human mirror system during the observation of dance movement .
neural activation is greater in those people who have direct motor experience of the observed dance movement .
therefore , observing other people s actions is imitative motor learning by a covert simulation of the very same action ( calvo - merino et al . , 2006 ) .
in addition , aglioti and colleagues ( 2008 ) investigated the dynamics of action anticipation and its underlying neural correlates in professional basketball players , sports journalists , and novices .
their results showed that both visuomotor and visual experts demonstrated a selective increase of motor - evoked potentials during the observation of basket shots .
however , only athletes show a time - specific motor activation during the observation of erroneous basket throws .
this suggests that achieving excellence in sports is related to the fine - tuning of specific anticipatory resonance mechanisms that endow elite athletes brains with the ability to predict others actions ahead of their realization .
this study shows that tuning of resonant action systems is important in the perceptual abilities shown by the movers engaging in the action .
in humans , mirror neurons have a part in understanding not only other people s actions but also their emotions .
many neuroscience studies have shown that the mirror neuron system involving visceral - motor centers allows people to recognize each other s emotions , like one involving visual - motor centers allows people to recognize each other s actions ( gallese et al . , 2004 ) .
decety and colleagues ( 2004 ) used fmri brain scans to compare how people perceive pain .
they found that perceiving photographs of situations likely to cause pain was associated with significant bilateral changes in activity in several regions , notably the anterior cingulate , the anterior insula , the cerebellum , and to a lesser extent the thalamus , which are known to play a significant part in pain processing .
the results suggest a direct linkage between perceiving emotion in another individual and experiencing it oneself .
developmental studies have shown that young children can imitate various body movements in the sense that they appear to view human actions in terms of the relation between the agent , the means , and the goal of the action ( gergely et al . , 2002 ) . however , children with autism have often been found to be impaired at imitation due to a deficit in embodied simulation ( carpenter et al . , 2001 ) .
in addition , some researchers showed that adults with asperger s syndrome or high - functioning autism were impaired at imitating in a mirror - image manner ( avikainen et al .
empathy is defined as a sense of similarity between the feelings one experiences and those expressed by others .
empathy extends beyond simply understanding other people s emotional state to embodying the experience of that state ( singer et al . ,
empathy is an embodied affective resonance that involves some level of cognitive processing ( decety and jackson , 2004 ; lamm et al . , 2007 ) .
the presumption is that neuronal connectivity between the prefrontal cortex and the limbic system function are vital units .
researchers in the field of body psychotherapy have stressed that therapeutic interventions based on bodily expression can foster empathy and , more generally , develop interpersonal coordination ( mcgarry and russo , 2011 ) .
specifically , movement therapy research has focused on special - needs populations like people with autism ( berrol , 2006 ) .
it is known that the core deficits of autism , which are motor deficits , language impairment , and social impairment , are indicative of dysfunction of the mirror neuron system ( iacoboni and dapretto , 2006 ) .
dysfunction of the mirror neuron system could account for the language disorders that autism is characterized by .
furthermore , the links between the human mirror neuron system and social cognition reviewed in the previous section of this paper suggest that mirror neuron dysfunction could also account for the social deficits in autism .
dapretto and colleagues ( 2005 ) examined mirror neuron abnormalities in autism through observation and imitation of facial emotional expressions .
this study has demonstrated strong correlations between the reduced activity in mirror neuron areas and the severity of the autism .
they suggested that a dysfunctional mirror neuron system underlies the social deficits observed in autism .
the activation of premotor neurons during mere observation of actions is a highly appealing feature for motor functions rehabilitation .
patients with severe stroke show chronic motor disorder ; therefore , the use of movement learning by observation as a form of rehabilitation is highly suitable for stroke patients ( iacoboni and mazziotta , 2007 ) .
ertelt and colleagues ( 2007 ) examined a new neuro - rehabilitative program , called
results showed that the experimental group demonstrated a significant improvement of motor functions compared with both the pretreatment baseline and control groups .
additionally , the effects of action observation therapy on the reorganization of motor system were investigated in this fmri study , using an independent sensorimotor task consisting of object manipulation . furthermore , celnik and colleagues ( 2008 ) assessed the effect of action observation therapy on motor memory formation in patients with chronic stroke , using transcranial magnetic stimulation ( tms ) .
results indicated that the magnitude of motor memory formation was larger in the action observation group than in the group receiving physical training only . to date
, few researchers have focused on the mirror neuron system in rehabilitation ; however , existing studies have impressive results that are worthy of further investigation .
empathy is defined as a sense of similarity between the feelings one experiences and those expressed by others .
empathy extends beyond simply understanding other people s emotional state to embodying the experience of that state ( singer et al . ,
empathy is an embodied affective resonance that involves some level of cognitive processing ( decety and jackson , 2004 ; lamm et al . , 2007 ) .
the presumption is that neuronal connectivity between the prefrontal cortex and the limbic system function are vital units .
researchers in the field of body psychotherapy have stressed that therapeutic interventions based on bodily expression can foster empathy and , more generally , develop interpersonal coordination ( mcgarry and russo , 2011 ) .
specifically , movement therapy research has focused on special - needs populations like people with autism ( berrol , 2006 ) .
it is known that the core deficits of autism , which are motor deficits , language impairment , and social impairment , are indicative of dysfunction of the mirror neuron system ( iacoboni and dapretto , 2006 ) .
dysfunction of the mirror neuron system could account for the language disorders that autism is characterized by .
furthermore , the links between the human mirror neuron system and social cognition reviewed in the previous section of this paper suggest that mirror neuron dysfunction could also account for the social deficits in autism .
dapretto and colleagues ( 2005 ) examined mirror neuron abnormalities in autism through observation and imitation of facial emotional expressions .
this study has demonstrated strong correlations between the reduced activity in mirror neuron areas and the severity of the autism .
they suggested that a dysfunctional mirror neuron system underlies the social deficits observed in autism .
the activation of premotor neurons during mere observation of actions is a highly appealing feature for motor functions rehabilitation .
patients with severe stroke show chronic motor disorder ; therefore , the use of movement learning by observation as a form of rehabilitation is highly suitable for stroke patients ( iacoboni and mazziotta , 2007 ) .
ertelt and colleagues ( 2007 ) examined a new neuro - rehabilitative program , called
results showed that the experimental group demonstrated a significant improvement of motor functions compared with both the pretreatment baseline and control groups .
additionally , the effects of action observation therapy on the reorganization of motor system were investigated in this fmri study , using an independent sensorimotor task consisting of object manipulation .
furthermore , celnik and colleagues ( 2008 ) assessed the effect of action observation therapy on motor memory formation in patients with chronic stroke , using transcranial magnetic stimulation ( tms ) .
results indicated that the magnitude of motor memory formation was larger in the action observation group than in the group receiving physical training only . to date , few researchers have focused on the mirror neuron system in rehabilitation ; however , existing studies have impressive results that are worthy of further investigation .
the discovery of mirror neurons has been recognized as one of the major developments in neuroscience , with possible implications for the explanation of many important cognitive functions , including action and perception understanding , imitation , and empathy . in the mirror neuron system ,
action observation , imitation , and empathy are represented in the same basic motor circuit as action execution .
providing an additional source of information to motor training could be useful in promoting recovery not only mentally but also physically in stroke patients . through successfully translating basic concepts of mirror neuron to initial clinical applications in the exercise rehabilitation field
, future work on the mirror neuron system could consolidate and expand the clinical applications . | the discovery of mirror neurons has been recognized as one of the major developments in neuroscience , with possible implications for the explanation of many important cognitive functions , including action and perception understanding , imitation , and empathy . in the mirror neuron system ,
action observation , imitation , and empathy are represented in the same basic motor circuit as action execution .
the present paper presents basic neurophysiological findings about mirror neuron system and discusses the effects of perceptual and motor resonance on other people s actions .
finally , this paper examines strategies informed by mirror neuron research in an empathy - fostering program for autism and in a motor rehabilitation program for stroke patients . |
veno - arterial extra corporeal membrane oxygenation ( va ecmo ) is a common clinical aid for cardiopulmonary surgery [ 14 ] .
based on the different cannular position , va ecmo is classified as peripheral ecmo ( pecmo ) and central ecmo ( cecmo ) . for pecmo cannulas
in contrast , cecmo usually has an arterial cannula placed into the ascending aorta and a venous cannula placed in the right atrium .
along with the widely uses of peripheral and cecmo , their clinical complications have been studied by more and more researchers .
for instance , compared with pecmo , the cecmo was found that it could lead more required re - exploration for bleeding and higher risk of infection .
in contrast with cecmo , the pecmo has more complications on the blood perfusion and vascular impair , including lower extremity ischemia , amputation and vascular complication .
in addition , more than half of patients appeared complication of limb ischemic with the treatment of pecmo [ 1214 ] . and there is also report that patients undergoing pecmo support through femoral vessels are prone to vascular complications .
although the precise reason on the difference in the complications caused by both types of ecmo , the differences in the hemodynamic states resulted from the different cannular location of both types of ecmo are considered as a key factors .
computational fluid dynamics ( cfd ) as an effective method widely used to evaluate the hemodynamic effects of ecmo on cardiovascular system .
similarly , neidlin et al . conducted cfd simulation to study the hemodynamic effect of the distance between the cannula tip and the vertebral artery branch on the cerebral perfusion .
proposed a multi - scale model , involving the cerebral autoregulation characteristic , to study the effect of ecmo on cerebral perfusion .
although there are many studies on the hemodynamic effects of ecmo on the cardiovascular system , the hemodynamic difference between peripheral and cecmo is still under investigation . in order to clarify this problem ,
the aortic pressure and flow rate , derived from previous work [ 1922 ] , were chosen as the boundary conditions . the flow pattern in the aorta and femoral branches , the distribution of blood flow rate from the aorta to brain and limbs , the distribution of blood flow rate between arterial bifurcations , flow junction , harmonic index ( hi ) of blood flow ( hi is used to evaluate the pulsatility of blood flow in the aorta ) , wall shear stress ( wss ) , and the oscillatory shear index ( osi , which is used to evaluate the effects of blood flow on the endotheliocyte in the aorta ) were used as the factors to evaluate the hemodynamic states .
the ideal 3-dimensional geometry model with peripheral and cecmo ( figure 1 ) was constructed by using the commercial software solidworks .
the models consist of the ascending aorta , the innominate artery ( ia ) , left common carotid artery ( lcca ) , left subclavian artery ( lsa ) , left femoral artery ( lfa ) , right femoral artery ( rfa ) , and the cannula of ecmo .
the geometric characteristics of models are derived from the literature , as shown in table 1 . a standard arterial cannula ( 24 fr )
was added to the ascending aorta on the site that is routinely used in clinical practice ( figure 1b ) .
the cannular anastomotic position is about 2 cm below the inlet of the innominate artery on the anterior wall of the aorta , at a 45 angle to the x y plane .
both geometric models were meshed by using hypermesh commercial software to generate the computational models .
both models are meshed by using tetrahedral elements , in which there are 908 666 elements and 973 949 elements , respectively .
mesh independence tests were conducted for mass flow rate and the maximal error is within 5% ; the reasonable accuracy of the computing was confirmed .
the blood motion is governed by 3-dimensional navier - stokes equations : where u represents the blood velocity .
represents the blood density and f is the volume force field . in all simulation , the blood was set up as a homogeneous , incompressible , and newtonian fluid flow with =1020 kg / m and =0.0035kg / m.s . as the mean reynolds s number is less than 2300 , the blood flow here was assumed as laminar flow .
the finite volume method was used to solve navier - stokes equations by the transient simulation . for studying the hemodynamic effects
, the continuous flow of 4 l / min was used as the inlet boundary condition delivered from inlet cannula of both ecmo .
pulsatile flow rates ( average value 1 l / min ) , derived from a validated lumped parameter model ( lmp ) [ 2426 ] , were imposed on inlet of aorta as the inlet boundary conditions ( figure 2 ) .
thus , the total blood perfusion was 5 l / min , which satisfies the physical requirement .
the mean blood pressure ( 70 mmhg ) was imposed to the outlets of aorta as the outlet boundary condition . to assess the difference of perfusion condition under both ecmo
, blood flow rate ratios ( r ) of arterial bifurcations was defined as the equation ( 3 ) : where qwessel was the flow rate of the concerning vessel in one cardiac cycle , qaorta was the flow rate of the cardiac output . specially , rup was the ratio of the upper limb and brain blood supply to total blood supply .
they were defined as equations ( 4 ) and ( 5 ) . to evaluate the pulsatility of the flow rate , the harmonic index ( hi )
hi was a measure of the relative contribution of non - static intensity to the overall signal intensity , and this parameter ranges from 0 ( in the case of a steady nonzero flow rate signal ) to 1 ( in the case of a purely oscillatory signal with a time average of zero ) .
hi was dened as the equation ( 6 ) : where t[nwo ] was the magnitude of the transformed flow rate signal . to quantify the magnitude of flow oscillation during cardiac cycle , the oscillatory shear index ( osi )
was calculated as the equation ( 7 ) : where w was the wall shear stress , t was 1 cardiac cycle .
the osi value can vary from 0 to 0.5 , with 0 corresponding to a totally unidirectional wss and 0.5 to a purely oscillatory wss .
the ideal 3-dimensional geometry model with peripheral and cecmo ( figure 1 ) was constructed by using the commercial software solidworks .
the models consist of the ascending aorta , the innominate artery ( ia ) , left common carotid artery ( lcca ) , left subclavian artery ( lsa ) , left femoral artery ( lfa ) , right femoral artery ( rfa ) , and the cannula of ecmo .
the geometric characteristics of models are derived from the literature , as shown in table 1 . a standard arterial cannula ( 24 fr )
was added to the ascending aorta on the site that is routinely used in clinical practice ( figure 1b ) .
the cannular anastomotic position is about 2 cm below the inlet of the innominate artery on the anterior wall of the aorta , at a 45 angle to the x y plane .
both geometric models were meshed by using hypermesh commercial software to generate the computational models .
both models are meshed by using tetrahedral elements , in which there are 908 666 elements and 973 949 elements , respectively .
mesh independence tests were conducted for mass flow rate and the maximal error is within 5% ; the reasonable accuracy of the computing was confirmed .
the blood motion is governed by 3-dimensional navier - stokes equations : where u represents the blood velocity .
represents the blood density and f is the volume force field . in all simulation , the blood was set up as a homogeneous , incompressible , and newtonian fluid flow with =1020 kg / m and =0.0035kg / m.s . as the mean reynolds s number is less than 2300 , the blood flow here was assumed as laminar flow .
the finite volume method was used to solve navier - stokes equations by the transient simulation .
for studying the hemodynamic effects , the continuous flow of 4 l / min was used as the inlet boundary condition delivered from inlet cannula of both ecmo .
pulsatile flow rates ( average value 1 l / min ) , derived from a validated lumped parameter model ( lmp ) [ 2426 ] , were imposed on inlet of aorta as the inlet boundary conditions ( figure 2 ) .
thus , the total blood perfusion was 5 l / min , which satisfies the physical requirement .
the mean blood pressure ( 70 mmhg ) was imposed to the outlets of aorta as the outlet boundary condition .
to assess the difference of perfusion condition under both ecmo , blood flow rate ratios ( r ) of arterial bifurcations was defined as the equation ( 3 ) : where qwessel was the flow rate of the concerning vessel in one cardiac cycle , qaorta was the flow rate of the cardiac output . specially , rup was the ratio of the upper limb and brain blood supply to total blood supply .
they were defined as equations ( 4 ) and ( 5 ) . to evaluate the pulsatility of the flow rate , the harmonic index ( hi )
hi was a measure of the relative contribution of non - static intensity to the overall signal intensity , and this parameter ranges from 0 ( in the case of a steady nonzero flow rate signal ) to 1 ( in the case of a purely oscillatory signal with a time average of zero ) .
hi was dened as the equation ( 6 ) : where t[nwo ] was the magnitude of the transformed flow rate signal . to quantify the magnitude of flow oscillation during cardiac cycle , the oscillatory shear index ( osi )
was calculated as the equation ( 7 ) : where w was the wall shear stress , t was 1 cardiac cycle .
the osi value can vary from 0 to 0.5 , with 0 corresponding to a totally unidirectional wss and 0.5 to a purely oscillatory wss .
figure 3 shows the flow rate of all the outlets of the cases . figure 3a shows the mass flow rate of the pecmo .
figure 3b shows the mass flow rate of the cecmo . for both types of ecmo , when the blood is injected from left ventricle into the ascending aorta , the blood flow entering into ia was much higher than at other outlets .
the average flow rate values of ia and lfa under pecmo were higher than that of cecmo , but the rest of the outlets were the reverse .
in addition , for pecmo , rfa was almost blocked by the cannula inlet , so most of the downstream blood went to the lfa .
compared with cecmo , the average flow rate of lfa of pecmo was lower than the sum of average flow rate of lfa and rfa of cecmo(0.03067 < 0.03401 kg / s ) , but the sum average flow rate of ia , lcca , and lsa under pecmo were higher than that of cecmo ( 0.05458 > 0.05062 kg / s ) .
that is , in this study , pecmo has a better perfusion to the upper limb and brain than cecmo , while the downstream perfusion cecmo overshadowed pecmo .
table 3 shows the hi of all the flow rate waves of the 2 modes of ecmo .
the hi of flow rate of ia and lsa under cecmo was higher than that under pecmo , while hi of flow rate of lfa , lcca , and lsa under cecmo was lower .
figure 5 shows the velocity vector of the aorta arch and femoral braches at different times under both types of ecmos .
figure 5a and 5c show the flow pattern under pecmo ; similarly , figure 5b and 5d show the flow pattern under cecmo .
figure 5a shows the obvious blood interface ( the circle region ) at the aortic arch , due to the different direction of blood injected from heart and cannula of ecmo .
moreover , the location of the interface is changed along with the fluctuation of instantaneous blood flow rate from both inlets .
retrograde blood flow from the cannula of ecmo crash with the antegrade blood flow from left ventricle at the aortic arch forming the interface . for cecmo ,
the velocity of the blood injected from the cannula was much higher than in other regions ( figure 5b ) , which causes the vertexes ( the arrow location ) . at the femoral branch , as the high - speed blood flow is injected from the pecmo cannula , there are vertexes ( the arrow location ) . for cecmo , the blood flow in femoral
the results indicate that pecmo has worse hemodynamic conditions at the femoral bifurcation than that supported by cecmo , and there is blood flow junction also observed under pecmo .
figure 6a and 6c show the wss distribution under pecmo , while figure 6b and 6d show the wss distribution under cecmo . in the aortic arch ,
the wss under pecmo is significantly lower than that under cecmo , as the high velocity blood flow is injected from the cannula of cecmo .
in contrast , wss at the femoral branch was higher under pecmo compared with that under cecmo , as the blood flow jetted from lfa . moreover , the high wss region under both types of ecmo is different . for pecmo , the inner wall of the aortic arch and femoral branch were high wss regions ( reaching 5 pa and 10 pa , respectively ) , while the region appeared at the posterior wall of aortic arch for cecmo ( reaching 10 pa ) .
figure 7 illustrates the change in wss at special areas along with time during the whole cardiac cycle . at the inside wall of the aortic arch ( region 1 ) ,
in contrast , at the posterior wall of the aortic arch ( region 2 ) , wss under cecmo was higher than that under pecmo . for the femoral branch ( region 3 ) ,
table 4 lists the mean values of wss at regions 1 , 2 , and 3 under both types of ecmos .
figure 8b shows the distribution of osi under cecmo . for both types of ecmo ,
osi were high ( max value is 0.45 ) at the inner wall of the aortic arch , the descending aorta , and the femoral branch .
however , the high osi regions under both types of ecmo were different from each other . for pecmo ,
the high osi region was at the aortic arch closing with the ascending aorta and femoral branch .
for cecmo , however , the region was at the aortic arch closing with the descending aorta , showing that high osis were usually located in the regions where wss was low , which agrees with other studies .
for instance , chen et al . reported that ecmo had become an important method for organ transplants in clinical practice .
. reported that the ecmo is potentially a reliable bridge - to - diagnosis and bridge - to - decision in cardiac amyloidosis patients .
doctors could determine the optimal operation for the patients according to the treatment performance of ecmo .
. found that the short - term support by ecmo is a useful operation to improve the survival rate of heart failure patients .
mancio silva et al . reported that an experienced ecmo team , careful patient selection , and rigorous management protocols with objective criteria to wean or stop ecmo are needed for improving the performance of va ecmo clinical application .
similarly , religa et al . applied a short - term ecmo as a bridge - to - bridge helped save the patient from severe cardiogenic shock caused by increased left ventricular afterload .
along with va ecmo was widely applied in the clinical practice , the complications , including ischemia - reperfused injury , hypoxemia , hyper perfusion and vascular complication , attracted more and more attention .
although many studies focus on the hemodynamic effects of ecmo on cardiovascular system , there is no study on the hemodynamic differences between pecmo and cecmo .
this work focuses on the hemodynamic difference between the pecmo and cecmo and explores the relationship of hemodynamic factors and perfusion condition to provide some advice on reducing the risk of hemodynamic complications .
limb ischemia is a general complication of ecmo leading to limb loss even death in serious cases .
and the risk of limb ischemia caused by pecmo is higher than that caused by cecmo .
stated that 16.9% of patients develop lower extremity ischemia and 4.7% of patients have lower extremity amputation for pecmo .
distal perfusion catheters were used to improve this situation , while there is still 3.2% of limb ischemia .
in addition , the cerebral blood vessels and upper limbs are also at risk of hypoxemia for the patients undergoing pecmo , which receive predominantly deoxygenated blood from the left heart .
our results suggest that , pecmo has a better perfusion to the upper limb and brain than cecmo ( figure 4a ) , while for the downstream perfusion cecmo has advantage over pecmo ( figure 4b ) .
the results of this study are consistence with the previous literatures . and the difference in the distribution of blood flow in each vessel maybe result from the different types of ecmo . consequently , if the patient supported by the pecmo , the lower extremity ischemia is a complication that must be concerned . for heart mechanism ,
the ecmo , however , generated the nonpulsatile blood flow , which may have the negative effects on heart and aorta .
shown that va ecmo altered pulsatile blood ow and cerebral autoregulation have the effect on endothelial reactivity in animal models .
hi as index evaluating the pulsatility of the flow rate has been recommended that change the flow pattern from nonpulsatile flow to pulsatile flow .
mean and maximum values of wss were major factors for endothelial pathology and intimal disease .
adel et al . reported that arterial - level shear stress ( about 2 pa ) is suitable for maintaining the endothelial quiescence and an atheroprotective gene expression profile , while low shear stress ( < 0.4 pa ) that is prevalent at atherosclerosis - prone sites stimulates an atherogenic phenotype .
as seen in table 4 , for cecmo , the average wss of region 2 is much higher than 2 pa .
for pecmo , the average wss of region1 and region 2 is lower than 0.4 pa , while the average wss of region 3 is much higher than 2 pa .
there exist low shear stress ( < 0.4 pa ) at region 1 and region 2 under pecmo .
the osi of the aorta arch and access of femoral artery is higher than cecmo .
the duration of the ecmo was conducted was a very important factor to its effects on the vessel . for pecmo ,
the duration is usually one or two weeks . according to the study on the biomechanical effect of blood flow on the endothelial cell , the duration of pecmo was sufficient to active the endothelial function and result in the abnormal function of vessel
therefore , the abnormal distribution of wss under pecmo may contribute to the endothelial and vascular dysfunction .
in contrast , the cecmo usually was used in the cardiac surgery , which is usually less than 12 hours .
thus , the distribution of wss under cecmo may have little effects on the vascular disease .
moreover , according to hemodynamic theory , the distribution of wss is regulated by both the type and support level of ecmo .
hence , surgeons should carefully choose the appropriate type and support level of ecmo according to the states of patients .
meanwhile , surgeons should reduce the duration of ecmo as well to reduce the risk of endothelial and vascular dysfunction .
the present study is based on an ideal geometric model in which the parameters were from derived from the literature . because the differences among patients are huge , the ideal geometric model may not be sufficient to clarify the changes in hemodynamic effects of the 2 types of va ecmo . in the future
, the patient - specific models will be established and the realistic boundary conditions collected from clinical practice also would be used to study the hemodynamic differences under the 2 types of va ecmo support . in addition , the related statistical analysis would be conducted to provide guidance to surgeons .
the present study is based on an ideal geometric model in which the parameters were from derived from the literature . because the differences among patients are huge , the ideal geometric model may not be sufficient to clarify the changes in hemodynamic effects of the 2 types of va ecmo . in the future
, the patient - specific models will be established and the realistic boundary conditions collected from clinical practice also would be used to study the hemodynamic differences under the 2 types of va ecmo support . in addition , the related statistical analysis would be conducted to provide guidance to surgeons .
results demonstrated that pecmo had lower perfusion to lower limb than cecmo , which may contribute to the lower extremity ischemia .
in addition , the pecmo also has a higher wss at the bifurcation of the femoral artery , which may induce vascular complications . and for pecmo , osi of the aorta arch and access of femoral artery are higher than cecmo .
. these results may contribute to the findings that pecmo is prone to vascular complications .
moreover , for pecmo , there exists a flow junction in the aorta arch , and it may result in severe flow condition related to disease development .
in addition , pecmo has a lower hi than cecmo , and it decreases the pulsatility of the blood flow .
therefore , for peripheral emco , the lower - extremity ischemia is a complication that must be considered .
the type , support level , and duration of ecmo should also be carefully regulated according to the patients condition , as they are important factors related to vascular complications . | backgroundveno - arterial extracorporeal membrane oxygenation ( vaecmo ) , including central ecmo ( cecmo ) and peripheral ecmo ( pecmo ) , is widely used in cardiopulmonary surgery .
the outcomes and complications of both types of ecmo are quite different from each other .
the hemodynamic differences among them are hypothesized as a key factor .
hence , a numerical study was conducted to test this hypothesis.material/methodsideal cardiovascular models with pecmo and cecmo were established .
the aortic pressure and flow rate were chosen as boundary conditions .
the flow pattern , blood flow distributions , flow junction , harmonic index ( hi ) of blood flow , wall shear stress ( wss ) , and the oscillatory shear index ( osi ) were calculated to evaluate the hemodynamic states.resultspecmo could achieve better upper limb and brain perfusion ( 0.05458 vs. 0.05062 kg / s ) , and worse lower limb perfusion ( 0.03067 vs. 0.03401 kg / s ) .
there exist low wss ( < 0.4 pa ) regions at the inner and posterior wall of the aorta , and high wss ( > 2 pa ) region at the access of the femoral artery .
these regions also have relatively high osi value ( reaching 0.45 ) .
in contrast , for cecmo , there exist high wss at the posterior wall of the aortic arch.conclusionsthe hemodynamic performances of various types of ecmo are different from each other , which maybe the key reasons for the differences in the outcomes and complications .
therefore , for pemco , the lower - extremity ischemia is a complication that must be considered .
the type , support level , and duration of ecmo should also be carefully regulated according to the patients condition , as they are the important factors related to vascular complications . |
dystonia , one of the most prevalent forms of movement disorder , is defined as sustained or intermittent muscle contractions usually producing twisting and repetitive movement or abnormal posture ( 12 ) .
oral medications and botulinum toxin injections have been the mainstays of treatment for a time , but are not sufficiently effective in many patients .
internal globus pallidus ( gpi ) deep brain stimulation ( dbs ) has been widely accepted as an effective treatment modality of medically refractory dystonia ( 34 ) .
however , few studies have been reported regarding the safety issue of pregnancy and childbirth and its long term outcome of the gpi dbs during the pregnancies .
this report describes a female patient with generalized dystonia implanted with gpi dbs who delivered a baby during 84-month follow - up period after dbs surgery .
a 33-year - old female patient , a mother of one child , developed a left upper limb dystonia after being asphyxiated under blankets at the age of 4 weeks .
the dystonia in both lower limbs progressively developed into a generalized form , accompanied by a left facial spasm .
she had undergone orthopedic surgery because of right ankle eversion and lateral deviation of the right great toe 2 years ago .
the most painful symptom to her was a dystonia of left upper limb accompanied by tremor occurred in both resting and exercise .
the symptoms were not improved by medical treatment including nortriptyline , levodopa , clonazepam , baclofen , and biperiden .
the patient was referred to our institution for evaluation of the dbs ( november 14 , 2006 ) .
after orthopedic surgery , the right ankle was nearly corrected but the right great toe was still deviated laterally .
the left wrist was hyperextended , with continuing rhythmic clenching and opening of the fist ( fig .
the dystonia severity was measured using burke - fahn - marsden dystonia rating scale ( bfmdrs ) ; her preoperative movement score was 33 points , and functional disability score was 10 points .
mini - mental state examination ( mmse ) was 29 points , and beck s depression inventory ( bdi ) scale was 3 points .
on magnetic resonance imaging ( mri ) , there were no definite focal lesions in the brain parenchyma .
( a ) movie frames obtained from a preoperative video showing the patient lifting both arms and walking .
( b ) movie frame obtained from a postoperative video showing the same patient 7 years after deep brain stimulation ( dbs ) surgery . in november 2006 , she underwent bilateral gpi implantation .
a leksell stereotactic frame was secured to the patient s head after application of a local anesthetic and she was transferred to the mr suite .
the posteroventral portion of the gpi was targeted by means of axial , sagittal , and coronal mri images ( 5 ) .
the pallidal target was 12.4 mm anterior to the midcommisural point , 20 mm lateral to the midline , and 3 mm below the intercommisural line in both side .
the procedure was performed under general anesthesia , with the assistance of microelectrode recording ( mer ) .
a set of four microelectrodes ( differential microtargeting electrodes ; fhc , chemnitz , germany ; 1.5 m impedance ) were sequentially inserted toward the anatomical target within the gpi , which was vertically on the axial slice at the level of anterior commissure and horizontally at the junction between the two posterior quarters of the gpi ( 6 ) .
permanent dbs electrodes ( dbs 3387 ; medtronic , minneapolis , mn , usa ) placements were determined without interpreting vessels , ventricles , and sulci .
the electrode of the left side was inserted earlier than that of the right side , to minimize the error of the dominant side by brain shifting after cerebrospinal leakage .
the electrodes were fixed to the burr hole and connected to pulse generators ( ipg , soletra 7426 ; medtronic ) implanted on both subclavicular pouch .
one day after surgery , stimulation was begun using an nvision programmer ( medtronic ) .
the initial setting was as follows : monopolar stimulation by using contact 1 as the negative and the internal pulse generator case as the positive pole with an amplitude of 3.72 v , pulse width 60 sec , and frequency 130 hz .
six months later , we performed a repeat computed tomography ( ct ) scan and fused it to the preoperative mri to confirm the locations of the leads ( fig .
( a ) postoperative magnetic resonance imaging ( mri ) scans demonstrating the bilateral deep brain stimulation electrodes in the posteroventral internal globus pallidus ( gpi ) axial fluid - attenuated inversion recovery ( axial flair ) .
( b ) postoperative assessment of implanted electrodes by image fusion of a postoperative computed tomography ( ct ) scan with the corresponding preoperative inversion - recovery image .
the movement and disability scores and the stimulation parameters were recorded , as described in tables 1 and 2 .
three months later , she showed improvement in dystonic dyskinesia , but facial dyskinesia on left eyelid was still noted .
her movement score was decreased to 29 points and disability score to 5 points , but karnofsky performance scale ( kps ) score was still checked as 80 points at 6-month follow - up .
she found it easier to clean , but still had difficulty in washing . at 12-month follow - up
, the movement score decreased to 28 points ( improvement rates , 15% ) and disability score to 5 points ( improvement rates , 50% ) were checked , and she had sustained such improved state .
the maternal serum triple test and amniocentesis were performed because of her old age , and revealed low risk of congenital anomaly .
after induction using thiopental and succinylcholine , intubation was done quickly , followed by dbs turn off .
the surgery lasted for one hour , and the blood pressure was maintained within the normal range during the surgery . for hemostasis , only bipolar electrocautery was used . before awakening from the anesthesia ,
after delivery , she could feed her baby by herself , because the dystonia of left upper extremity and hand was improved .
six years after dbs surgery , her general condition was more improved , and she started to play ping - pong and billiards .
until now , she has been showing continual improvement and being good at housework , carrying for children , with no trouble in daily life based on her kps score of 90 points and mmse score of 30 points .
she even obtained a driver s license in april 2014 and now drives a car by herself .
a 33-year - old female patient , a mother of one child , developed a left upper limb dystonia after being asphyxiated under blankets at the age of 4 weeks .
the dystonia in both lower limbs progressively developed into a generalized form , accompanied by a left facial spasm .
she had undergone orthopedic surgery because of right ankle eversion and lateral deviation of the right great toe 2 years ago .
the most painful symptom to her was a dystonia of left upper limb accompanied by tremor occurred in both resting and exercise .
the symptoms were not improved by medical treatment including nortriptyline , levodopa , clonazepam , baclofen , and biperiden .
the patient was referred to our institution for evaluation of the dbs ( november 14 , 2006 ) .
after orthopedic surgery , the right ankle was nearly corrected but the right great toe was still deviated laterally .
the left wrist was hyperextended , with continuing rhythmic clenching and opening of the fist ( fig .
the dystonia severity was measured using burke - fahn - marsden dystonia rating scale ( bfmdrs ) ; her preoperative movement score was 33 points , and functional disability score was 10 points .
mini - mental state examination ( mmse ) was 29 points , and beck s depression inventory ( bdi ) scale was 3 points .
on magnetic resonance imaging ( mri ) , there were no definite focal lesions in the brain parenchyma .
movie frames obtained from preoperative and postoperative video . ( a ) movie frames obtained from a preoperative video showing the patient lifting both arms and walking .
( b ) movie frame obtained from a postoperative video showing the same patient 7 years after deep brain stimulation ( dbs ) surgery .
a leksell stereotactic frame was secured to the patient s head after application of a local anesthetic and she was transferred to the mr suite .
the posteroventral portion of the gpi was targeted by means of axial , sagittal , and coronal mri images ( 5 ) .
the pallidal target was 12.4 mm anterior to the midcommisural point , 20 mm lateral to the midline , and 3 mm below the intercommisural line in both side .
the procedure was performed under general anesthesia , with the assistance of microelectrode recording ( mer ) .
a set of four microelectrodes ( differential microtargeting electrodes ; fhc , chemnitz , germany ; 1.5 m impedance ) were sequentially inserted toward the anatomical target within the gpi , which was vertically on the axial slice at the level of anterior commissure and horizontally at the junction between the two posterior quarters of the gpi ( 6 ) .
permanent dbs electrodes ( dbs 3387 ; medtronic , minneapolis , mn , usa ) placements were determined without interpreting vessels , ventricles , and sulci .
the electrode of the left side was inserted earlier than that of the right side , to minimize the error of the dominant side by brain shifting after cerebrospinal leakage .
the electrodes were fixed to the burr hole and connected to pulse generators ( ipg , soletra 7426 ; medtronic ) implanted on both subclavicular pouch .
one day after surgery , stimulation was begun using an nvision programmer ( medtronic ) .
the initial setting was as follows : monopolar stimulation by using contact 1 as the negative and the internal pulse generator case as the positive pole with an amplitude of 3.72 v , pulse width 60 sec , and frequency 130 hz .
six months later , we performed a repeat computed tomography ( ct ) scan and fused it to the preoperative mri to confirm the locations of the leads ( fig .
( a ) postoperative magnetic resonance imaging ( mri ) scans demonstrating the bilateral deep brain stimulation electrodes in the posteroventral internal globus pallidus ( gpi ) axial fluid - attenuated inversion recovery ( axial flair ) .
( b ) postoperative assessment of implanted electrodes by image fusion of a postoperative computed tomography ( ct ) scan with the corresponding preoperative inversion - recovery image .
the movement and disability scores and the stimulation parameters were recorded , as described in tables 1 and 2 . after dbs implantation , considerable improvement was noted .
three months later , she showed improvement in dystonic dyskinesia , but facial dyskinesia on left eyelid was still noted .
her movement score was decreased to 29 points and disability score to 5 points , but karnofsky performance scale ( kps ) score was still checked as 80 points at 6-month follow - up .
she found it easier to clean , but still had difficulty in washing . at 12-month follow - up
, the movement score decreased to 28 points ( improvement rates , 15% ) and disability score to 5 points ( improvement rates , 50% ) were checked , and she had sustained such improved state .
the maternal serum triple test and amniocentesis were performed because of her old age , and revealed low risk of congenital anomaly .
after induction using thiopental and succinylcholine , intubation was done quickly , followed by dbs turn off .
the surgery lasted for one hour , and the blood pressure was maintained within the normal range during the surgery . for hemostasis , only bipolar electrocautery was used . before awakening from the anesthesia , dbs
after delivery , she could feed her baby by herself , because the dystonia of left upper extremity and hand was improved .
six years after dbs surgery , her general condition was more improved , and she started to play ping - pong and billiards . until now
, she has been showing continual improvement and being good at housework , carrying for children , with no trouble in daily life based on her kps score of 90 points and mmse score of 30 points .
she even obtained a driver s license in april 2014 and now drives a car by herself .
this report is one of the rare reports on gpi dbs for dystonia with long - term follow up over 7 years .
dystonia can be classified according to the involved body distribution : focal , segmental , multifocal , generalized , and hemidystonia , or according to the etiology : inherited dystonia of proven genetic origin , acquired dystonia with a known specific cause ( e.g. , perinatal brain injury , infection , drugs , toxicity , vascular , neoplastic , or brain injury ) , and idiopathic dystonia of unknown cause .
previous reports have shown promising results of gpi dbs especially in the patients diagnosed as primary generalized dystonia ( pgd ) with dyt-1 positive , focal , and tardive dystonia ( 78910 ) .
the patient was diagnosed as a patient with acquired dystonia that has been known to show less response to gpi dbs ( 111213 ) . however , vercueil et al . ( 14 ) reported a few cases with secondary dystonia showing successful response .
( 15 ) also reported that gpi dbs was useful in some secondary dystonia patients , but many patients diagnosed as tardive dystonia and hallervorden - spatz disease ( hsd ) were included into their secondary dystonia group .
since march 2005 , the author experienced 12 cases of acquired dystonia including this patient .
the improvement rate of the patients diagnosed as acquired dystonia was about 29% at 12-month follow up according to our unpublished study . although the improvement rate was not high as much as those of pgd with or without dyt-1 positive , the patients diagnosed as acquired dystonia also gained benefit from gpi dbs .
the reason for favorable outcome in this patient might be an absence of the structural abnormality on preoperative mri .
mer facilitates the selection of the final target in dbs in our experience , although some authors reported no benefit from mer ( 16 ) .
all procedures of gpi dbs for dystonia were performed under general anesthesia because of the patients abnormal posture and muscle contractions .
the typical bursting pattern could be identified , whereas amplitude was decreased and bursting pattern was emphasized more than in awaken surgery .
one notable finding was that this patient revealed improved outcome despite the bilateral electrodes located in the external globus pallidus ( gpe ) , as shown in fig .
another assumption is that the gamma - aminobutyric acid - ergic ( gabaergic ) pathway from striatum to gpi could be stimulated .
few authors reported several cases about pregnancy and delivery in patients who underwent dbs surgery .
( 17 ) reported three women who were pregnant and had babies by vaginal delivery after bilateral gpi dbs surgery .
these women showed no worsening of dystonic symptoms during the pregnancy and labor periods , and had no problems in breast feeding .
there is no evidence of that the pregnancy and delivery should not be allowed to the patients who underwent dbs implantation .
gwinn - hardy et al . ( 18 ) tried to figure out the correlation between hormones and dystonia in 279 female patients , but they found no clear - cut relationship between pregnancy , menopause , postmenopausal hormone replacement therapy , and worsening of dystonic symptoms .
if the surgeon is careful not to use short wave ( around 2,727 mhz ) , microwave ( 2.45 ghz ) diathermy , or therapeutic ultrasound ( 15 mhz ) diathermy , delivery can be performed safely .
the bipolar electrocautery should be used at least 15 cm away from the ipg device , extension cable , and lead ( 17 ) . as the indication for dbs increases
a standardized protocol for dbs implanted patients who undergo pregnancy , delivery , and surgery irrelevant to neurosurgery would be required . in conclusion ,
favorable outcomes could be expected even for the patients with not only pgd , but also acquired dystonia . | internal globus pallidus ( gpi ) deep brain stimulation ( dbs ) has been widely accepted as an effective treatment modality of medically refractory dystonia .
however , there have been few studies regarding the safety issue of pregnancy and childbirth related with dbs .
this report describes a female patient who was pregnant and delivered a baby after gpi dbs surgery . a 33-year - old female patient with acquired generalized dystonia underwent bilateral gpi dbs implantation .
she obtained considerable improvement in both movement and disability after dbs implantation .
four years later , she was pregnant and the obstetricians consulted us about the safety of the delivery . at 38-weeks into pregnancy , a scheduled caesarian section was carried out under general anesthesia .
after induction using thiopental and succinylcholine , intubation was done quickly , followed by dbs turn off . for hemostasis ,
only bipolar electrocautery was used . before awakening from the anesthesia ,
dbs was turned on as the same parameters previously adjusted .
after delivery , she could feed her baby by herself , because the dystonia of left upper extremity and hand was improved . until now
, she has been showing continual improvement and being good at housework , carrying for children , with no trouble in daily life .
this observation indicates that the patients who underwent dbs could safely be pregnant and deliver a baby . |
direct antiviral agent ( daa ) has been the standard of care for patients with hepatitis c virus ( hcv ) infection .
twelve weeks of paritaprevir / ritonavir / ombitasvir plus dasabuvir ( prod ) with or without ribavirin has shown to have a sustained virological response at post - treatment 12 weeks ( svr12 ) rate of > 90% in hcv genotype 1 ( hcv-1 ) patients .
it may attribute to low baseline viral loads and extraordinarily rapid suppression of hcv after treatment day1 .
the finding may shed light for possible response - guided - therapy for so - called ultra - super - responders in the daa era .
whether the dengue virus , the flaviviridae family as with hcv , enhanced the hcv clearance remains unclear and needs further exploration .
direct antiviral agent ( daa ) has been the standard of care for patients with hepatitis c virus ( hcv ) infection .
the mainstream treatment duration are 12 to 24 weeks depending on regimens and patient characteristics .
paritaprevir / ritonavir / ombitasvir plus dasabuvir ( prod ) with or without ribavirin has shown to have a sustained virological response at post - treatment 12 weeks ( svr12 ) rate of > 90% in hcv genotype 1 ( hcv-1 ) patients .
in addition to the development of pangenotypic , high potency daas , efforts now are made trying to abbreviate treatment duration to 4 to 8 weeks without compromising efficacy .
the 81-year - old female had the history of chronic hcv infection for > 30 years and co - morbidity of depressive disorder and dementia under regular treatment with quetiapine 50 mg / day .
due to fear of adverse events and underlying psychiatric disorder , she was ineligible to pegylated interferon / ribavirin , the current reimbursed regimen in taiwan .
the genotype and pretreatment viral loads was hcv-1b and 17,019 iu / ml ( real - time hcv ; abbott molecular , des plaines il ; detection limit : 12 iu / ml ) .
the result of the acoustic radiation force impulse ( arfi ) was 1.3 m / s , which corresponded to fibrosis stage 2 .
daily fixed - dose combination of paritaprevir / ritonavir / ombitasvir ( 150 mg/100 mg/25 mg ) plus twice - daily dasabuvir ( 250 mg ) was administered for the treatment of chronic hcv infection .
the hcv ribonucleic acid ( rna ) became rapidly undetectable on day 1 , followed by day 7 and day 14 after dosing .
unfortunately , she was admitted due to fever , progressive thrombocytopenia , and elevated liver enzymes .
dengue fever infection with dengue virus genotype 2 ( sybr green - based quantitative rt - pcr ) infection was impressed on day 25 of treatment .
she received supportive treatment with antipyretics ( paracetamol 500 mg / time as needed ) and intravenous fluids supply during hospitalization .
dramatically , the serial testing of serum hcv rna showed that the patient achieved svr4 , svr8 , and svr12 ( table 1 ) .
to our knowledge , this is the first report of daa prescription that encountered dengue fever , and the patient benefited from hcv eradication only by 4 weeks of all oral daa regimens .
the current evidence support 12 to 24 weeks of interferon - free daas in treating chc .
an 8-week sofosbuvir / ledipasvir is recommended for naive , noncirrhotic hcv-1 patients with baseline viral loads < 8 million iu / ml . as observed in a phase 2b study that 8 weeks of prod plus ribavirin had very high svr rates for naive , noncirrhotic patients of hcv-1b ( 96% , 23/24 ) or with low viral loads ( < 800,000 iu / ml , 100% , 9/9 ) .
the achievement of svr to only 25 days of prod in this patient may attribute to hcv-1b with low baseline viral loads and extraordinarily rapid suppression of hcv after treatment day 1 . as with interferon - based therapy ,
the finding may shed light for possible response - guided - therapy for so - called ultra - super - responders in the daa era
whether the dengue virus , the flaviviridae family as with hcv , enhances the hcv clearance remains unclear and needs further exploration . | abstractbackground : direct antiviral agent ( daa ) has been the standard of care for patients with hepatitis c virus ( hcv ) infection .
twelve weeks of paritaprevir / ritonavir / ombitasvir plus dasabuvir ( prod ) with or without ribavirin has shown to have a sustained virological response at post - treatment 12 weeks ( svr12 ) rate of > 90% in hcv genotype 1 ( hcv-1 ) patients.methods:we report a hcv-1b patient who received only 25 days of prod treatment.results:the patient early terminated treatment due to dengue fever but eventually achieved svr12 .
it may attribute to low baseline viral loads and extraordinarily rapid suppression of hcv after treatment day1.conclusions:the finding may shed light for possible response - guided - therapy for so - called ultra - super - responders in the daa era .
whether the dengue virus , the flaviviridae family as with hcv , enhanced the hcv clearance remains unclear and needs further exploration . |
a 67-year - old woman presented with a 10-day history of localized zoster in the right occipital area .
skin rash developed on the right occipital area of the second and third cervical dermatome .
three days after the appearance of the rash , she was diagnosed with hz by a dermatologist and was prescribed 5 mg of oxycontin twice a day , 650 mg of acetaminophen twice a day , 75 mg of pregabalin twice a day , and 500 mg of famciclovir 3 times a day , for 7 days , respectively . in spite of the 7-day administration of these medications , her pain was rated at an intensity of 7 on the visual analogue scales ( vas ) from 0 ( no pain ) to 10 ( worst pain imaginable ) at first visit to our department .
she suffered from constant aching pain along with intermittent , spontaneous , sore and shooting pain over the right occipital area , which was provoked by brushing .
therefore , right stellate ganglion block ( sgb ) using 7 ml of 1% lidocaine with ultrasound was performed but it did not reduce the symptoms .
30 minutes after sgb , 2 g of vitamin c was administered intravenously , but it also did not alleviate her pain . on the second day , a second attempt of right sgb was performed without any reduction in pain .
30 minutes after sgb , 4 g of vitamin c was administered intravenously , and then , the patient reported immediate pain relief from a vas of 7 to 2 . during the first 12 hours
, intermittent shooting pain and constant aching pain were maintained at a reduced intensity , but constant aching pain increased to a vas of 5 again after 12 hours , while shooting pain remained constant at a vas of 2 . on the third day after right sgb , the pain did not decrease in intensity .
but 30 minutes after sgb , 4 g of vitamin c administered intravenously sequentially reduced the constant aching pain from a vas of 5 to 2 , which was maintained for about 12 hours .
however , there was no intermittent shooting pain after the administration of the vitamin c on the third day . on the fourth day ,
right sgb and sequential intravenous injection of 4 g of vitamin c was done just like before . immediately after the administration of the vitamin c
, she rated her pain intensity from a vas of 4 to 1 , which was maintained for about 12 hours .
on the fifth day , intravenous injection of 4 g of vitamin c was done without sgb . immediately after the administration of the vitamin c
, she rated her pain intensity from a vas of 4 to 0 . since then , her pain intensity has been maintained at a vas of 0 - 1 .
the administration of 5 mg of oxycontin twice daily and 650 mg of acetaminophen twice daily was stopped .
then she was discharged with a prescription for 75 mg of pregabalin twice a day and 1 g of vitamin c twice a day .
five days after taking the pregabalin and vitamin c , she reported a complete resolution of the pain and stopped taking the medication . at 3 months follow - up
herpes zoster is a result of the reactivation of the latent varicella - zoster virus within the dorsal root ganglia ( drg ) or cranial nerve ganglia under various conditions related to a decrease in cell - mediated immunity .
replication of the virus results in nerve injury and produces debilitating pain preceding the skin eruptions such as rashes or vesicles in the corresponding dermatome .
the most common complication of hz is phn , which is defined as pain persisting for more than a month after healing of the rashes from acute hz .
phn is notoriously difficult to treat and often is accompanied by physical and social disabilities and even psychological distress .
the genesis of the pain during acute hz is thought to be from inflammation and damage to the drg and peripheral nerves .
the inflammatory changes in the drg can reduce intraneural blood flow , leading to hypoxia and endoneurial edema .
this process finally causes neural injuries that can lead to the development of neuropathic pain .
in addition , inflammatory changes in the dorsal horn produce nociceptor excitation and sensitization that cause central hyperexcitability . in other words , uncontrolled persistent pain in acute phase
may finally lead to chronic neuropathic pain ; therefore , faster resolution of inflammation and pain is important .
the incidence of phn increases with the increasing age of the patient . in this case , the 67-year - old patient was at risk of developing pnh .
in addition to the drug therapies , sgb was tried for symptomatic relief in the acute phase of hz and for the prevention of phn .
pregabalin medication has been shown to decrease significantly postherpetic neuralgia after the first day of treatment .
our patient reported pain relief immediately after intravenous administration of vitamin c despite no relief after 7-days of administrating pregabalin and other drugs .
therefore , we presume that the vitamin c might be responsible for the pain relief .
a community - based case control study revealed that those with low vitamin c intake were significantly at higher risk for hz .
plasma vitamin c concentrations have been suggested to be related to pain modulation for intractable phn .
in addition , vitamin c has been reported to reduce the prevalence of complex regional pain syndrome ( crps ) after foot and ankle surgery .
therefore , it has been proposed that it may be beneficial to supply and increase plasma concentrations of vitamin c for patients at high risk for crps .
the mechanisms of neuropathic pain like zoster - associated pain and crps include neuroinflammation , central sensitization , disinhibition , and reactive oxygen species ( ros ) .
recent studies have suggested that ros which are produced from peripheral inflammation will sensitize nociceptors so that they not only respond more vigorously to noxious stimuli but also start to respond to normally subthreshold stimuli .
this peripheral sensitization not only induces pain directly , but also induces central sensitization in the spinal cord , which indirectly contributes to pain as well .
ros which result from persistent abnormal afferent inputs produced in the spinal cord can lead to central sensitization , which in turn produces pain .
in other words , ros have been suggested to contribute to the development and maintenance of neuropathic pain that can be relieved by systemic injection of ros scavengers .
therefore , an ros scavenger such as vitamin c is suggested to be neuroprotective by scavenging excess ros .
vitamin c is an extracellular and intracellular antioxidant but also a major antioxidant in csf , and its effect is concentration dependent .
recently , previous reports showed that short - term intravenous administration of high - doses of vitamin c helped to reduce the pain in patients with phn and to treat patients with hz .
it was reported that 2.5 g of intravenous vitamin c reduced pain in a 78-year - old man with pnh .
in addition , 15 g of intravenous vitamin c was efficient in the treatment of two patients ( females aged 67 and 53 years ) with hz .
in this case , intravenous administration of 4 g of vitamin c was effective to reduce the pain in our patient with hz . in conclusion
, vitamin c may be an efficient adjuvant for multi - drug regimens to control pain in patients with hz .
formal studies are required to determine whether treatment with vitamin c may prove useful in patients with hz . | herpes zoster as a result of reactivated varicella - zoster virus is characterized by vesicular eruptions on skin and painful neuralgia in the dermatome distribution .
pain during an acute phase of herpes zoster has been associated with a higher risk of developing postherpetic neuralgia .
the current therapies for herpes zoster including analgesics and sympathetic nerve block as well as antiviral agents are important to alleviate pain and prevent postherpetic neuralgia .
however , in some cases , the pain does not respond well to these treatments .
we had a case in which a patient with herpes zoster did not respond to conventional therapy so we attempted to administer intravenous infusion of vitamin c which resulted in an immediate reduction in the pain . |
metabolic syndrome ( ms ) is a recently coined term for the designation of an aggregation of risk factors including visceral obesity , arterial hypertension , hyperglycemia , and dyslipidemia which in conjunction augment the probabilities of developing type 2 diabetes mellitus ( dm2 ) and cardiovascular disease ( cvd ) .
place the prevalence of ms in latin america at 24.9% in a previous meta - analysis .
meanwhile , in our country , the carmela study finds the city of barquisimeto to be parallel to mexico city , boasting the highest prevalence of ms in latin america in 2009 . in consequence ,
given its prominent morbidity and its importance in the ethiopathogenics of cvd , which in turn represents the main cause of mortality at a worldwide , national , and regional level [ 46 ] , the ms has been the object of numerous investigations focused on the search of associations with new risk factors , both in general and relating to each of its specific separate components . in this sense
, alterations linked to plasma lipoproteins , especially those regarding low - density lipoproteins ( ldl - c ) are particularly notable within the physiopathologic aspects of ms , showcasing its genetic implications .
therefore , in addition to protein molecules such as high sensitivity c - reactive protein ( hs - crp ) , homocysteine , and fibrinogen , lipoprotein(a ) [ lp(a ) ] represents a substantial target in the analysis of novel risk factors .
lp(a ) was initially isolated from human plasma by berg in 1963 , constituted by the association of an ldl - c particle covalently bound to a large glycoprotein , apolipoprotein(a ) [ apo(a ) ] to apolipoprotein b by a disulfide bridge .
the apo(a ) chain contains five cysteine - rich domains known as kringles , which are coded by a gene localized in the long arm of chromosome 6 ( 6q26 - 27 ) and is subject to multiple polymorphisms , particularly regarding the size of kringle iv [ 10 , 11 ] . in turn
, this feature characterizes the different isoforms of lp(a ) and is inversely associated with plasma lp(a ) levels .
these variations are outstandingly marked among races , as illustrated by the remarkably higher plasma lp(a ) concentrations in afrodescendants .
clinical interest in lp(a ) has grown exponentially in recent times , as an assortment of epidemiological studies has pinpointed the link between plasma lp(a ) concentrations ( reported as 300 mg / l or 30 mg / dl ) and the risk of suffering coronary events , peripheral artery disease , cerebrovascular disease , and the early development of atherosclerosis in children and adolescents [ 13 , 14 ] . despite this prominence , the interpretation and application of lp(a ) levels in clinical scenarios
remain a controversial issue , since no guidelines have been suggested outlining the profiles of patients whose lp(a ) concentration should be quantified . as a result , experimental studies are required for the clarification of its role as a cvd risk factor , as well as epidemiological studies evaluating the behavior of its plasma levels regarding other cvd risk factors across different latitudes in order to effectively direct genetic studies focused on highlighting the true role of the genetic intricacies underlying the greater variations reported among demographics .
stemming from this , along with the scarcity of great - scale studies detailing the epidemiological behavior of lp(a ) in latin america , the main objective of this research was to assess the influence of its plasma levels in the ms and its individual components in adult individuals in the city of maracaibo , venezuela .
the study was approved by the ethics committee of the endocrine and metabolic diseases research center .
the sample method has been already published in the maracaibo city metabolic syndrome prevalence study cross - sectional proposal , yet the main aspects will be mentioned .
it was a cross - sectional , descriptive , randomized , multistaged study which enrolled a total of 2,230 subjects .
for this research , 1,807 subjects were studied , representing the randomly selected subsample which had their serum lp(a ) concentrations quantified .
a full medical history was obtained using the venezuelan popular powers health ministry approved medical chart filled out by trained personnel . for the measurement of blood pressure ( bp ) ,
patients were sitting and at rest for a minimum of 15 minutes , with their feet on the ground and the arm used for the measurement at the level of the heart .
regarding anthropometric evaluation , waist circumference values were determined employing a tape measure graduated in centimeters and millimeters ( cm , mm ) , placing it at a point equidistant to the costal margin and the anterior superior iliac spine . for the diagnosis of ms ,
the criteria from the idf / aha / nhlbi / whf / iaso-2009 consensus were applied , and american diabetes association criteria were used for the definition of metabolic alterations concerning glycemic status .
serum levels of glucose , total cholesterol , tag , and hdl - c were determined employing commercial enzymatic - colorimetric kits ( human gesellschaft fr biochemica and diagnostica mbh ) and specialized computerized equipment .
ldl - c levels were calculated through friedewald 's formula , and its adjustment based on lp(a)-bound cholesterol [ lp(a)-c ] applying dahlen 's formula [ ldl - c = tc hdl - c vldl - c lp(a)-c ] [ 20 , 21 ] .
lp(a ) was estimated through the latex turbidimetric method , human gesellschaft fr biochemica and diagnostica , germany . in this method ,
the presence of lp(a ) in the sample causes agglutination of latex particles coated with antibodies against lp(a ) , the agglutination is proportional to the lp(a ) concentration in the sample and can be measured by turbidimetry .
the cut - off value for the consideration as elevated lp(a ) levels was 30 mg / dl .
likewise , serum hs - crp levels were quantified employing immunoturbidimetric essays ( human gesellschaft fr biochemica and diagnostica mbh ) , and basal insulin levels were determined after 8 to 12 hours of fasting using drg international inc .
insulin kits . for the evaluation of insulin resistance ( ir ) , the homa2-ir model proposed by levy et al . was utilized , determined through the homa - calculator v2.2.2 program .
normal distribution of continuous variables ( or lack thereof ) was evaluated by using kolmogorov - smirnov ( when n < 500 ) or geary 's ( when n 500 ) test , accordingly .
for normally distributed variables , the results were expressed as arithmetic mean sd ( standard deviation ) .
differences between arithmetic means were assessed using student 's t - test ( when two groups were compared ) or anova ( when three or more groups were compared ) .
qualitative variables were expressed as absolute and relative frequencies , considering the results statistically significant when p < 0.05 in the z test for proportions or test when applied .
likewise , logistic regression models were designed , estimating odds ratios ( ic 95% ) .
the first model estimated odds ratios ( ors ) for elevated lp(a ) adjusted by gender , ethnic groups , age groups , and diagnostic criteria for metabolic syndrome and hs - crp tertiles ( tertile 1 : < 0.25 , tertile 2 : 0.250.61 , tertile 3 : 0.62
the same covariates were employed , with the addition of the glycemic status and ldl - c tertiles ( tertile 1 : < 100.67 , tertile 2 : 100.67131.99 , tertile 3 : 132.0
a third model was constructed using corrected ldl - c tertiles ( tertile 1 : < 93.2 , tertile 2 : 93.2123.61 , tertile 3 : 123.62 mg / dl ) .
lastly , a fourth model includes risk factors for metabolic syndrome and is adjusted for gender , ethnic groups , age groups , hs - crp tertiles , ldl - c tertiles , and lp(a ) classification by reference intervals previously reported for our population and a tertile model for corrected ldl - c . the database analyses were performed using the statistical package for the social science ( spss ) v. 19 for windows ( ibm inc .
chicago , il , usa ) , considering significant results as values p < 0.05 .
general characteristics of the studied population are presented in table 1 , while anthropometric and laboratory variables are observed in table 2 .
a total of 1,807 subjects were studied , of which 55.3% ( n = 999 ) belonged to the female gender and 44.7% ( n = 808 ) to the male gender .
the mean values and percentile distribution of serum lp(a ) concentration in the general population and by gender are presented in table 3 .
no differences were found when comparing males and females , resembling the behavior of homa2-ir , insulin , and hs - crp concentration . regarding distribution of subjects with elevated lp(a )
levels , 51.2% ( n = 339 ) presented a diagnosis of ms , in contrast to the proportion of individuals with normal lp(a ) levels : 38.3% ( n = 439 ) ; p < 0.05 . the association between the presence of ms and this lipid alteration was found to be significant ( = 28.33 ; p < 0,0001 ) ( figure 1 ) . when analyzing the behavior of the serum lp(a ) concentration according to presence of ms , individuals with the diagnosis appeared to have higher levels than those without the diagnosis ( with ms : 29.16 13.19 versus without ms : 26.09 11.84 mg / dl ; p = 1.19 10 ) .
moreover , in figure 2 a progressive increase in lp(a ) levels was observed as the number of criteria for ms rose , with values 24.54 12.07 mg / dl in subjects without any criteria , ascending to 28.95 12.78 mg / dl in subjects with all criteria . in the specific analysis of the components of ms ,
a similar behavior was observed for all criteria except elevated glycemia : lp(a ) concentrations were greater in subjects with each component when comparing individuals with and without each of the criteria ( table 4 ) .
furthermore , subjects with hypertriacylglyceridemia displayed the most elevated lp(a ) levels ( 29.57 13.02 mg / dl ) , and the greatest mean difference was found when comparing subjects with and without a high waist circumference .
lp(a ) levels in the general population and for each gender according to the different specific diagnostic combinations for the ms are shown in table 5 .
the greatest values were exhibited by subjects with the high basal glucose - low hdl - c - hypertriacylglyceridemia combination ( 36.96 29.85 mg / dl ) . when comparing the means between genders ,
the sole statistically significant difference was found in subjects with the high waist circumference - high blood pressure - hypertriacylglyceridemia - low hdl - c combination , displaying higher serum lp(a ) concentrations in women ( 34.42 11.69 versus 26.92 11.52 mg / dl ; p = 0.004 ) .
the main risk factors for presenting elevated lp(a ) concentrations were initially determined in the multivariate analysis ( table 6 ) . in model 1 , age , hypertriacylglyceridemia , hs - crp , and elevated basal glycemia
were the variables with statistical significance , where subjects aged 60 years or older presented the highest risk estimation ( or : 3.91 ; ic 95% : 1.977.76 ; p < 0.01 ) , while elevated basal glycemia behaved as a protecting factor ( or : 0.73 ; ic 95% : 0.540.98 ; p = 0.04 ) .
stemming from this , in model 2 the adjustment included ldl - c tertiles and the specific glycemic status of subjects , amongst which individuals with impaired fasting glucose ( ifg ) had the lowest risk of presenting elevated lp(a ) levels ( or : 0.69 ; ic 95% : 0.480.98 ; p = 0.04 ) ; this pattern is still observed after the adjustment of ldl - c to lp(a)-c in the resultant tertiles .
furthermore , the main metabolic risk factors for ms are analyzed in table 7 , unveiling subjects classified in the highest ldl - c or hs - crp tertiles to be the most associated with the diagnosis of ms , while individuals categorized in the normal interval for lp(a ) in our population displayed the lowest risk of presenting ms ( or : 0.65 ; ic 95% : 0.450.94 ; p = 0.03 ) ; after the ldl - c adjustment , the risk remains in a similar manner .
the proportion of individuals affected by the ms worldwide shows the current pandemic magnitude of this endocrine - metabolic disorder , reaching prevalence figures as high as 40% in our city as contemplated by our research group ( unpublished data ) , similar to the values obtained in this report ( 47% ) . due to this
, it has become a necessity to identify new risk factors involved in the physiopathology of ms , which may serve as predictors of its onset and as new therapeutic targets which may in turn be linked to the development of cardiovascular events . as a component of ms
, dyslipidemia represents one of the fundamental pillars in its ethiopathogenics , being directly related to the degree of ir and representing a series of molecular disturbances comprising the increase of the serum concentrations of apolipoprotein b , ldl - c , and vldl - c , as well as an augmented flux of free fatty acids . in the clinical setting , these disorders translate into the widely known criteria for elevated tag and low hdl - c .
furthermore , these lipid alterations are intimately associated with a chronic inflammatory state , which represents the essential mechanism from which atherosclerosis and cvd stem [ 29 , 30 ] .
based on these premises , dyslipidemia , and inflammation , lp(a ) plays an important role at the molecular level both for cvd and ms when its plasmatic concentration is elevated , being able to generate both of the aforementioned basic disturbances [ 3133 ] .
a great deal of these studies have been executed in european and asian populations , showing proportions of individuals with ms and high lp(a ) similar to ours , with prevalence figures as elevated as 51.4% in a small turkish study .
it is important to highlight the lack of differences of lp(a ) levels between genders in this report , as has been outlined in previous investigations [ 31 , 35 ] ; therefore , most comparisons were done utilizing the general population . exhibiting a qualitative association with lp(a )
, subjects with ms also showed higher levels than healthy subjects , similar to the results of bozba et al .
. nevertheless , this behavior differs from that described for older japanese adults , whose plasmatic concentrations were not statistically different .
notably , notwithstanding the escalating tendency of lp(a ) levels as the number of criteria increased , it is not the amount of criteria expressed but the actual diagnosis of ms that appears relevant regarding the presence of elevated lp(a ) concentrations . with reference to the analysis by individual diagnostic criteria ,
previous studies evaluating the relationship between lp(a ) and the isolated components ms are not abundant , and very few include all criteria in their analyses [ 3740 ] . in our univariate estimations , subjects displaying each of the components appeared to have higher serum lp(a ) concentrations in contrast to those without these conditions , except those with elevated glycemia , where differences were not statistically significant .
these results differ from those depicted by cndido et al . in 400 brazilian individuals , who did not find such association with these criteria in an analysis akin to ours .
it is important to acknowledge that the variables demonstrating the greater differences in lp(a ) levels ( waist circumference and elevated tag ) are the most associated with systemic inflammatory state characteristic of ms [ 42 , 43 ] .
these findings may underline the role of lp(a ) in this process , whether as an active molecule or as a potential proinflammatory
likewise , when assessing its plasmatic concentration according to the possible specific diagnostic combinations for ms criteria , a large heterogeneity was found concerning these levels and the amount of criteria ; yet , the greatest values were found in subjects with more than 3 alterations .
notoriously , the high basal glucose / low hdl - c / hypertriacylglyceridemia combination displayed the highest lp(a ) values , and females only showed larger figures only within the subset of subjects with high waist circumference / high blood pressure / hypertriacylglyceridemia / low hdl - c combination ; in addition , these women also had higher ldl - c levels .
these phenomena turn both of these groups of patients into potential candidates for the application of therapeutic measures aimed to the decrease of lp(a ) values , particularly with an increment in the degree of physical activity performed , since it has been associated with normal levels of this lipoprotein in our demography .
these patients are also ideal candidates for the investigation of genetic disorders which may be responsible for this dyslipidemia .
indeed , the decisive role played by genetic factors regarding lp(a ) is broadly known [ 31 , 48 ] ; nonetheless , several conditions , alterations , and molecules can influence and generate important variations in its plasmatic concentration . in our population
, age appears to be one of the main risk factors for presenting elevated lp(a ) , resembling previous reports on the taiwanese population and on swedish subjects from the monica study .
moreover , despite the cardiovascular consequences generated by high levels of this molecule , when it coexists with specific apo(a ) isoforms , it has been associated with longevity . on the other hand , in the multivariate analysis of all diagnostic criteria for ms , only patients with hypertriacylglyceridemia exhibited a greater risk of presenting elevated lp(a ) . however , after adjusting the model for ldl - c categories , not only is it apparent that this lipoprotein boasts the closest association with high levels of lp(a ) , but the effects of tag seem to disappear ; it is important to highlight that this tendency was only observed with ldl - c adjusted for lp(a)-c , not priorly .
this pattern deviates from those portrayed by rainwater in healthy subjects and hernndez et al . in diabetic patients , who both found a positive ( lp(a ) ldl - c ) relationship and an inverse ( lp(a ) tag ) relationship .
therefore , future studies should focus on the evaluation of the behavior of lp(a ) with respect to the various types of dyslipidemia , the understanding of molecular mechanisms explaining the proportionality of ldl - c / lp(a ) concentrations , and the therapeutic considerations that may be established for these patients .
property displayed by elevated glycemia , a complex ms diagnostic criterion which required further more detailed categorization due to its overwhelming heterogeneity ( table 6 , model 3 ) .
subjects with ifg yielded a lower risk ( 29% ) of presenting elevated lp(a ) values in comparison to normoglycemic individuals .
this behavior is intimately linked to the impact of insulin in the metabolism of lp(a ) , where it has been attributed an inhibiting effect in the synthesis of apo(a ) in animal models , supported by inverse relationships observed between both molecules in population studies [ 57 , 58 ] . of all
glycemic status subgroups , subjects with ifg presented the most augmented values of insulinemia , statistically different to those of the normoglycemics ( 19.23 12.84 versus 14.17 8.45 mg / dl ; p < 0.05 ) . despite the fact that the group of diabetics showed high levels of insulin ( 18.93 12.58 )
, its effect may have been attenuated due to their inferior beta cell functionality and higher levels of ir when compared to subjects who only presented ifg .
although few studies have shown an inverse relationship between lp(a ) concentration and the presence of dm [ 59 , 60 ] , such an association has not been reported in the context of a premorbid state .
another interesting finding from this study is that the subjects with the highest hs - pcr and lp(a ) levels were the ones obtaining the highest cardiovascular risk , which could be attributed to the inflammatory properties that both molecules have [ 31 , 44 ] .
even though the particular characteristics of hs - pcr have been previously characterized in our population , other investigations should be undertaken to properly evaluate the interaction between these two .
finally , when exploring the factors that exhibited the greatest association with the diagnosis of ms , subjects with high ldl - c and hs - crp displayed the most substantial risk of presenting it .
concerning the dyslipidemia , up to 1.8 times more risk was ascertained in individuals with values higher than 132 mg / dl , confirming the position performed by these molecules in the physiopathology of ms ; even after the adjustment of ldl - c , the risk of presenting ms is similar ( or : 1.7 ) . in this light , it becomes relevant to determine the proportion of ldl - c that is already oxidized , as it may unveil the link between ms and cvd , since they are considered powerful inflammatory products . at any rate ,
with respect to elevated hs - crp values , findings were similar , albeit exhibiting a greater risk : 2.4 times more probability of developing ms , showcasing the elementary inflammatory component underlying ms and the independent effect of this protein in relation to other risk factors [ 64 , 65 ] .
notably , despite lp(a ) not being related to higher risk of ms as its concentration increased , individuals classified in the normal interval of lp(a ) by reference values specific to our population depicted a lower risk of developing ms when adjusted by other inflammatory factors .
this reinforces the importance of each of these metabolic disturbances in the integral management of subjects in risk and patients with ms
. however , this is a cross - sectional study , which makes it difficult to make decisions concerning causality .
this analysis demonstrates that ms is yet another disease to consider among disorders involving high lp(a ) levels ; future studies are required for discerning whether this relationship represents a state previous to the widely recognized cardiovascular consequences of this molecule , or if they each stand as independent outcomes .
likewise , the presence of ms influences the plasmatic concentration of lp(a ) , but this effect is irrespective of the amount of diagnostic criteria collected once the individual is ill . although these criteria seem to modify levels when they are present , when assessed in conjunction , their effects appear to be attenuated .
the only component to show an association despite several statistical adjustments is impaired fasting glucose , which , by virtue of being related to a hyperinsulinemic state , appears to diminish the probability of presenting elevated lp(a ) , an association that had previously only been suggested for dm2 . | background .
lipoprotein(a ) [ lp(a ) ] is a known risk factor for cardiovascular disease , yet its influence on metabolic syndrome ( ms ) is still controversial . the purpose of this study was to assess the impact generated by this diagnosis in serum lp(a ) concentrations .
materials and methods . a total of 1807 subjects of both genders ( 55.3% women and 44.7% men ) belonging to the maracaibo city metabolic syndrome prevalence study were evaluated .
results were expressed as mean sd , determining differences through student 's t - test and one - way anova test .
multiple logistic regression models were utilized for analyzing factors associated with elevated serum lp(a ) levels and ms .
total cholesterol and ldl - c were corrected according to lp(a)-cholesterol when necessary .
results .
no differences were found in lp(a ) values between genders ; p = 0,292 .
the association between ms and the classification of lp(a ) was statistically significant ( 2 = 28.33 ; p < 0,0001 ) , with greater levels in subjects with this diagnosis . in the univariate analysis
, subjects with each of the separate diagnostic criteria showed higher serum lp(a ) concentrations , except for hyperglycemia .
conclusions .
lp(a ) values exhibit important variations regarding ms and each of its components .
impaired fasting glucose appeared as a protecting factor against elevated lp(a ) concentrations , whereas its association with ldl - c and hs - crp suggests a potential pro - inflammatory role . |
health - related quality of life ( hrqol ) is generally conceptualized as a multidimensional construct encompassing domains including the psychological , mental , social , and spiritual areas of life ( 1 - 3 ) .
this conceptualization is in line with the world health organization definition of health as the state of complete physical , mental , and social wellbeing , and not merely the absence of disease or infirmity ( 1 ) . in spite of various definitions of hrqol in the current literature ,
an international consensus about the above - mentioned domains of hrqol has evolved ( 3 ) .
for example , ravens - sieberer and bullinger ( 4 ) have proposed the following definition , " hrqol can be viewed as a psychological construct , which describes the physical , mental , social , psychological , and functional aspects of wellbeing and function from the patient perspective . " because hrqol has become an important outcome measure in health care , valid , reliable , and simple instruments for measurement are needed . until now , hrqol research has focused mainly on adults , and thus only a limited number of instruments that measure the quality of children 's and adolescents ' health and wellbeing currently exist ( 5 - 7 ) .
only recently have health professionals focused on the importance of quality - of - life assessment in children and adolescents ( 8) .
some dimensions of the adult hrqol are not relevant for children ( e.g. family , school , and peers ) .
changes in children 's emotional and cognitive development must be recognized and addressed , and reading skills have to be considered ( 9 ) . in general , children are often regarded as unreliable respondents ( 10 - 12 ) .
therefore , early attempts to rate the hrqol in children were based on data provided by mothers and other proxy reports .
recently , studies have shown that children , and adolescents are able to answer the hrqol questionnaires reliably if their emotional development , cognitive capacity , and reading skills are taken into account ( 12 ) .
kidscreen-52 quality - of - life measure for children and adolescents ( kidscreen-52 hrqol ) was funded by the european commission and developed as a self - report measure applicable to healthy and chronically ill children and adolescents eight to eighteen years of age .
various psychometric aspects of the kidscreen-52-hrqol had been studied by ravens - sieberer et al .
in 12 european countries ( 9 ) . the aim of this study was to translate and validate the korean version of the kidscreen-52 quality - of- life measure ( k - kidscreen-52 hrqol )
the k - kidscreen-52 hrqol was developed from the english kidscreen-52 , which was funded by the european kidscreen group ( 9 ) .
the kidscreen-52 generic hrqol questionnaire was translated by a forward - backward - forward translation technique . in a first step two
thus two different versions of translation were acquired ( forward translation 1 and forward translation 2 ) . in the following reconciliation
step the two forward translators and one project member reviewed the respective two forward translations in order to create the respective reconciled forward translation , meeting all demands of conceptual equivalence with the original english draft . afterwards
the respective reconciled forward translation was back translated into english by a third translator . in the next step two research members as well as
the forward translator compared the respective backward translation with the draft , thus reviewing the respective reconciled forward translation and thereby generating the respective final forward translation .
subsequent to the generation of the respective final forward translation , all reviews and translation data was send to the german study center for documentation .
the objective of the following telephone conference was to resolve inadequate concepts of translation as well as all discrepancies between alternative versions .
the kidscreen-52 questionnaire consisted of 52 items assessing ten hrqol dimensions ( table 1 ) .
both possible item formats use a 5-point likert response scale , and the recall period is one week .
scores are computed for each dimension ( i.e. items are equally weighted ) and are transformed into t - values with a mean of 50 and a standard deviation of 10 ; higher scores indicate higher hrqol and wellbeing ( 14 ) .
all students from the seventh to ninth grade ( age 13 - 15 yr ) at one middle school in seoul , korea , were invited to take part in the study , provided that their understanding of the korean language and their reading and writing skills were sufficient .
the sample consisted of 405 adolescents , of which 204 were boys and 201 were girls .
the mean age for the sample was 13.83 yr . the students were told that the purpose of the study was to attain knowledge about the quality of life and health , in general , among teenagers .
they were further informed that their responses would be treated anonymously and that there was no right or wrong answer .
the scale instructions were given in written form , and the test was performed during school hours and lasted 30 to 40 min .
the rights of all participants were safeguarded through informed consent and confidentiality . to address the properties of the measure in terms of convergent and construct validity , several other measures
convergent validity was assessed by comparison of k - kidscreen-52 dimensions scores with the pedsql 4.0 , a known and validated questionnaire measuring similar concepts .
the pedsql 4.0 generic scales consists of an overall hrqol scale , a 23-item total score , and eight - item physical health summary subscale and a 15-item psychosocial health summary subscale .
the psychosocial health summary subscale was further composed of a five - item emotional functioning subscale , a five - item social functioning subscale , and a five - item school functioning subscale .
the physical health summary subscale was equivalent to and also referred to as the physical functioning subscale ( 15 - 17 ) .
the internal consistency coefficient of the korean version of the pedsql 4.0 has been reported previously to be 0.93 ( 18 ) .
the children 's depression scale ( cdi ) ( 19 , 20 ) and the revised children 's manifest anxiety scale ( rcmas ) ( 21 , 22 ) , which have been widely used for self- report to measure depressive and anxiety symptoms , were included . the statistical analyses for the reliability and validity of the k - kidscreen were performed using spss for windows ( version 10.0 ; spss , chicago , il , u.s.a . ) .
the internal consistency of each subscale was estimated by using the cronbach 's alpha coefficient .
the test - retest reliability was estimated via a comparison of the scores achieved when add ministered on two separate occasions , at interval of one week .
pearson correlation coefficients were computed to analyze convergent validity between k - kidscreen-52 dimensions and pedsql 4.0 , cdi , and rcmas .
convergent validity was considered to be demonstrated when correlations between comparable dimensions were significantly higher than those between theoretically different dimensions and were of a reasonable magnitude .
correlation coefficients between 0.1 and 0.3 were considered low , those between 0.31 and 0.5 moderate , and those over 0.5 were considered high ( 9 , 23 ) .
the k - kidscreen-52 hrqol was developed from the english kidscreen-52 , which was funded by the european kidscreen group ( 9 ) .
the kidscreen-52 generic hrqol questionnaire was translated by a forward - backward - forward translation technique . in a first step two
thus two different versions of translation were acquired ( forward translation 1 and forward translation 2 ) . in the following reconciliation
step the two forward translators and one project member reviewed the respective two forward translations in order to create the respective reconciled forward translation , meeting all demands of conceptual equivalence with the original english draft . afterwards
the respective reconciled forward translation was back translated into english by a third translator . in the next step two research members as well as
the forward translator compared the respective backward translation with the draft , thus reviewing the respective reconciled forward translation and thereby generating the respective final forward translation .
subsequent to the generation of the respective final forward translation , all reviews and translation data was send to the german study center for documentation .
the objective of the following telephone conference was to resolve inadequate concepts of translation as well as all discrepancies between alternative versions .
the kidscreen-52 questionnaire consisted of 52 items assessing ten hrqol dimensions ( table 1 ) .
both possible item formats use a 5-point likert response scale , and the recall period is one week .
scores are computed for each dimension ( i.e. items are equally weighted ) and are transformed into t - values with a mean of 50 and a standard deviation of 10 ; higher scores indicate higher hrqol and wellbeing ( 14 ) .
all students from the seventh to ninth grade ( age 13 - 15 yr ) at one middle school in seoul , korea , were invited to take part in the study , provided that their understanding of the korean language and their reading and writing skills were sufficient .
the sample consisted of 405 adolescents , of which 204 were boys and 201 were girls .
the mean age for the sample was 13.83 yr . the students were told that the purpose of the study was to attain knowledge about the quality of life and health , in general , among teenagers .
they were further informed that their responses would be treated anonymously and that there was no right or wrong answer .
the scale instructions were given in written form , and the test was performed during school hours and lasted 30 to 40 min .
to address the properties of the measure in terms of convergent and construct validity , several other measures were included , in addition to the k - kidscreen-52 hrqol .
convergent validity was assessed by comparison of k - kidscreen-52 dimensions scores with the pedsql 4.0 , a known and validated questionnaire measuring similar concepts .
the pedsql 4.0 generic scales consists of an overall hrqol scale , a 23-item total score , and eight - item physical health summary subscale and a 15-item psychosocial health summary subscale .
the psychosocial health summary subscale was further composed of a five - item emotional functioning subscale , a five - item social functioning subscale , and a five - item school functioning subscale .
the physical health summary subscale was equivalent to and also referred to as the physical functioning subscale ( 15 - 17 ) .
the internal consistency coefficient of the korean version of the pedsql 4.0 has been reported previously to be 0.93 ( 18 ) .
the children 's depression scale ( cdi ) ( 19 , 20 ) and the revised children 's manifest anxiety scale ( rcmas ) ( 21 , 22 ) , which have been widely used for self- report to measure depressive and anxiety symptoms , were included .
the statistical analyses for the reliability and validity of the k - kidscreen were performed using spss for windows ( version 10.0 ; spss , chicago , il , u.s.a . ) .
the internal consistency of each subscale was estimated by using the cronbach 's alpha coefficient .
the test - retest reliability was estimated via a comparison of the scores achieved when add ministered on two separate occasions , at interval of one week .
pearson correlation coefficients were computed to analyze convergent validity between k - kidscreen-52 dimensions and pedsql 4.0 , cdi , and rcmas .
convergent validity was considered to be demonstrated when correlations between comparable dimensions were significantly higher than those between theoretically different dimensions and were of a reasonable magnitude .
correlation coefficients between 0.1 and 0.3 were considered low , those between 0.31 and 0.5 moderate , and those over 0.5 were considered high ( 9 , 23 ) .
the mean score of the k - kidscreen 52 subscales , along with cronbach 's alpha coefficients , are presented in table 2 .
the k - kidscreen-52 hrqol and the pedsql 4.0 dimensions showed a moderate level of correlation , but the expected relationships .
the pedsql 4.0 physical functioning correlated highest with the k - kidscreen-52 hrqol physical wellbeing dimension ( r=0.32 ) .
the pedsql 4.0 emotional functioning and relational functioning scale correlated highest with kidscreen-52 moods and emotions ( r=0.57 , r=0.32 ) .
rcmas and cdi were the most negatively correlated with the kidscreen-52 moods and emotions dimension ( r=-0.42 , r=-0.34 ) .
however , the low correlation between the k - kidscreen-52 hrqol dimension school environment and the school functioning scale of the pedsql 4.0 ( r=0.14 ) , and the k - kidscreen-52 hrqol dimension social support and peers and the relational functioning of the pedsql 4.0 ( r=0.22 ) requires further explanation .
table 4 shows the inter - subscale correlations . in the k - kidscreen 52 subscales , the highest correlation was observed between psychological wellbeing and social support and peers subscales , but the correlation was modest ( r=0.59 ) .
the autonomy subscale was highly correlated with psychological wellbeing , social support and peers , and parent relation and home life subscales ( r=0.45 , 0.40 , 0.50 ) .
the school environment subscale was highly correlated with the parent relation and home life , self - perception , and autonomy subscales ( r=0.45 , 0.41 , 0.47 ) .
table 5 shows mean t - values for the k - kidscreen-52 dimensions stratified by gender .
differences by gender were found only in the physical wellbeing and self - perception dimensions . according to the kidscreen-52 hrqol ,
scores are computed for each dimension and are transformed into t - values with a mean of 50 and a standard deviation of 10 ; higher scores indicate higher hrqol and wellbeing ( appendix 1 ) .
the k - kidscreen-52 hrqol questionnaire includes ten dimensions covering physical , psychologic , and social aspects of the hrqol .
the importance of perceived psychologic wellbeing was underscored by three dimensions ; psychologic wellbeing , moods and emotions , and self - perception .
this dimension explored whether the adolescent felt that they have enough financial resources to allow them to live a lifestyle that was comparable with that of other adolescents and provided them with the opportunity to do things together with their peers .
classic psychometric analysis confirmed the instrument 's ability for sound measurement with sufficient psychometric properties .
the instrument reliability was good , with a cronbach ' alpha coefficient of 0.76 or above for all dimensions .
we analyzed the validity of the physical , psychological , and social constructs of health by correlating the k - kidscreen-52 with a similar instrument to assess hrqol , the pedsql 4.0 .
a comparison of k - kidscreen-52 dimensions with pedsql 4.0 scales showed the highest correlations for all similar concepts / dimensions ( e.g. , k - kidscreen-52 hrqol physical wellbeing dimension and physical functioning scale of the pedsql 4.0 ) .
this refers to a satisfying convergent validity , which means that measures that should be related were in reality related .
theoretically expected low correlations ( divergent validity ) were , in fact , found for the k - kidscreen-52 hrqol financial resources dimension with all pedql 4.0 scales . however , the low correlation between the k - kidscreen-52 hrqol dimension for school environment with the school functioning scale of the pedsql 4.0 , and the k - kidscreen-52 hrqol dimension for social support and peers and the relational functioning of the pedsql 4.0 requires further explanation . in general
, girls were found to have a lower hrqol in comparison with boys ( 24 ) . in the present study , this
was supported in only two dimensions : girls perception of their own body appearance was more negatively , and they were more concerned about their looks as well as their clothes ( self - perception ) .
overall , the good reliability and validity determined for kidscreen-52 hrqol in the original study was also found in this study , therefore , the kidscreen-52 hrqol was cross - culturally validated in this study .
using a hrqol profile , based on dimension scores , can provide detailed information about impairments in certain hrqol domains and can help suggest intervention strategies and , in the long run , prevention activities ( 9 ) .
problematic hrqol domains can be identified ; whereas an overall score is difficult to interpret since different health patterns may result in similar overall scores ( 25 , 26 ) .
furthermore , different dimensions or scales of a hrqol instrument display different degrees of sensitivity towards changes in hrqol following therapeutic intervention .
thus , the use of a profile instrument is more valid for pre- and post - interventional measurements than a global valuation score ( 27 ) .
first , the k - kidscreen-52 hrqol questionnaire was not tested in a clinical setting , therefore it needs to be tested in a clinical setting where clinical diagnoses and information about the severity of conditions are available .
further analyses will allow response patterns associated with those conditions to be identified and established ( 9 ) .
second , the european kidscreen-52 generic health - related quality of life questionnaire was developed for children and adolescents .
therefore , the k - kidscreen-52 questionnaire needs to be tested in children , in several studies , a lower hrqol was found in adolescents compared to children ( 28 ) .
third , exploratory and confirmatory factor analysis could be helpful to understand the factor structure of the kidscreen-52 questionnaire . in conclusion , a korean translation of the kidscreen-52 questionnaire was developed and verified for its reliability and validity .
this instrument can be used as a reliable self - administered scale for assessing hrqol in adolescents . | the kidscreen-52 quality - of - life ( kidscreen-52-hrqol ) is a relevant , worldwide tool used for assessing the health - related quality of life in children and adolescents .
the purpose of this study was to define measurement properties of the korean version of the kidscreen-52 hrqol .
the original questionnaire was translated following international translation guidelines .
analysis regarding psychometric properties showed that the cronbach - alpha ranged from 0.77 to 0.95 .
the correlation coefficient between the pedql and kidscreen-52 dimensions were high for the assessments of similar constructs .
therefore , the korean version of the kidscreen-52 was found to be suitable for use in korean adolescents . |
cycloadditions are an important class of reactions for site - specific labeling with applications in super - resolution microscopy of cellular components.[15 ] key properties of these reactions are their bioorthogonality and high selectivity . by exploiting the concept of mutual orthogonality between several click - type cycloaddition reactions ,
a combination of particularly slow and fast reactions enables the simultaneous labeling of multiple sites in a kinetically controlled fashion in vivo and in vitro.[68 ] recently , a set of orthogonal reactions has been used by niki et al . to label the insulin receptor and influenza proteins on non - canonical amino acids bearing strained 8-membered rings in a time dependent manner .
the click reactions utilized in this work are n - propyl azide , 3-benzyl tetrazine ( h - tet ) and 3-benzyl-6-methyl tetrazine ( me - tet ) ligations to three types of eight - membered cyclic rings ( figure 1 a and supporting information figure s1 , iupac names in supporting information ) , namely i ) racemic equatorial trans - cyclooct-2-en-1-methylcarbamate ( tco*e ) and racemic axial trans - cyclooct-2-en-1-methylcarbamate ( tco*a ) , ii ) two enantiomers of strained cyclooct-2-yn-1-methylcarbamate ( sco ) , and iii ) endo- and exo - bicyclonon-5-yn-1-methylcarbamate ( bcn & bcn ) .
the tetrazine ligation , a 4 + 2-cycloaddition , is a strain - promoted inverse electron demand diels alder reaction ( spiedac),[1011 ] while the alkyne
azide ligation is a strain - promoted huisgen - type 1,3-dipolar 3 + 2-cycloaddition ( spaac).[1215 ] the large differences in reaction speed when employing supposedly similar ligation partners , such as h - tet versus me - tet , or tco * versus sco , render dual color labeling of biological systems possible.[14 , 16]1 kinetics of mutually orthogonal click reactions .
a ) overview of the investigated reaction set between tco*e / a isomers , bcn isomers and sco with azide , h - tet and me - tet .
b ) correlation between experimental rate constants k and the calculated activation energies eact for each pair of reactants .
for the reactions too slow to be measured , we only obtained an upper limit for k as indicated by the error lines and omitted such points for the linear fit .
quantitatively predicting such differences and understanding their origin by quantum chemical calculations would increase the ability to further enhance the mutual orthogonality and augment the available set of reactions for preferential labeling .
a large body of previous quantum - chemical computational work has characterized and thereby significantly advanced our understanding of a number of highly related strain - promoted click reactions.[1721 ] they mostly ascribed the differences in reactivity of these reactions , which have been shown to feature an inverse diels alder electron demand , to contributions from orbital interactions , distortion and pauli repulsion.[2223 ] the higher reactivity of tetrazines towards trans - cyclooctene was attributed to their higher electrophilicity as compared to azides , while introducing nitrogens into the diene was proposed to decrease pauli repulsion , rendering tetrazines the fastest reaction partner for cycloadditions with alkenes . notwithstanding these advances
, previous work has been focusing on highly simplified reactant scaffolds limiting the comparison of calculated activation energies to reaction rates determined experimentally for more complex scaffolds .
however , attaching handles to the functional groups of azides , tetrazines and the different eight - membered rings is the crucial requisite to label biomolecules , and may also critically affect the reactivity of the molecule , as experimental data increasingly certify that the speed of these reactions is highly susceptible to minor changes in substituents .
importantly , handles on cyclooctyne rings introduce a single stereocenter whereas handles on cyclooctene rings add an additional stereocenter .
this results in two sco enantiomers and two pairs of tco * enantiomers ( see figure s1 ) .
the configuration of the stereocenters emerge as a crucial determinant for this class of click reactions.[2425 ] considering the effect of the genuine handles used for functionalization is therefore indispensable for a direct validation of insights from quantum chemical calculations and , more importantly , for the application of these findings to the design of novel orthogonal reactions .
we addressed this fundamental challenge and herein present the first comprehensive set of measured rates and computed barriers for reactants with direct relevance for biological applications .
we systematically computed the conformations and energies for the spiedac and spaac reactions described above , using density functional theory ( dft ) .
this resulted in a total of 48 reactions with configurationally distinct transition states and products , out of which 24 are chemically distinct enantiomers .
the resulting system sizes required advanced sampling of the complex energy landscape , and the diminishing barriers of the high speed tco * reactions rendered it necessary to incorporate van der waals complexes into the reaction pathway . on this basis , unexpectedly , we find that spiedac reactions involving sco and tetrazines to follow normal electron demand , ascribed to the electrophilicity of the sco .
we thus term this class of reactions spinedac ( strain - promoted inherently normal electron demand diels alder cycloaddition ) .
we observe isomeric configurations to critically fine - tune the reactivity of the investigated click reactions .
our study aids the engineering of a currently widely used set of cycloadditions to further enhance their reactivity and mutual orthogonality for their applicability in biology .
we used the m06 - 2x density functional[2627 ] with the 6 - 311+g(d , p ) basis set and pcm solvent for the quantum chemical calculations carried out in gaussian 09 , and used the cclib library for post - analysis . as a measure for the reaction rates of any of the putative 48 reactions ( 3 reactants azide / me - tet / h - tet times eight different enantiomers and diastereomers of tco*/sco / bcn , times two different relative tail orientations , table s1 )
, we calculated the activation energy eact along the reaction pathway ( figure s2 ) . conventionally , and also as previously reported for this class of reactions , eact is defined as the difference between the transition state ( ts ) energy and the sum of the energies of geometry optimized reactants , eact,react = etsereact , opt . here
, we instead use the energy of the van der waals complex formed by the two reactants , evdw , as a reference , that is , defined eact = eact , vdw = ets evdw ( figure s2 ) , the reason for which was two - fold .
first , ts conformations and van der waals complexes are affected by basis set superposition errors ( bsse ) , whereas the conformations of the smaller single reactants are less affected .
thus , the bsse is inherently included in eact,react but largely cancels out in eact , vdw . in addition , we found that ereact , opt is larger than ets for some of the fastest reactions involving tco * , which would imply the absence of any activation barrier and is an artefact from overlooking the formation of a favourable van der waals complex prior to the reaction .
nevertheless , we found a correlation of eact , vdw with eact,react ( figure s4 ) , and report eact , vdw as activation energies in the following .
orbital energies were computed using the hf/6 - 311+g(d , p ) level of theory and we define efmo as the energy difference between two relevant interacting orbitals . for computational efficiency ,
smaller compounds compared to the experimental reactants[9 , 25 ] were used , but including a significant fraction of the handles and therefore still more detailed when compared to the highly simplified scaffolds of previous computations .
the tetrazines r - group features a benzylamine , the azide a n - propyl , and the eight - ring an n - methylcarbamate group . while for the dft calculations we considered all conformers separately ,
experimental rates were obtained with mixtures of the tco*a , tco*e and sco enantiomers , using stopped - flow spectroscopy ( see supporting information ) .
this resulted in a total of 15 measured reactions rates directly comparable to with our calculated barriers ( see supplemental material for details ) .
we used the m06 - 2x density functional[2627 ] with the 6 - 311+g(d , p ) basis set and pcm solvent for the quantum chemical calculations carried out in gaussian 09 , and used the cclib library for post - analysis . as a measure for the reaction rates of any of the putative 48 reactions ( 3 reactants azide / me - tet / h - tet times eight different enantiomers and diastereomers of tco*/sco / bcn , times two different relative tail orientations , table s1 ) ,
we calculated the activation energy eact along the reaction pathway ( figure s2 ) . conventionally , and also as previously reported for this class of reactions , eact is defined as the difference between the transition state ( ts ) energy and the sum of the energies of geometry optimized reactants , eact,react = etsereact , opt . here , we instead use the energy of the van der waals complex formed by the two reactants , evdw , as a reference , that is , defined eact = eact , vdw = ets evdw ( figure s2 ) , the reason for which was two - fold .
first , ts conformations and van der waals complexes are affected by basis set superposition errors ( bsse ) , whereas the conformations of the smaller single reactants are less affected .
thus , the bsse is inherently included in eact,react but largely cancels out in eact , vdw .
in addition , we found that ereact , opt is larger than ets for some of the fastest reactions involving tco * , which would imply the absence of any activation barrier and is an artefact from overlooking the formation of a favourable van der waals complex prior to the reaction .
nevertheless , we found a correlation of eact , vdw with eact,react ( figure s4 ) , and report eact , vdw as activation energies in the following .
orbital energies were computed using the hf/6 - 311+g(d , p ) level of theory and we define efmo as the energy difference between two relevant interacting orbitals . for computational efficiency ,
smaller compounds compared to the experimental reactants[9 , 25 ] were used , but including a significant fraction of the handles and therefore still more detailed when compared to the highly simplified scaffolds of previous computations .
the tetrazines r - group features a benzylamine , the azide a n - propyl , and the eight - ring an n - methylcarbamate group . while for the dft calculations we considered all conformers separately ,
experimental rates were obtained with mixtures of the tco*a , tco*e and sco enantiomers , using stopped - flow spectroscopy ( see supporting information ) .
this resulted in a total of 15 measured reactions rates directly comparable to with our calculated barriers ( see supplemental material for details ) .
the understanding of click - chemistry reactions at their highest level of detail is crucial to improve their applicability in biomolecule labeling at the base of single - molecule spectroscopy .
therefore , azide / tetrazine cycloadditions to cyclooctenes and cyclooctynes were herein investigated taking into account the effect of r - groups used for proteins functionalization .
we obtained m06 - 2x energies from optimization of all stereoisomers including enantiomeric pairs of the reactants set ( figure s1 ) .
these energies suggest tco*e to be more stable than tco*a by 1.1 kcal mol , which also is the less reactive molecule among the tetrazine cycloadditions ( figure 1 a ) , but tco*e undergoes more readily cis - isomerisation .
we next calculated conformations and energies of van der waals complexes and transition states for all possible reactions to estimate energy barriers eact .
we obtained a high correlation between measured rates and calculated energy barriers ( figure 1 b , r=0.89 ) , validating our quantum mechanical calculations .
due to the sterically demanding protein that the eight - membered rings are attached to , the azide and tetrazine substituents are prone to orient in an antiparallel fashion to the carbamate sidechain of the 8-ring .
although a similar correlation between experimental rates and calculated activation energies was obtained when the side - chain regioselectivity was ignored ( figure s3 ) , individual barriers can vary by up to 3 kcal mol when changing tail orientation ( table s1 ) .
this suggests the steric demand of the linkers including the bulky label and biomolecule to crucially determine the reaction kinetics . to identify the origin of the differences in reactivity , we analyzed the energy differences efmo between interacting frontier molecular orbitals ( fmo ) of all 15 reactant pairs ( for details see supporting information ) .
overall efmo correlates very well with eact ( r0.72 , figure 2 a ) . as expected
, efmo systematically underestimates the barrier for ligations with high distortion energies ( dot size in figure 2 a ) .
we obtained an improved correlation when comparing the sum of distortion energies and efmo with eact ( r=0.82 ) , implying that in a first approximation these two contributions can be considered additive and are both critically determining reactivitiy .
a ) correlation between fmo energy differences ( efmo ) of the 15 distinct reactions ( eact is the barrier for the two enantiomers obtained in antiparallel tail orientation ) .
b ) electron demand of the 15 different reactant pairs according to fmo energy gaps .
the main contribution to distortion energies generally comes from the azide or tetrazine , respectively ( figure 3 ) .
tetrazine ( me - tet and h - tet ) ligations to sco as well as any of the cycloadditions involving me - tet show significantly higher distortions than the other cycloadditions ( figures 2 a and 3 ) , suggesting that the methyl group of me - tet as well as the carbamate right next to the triple bond of sco creates a steric hindrance for the transition state formation.3 distortion energies edist from the vdw complex to the transition state conformation , decomposed for the ring and azide / tetrazine compounds ( see methods for details ) .
we also analyzed the origins for the different observed reactivity between the investigated reactions . as expected , efmo are overall higher in cycloadditions involving azides rather than tetrazines , explaining the well - known fact that n - propyl azide reacts less efficiently overall ( figure 2 a).[1314 ] our calculations and measurements also confirm the previously established higher preference of this azide with bcn over sco,[2 , 31 ] for which we predict a 3 kcal mol difference in barrier .
our results further agree with the higher reactivity of azide with bcn over bcn , which we find to similarly hold for h - tet and me - tet ( figure 2 a ) .
we can now ascribe the smaller barrier for the cycloaddition of the azide with bcn to a smaller homo lumo gap between the two reactants ( figure 2 b ) . according to both calculations and measurements ,
tco*a reacts faster than tco*e with both h - tet ( 3-fold ) and me - tet ( 700-fold , figure 1 b ) , again directly in line with the smaller fmo energy gap for the tco*a isomer ( figure 2 b ) .
the axial position increases the electron - withdrawing effect of the carbamate group , an effect that is further enhanced by the smaller distortion required for the ligation of tco*a to a tetrazine ( figure 3 ) .
reactions involving h - tet are generally faster than those with me - tet , a trend to be expected in this case of inverse electron demand of spiedac reactions , as the methyl group shifts electron density into the reacting 6-ring ( figure 4 a ) .
surprisingly , the so - called spiedac involving sco and a tetrazine we instead predict to proceed with normal electron demand and correspondingly term this reaction spinedac .
the carbamate group makes sco more electrophilic than bcn , rendering the interaction of its lumo with the tetrazine homo more favorable ( figure 4 b ) .
sco ligation to me - tet nevertheless is slower than to h - tet , because the sterically more demanding methyl group gives rise to a 6 kcal mol higher distortion of the transition state ( figures 2 and 4 c ) .
we therefore propose that this spinedac reaction can be sped up conversely to spiedac reactions , namely by more strongly electron - drawing cyclooctyne substituents and/or by further electron - donating tetrazine substituents , both with as little steric demand as possible .
our results , however , also emphasize that care must be taken as reactions can switch between inverse and normal electron demand upon allegedly minuscule chemical changes.4 origin of differences in electron demand and distortion a ) the methyl group of me - tet shifts electron density into the tetrazine ring , as evidenced by the differences in shape of the lumo and homo between h - tet and me - tet ( for clarity only the substituted tetrazine ring is shown ) .
b ) energy gaps between fmos of h - tet reacting with sco and bcn .
sco features lower fmo energies than bcn and undergoes cycloadditions with h- tet ( and me - tet , see table s3 ) with normal electron demand ( solid blue line ) instead of inverse electron demand as for bcn ( solid red line ) .
c ) the transition state of sco - me - tet ( orange ) shows a significantly higher distortion than the one of sco - h - tet ( green , compare orange and green dashed lines ) resulting in a larger asymmetry with regard to the forming of c
we here analyzed the determinants of reactivity for a set of strain - promoted cycloaddition reaction partners that is identical to those used for in vitro and in vivo labeling .
we could show that handles not only play the role to attach the reactive moieties to fluorescent labels and proteins .
instead , they also decisively influence the reaction rate of the cycloadditions , due to their electronic properties and steric demand .
our data explain how different isomeric states , methyl groups and carbamate linkers can independently and decisively alter the speed of spaac , spiedac and the newly defined spinedac reactions .
most importantly , the presence of the methyl and/or benzyl moieties of h - tet and me - tet together with the electron - withdrawing properties of the carbamate handle of the sco cyclooctyne can switch the electron demand from an inverse to a normal type , with direct consequences for the rational tuning of the reactivity of these reactions . a more strongly electron - donating group replacing the methyl or benzyl groups of h - tet /
me - tet will increase the reactivity of ligations with sco , but not with tco * and bcn .
even modifications of the aminogroup at the benzyl ring of h - tet / me - tet , although distant , can modulate the electron density of the benzene and thereby the reactivity of the tetrazines . likewise , replacing the carbamate handle with more strongly electron - withdrawing groups , is predicted to have a similar effect .
benzyl and methyl moieties at me - tet and h - tet are sterically highly demanding .
their substitution by smaller and/or electron - withdrawing groups should considerably enhance spiedac , but not spinedac reactions .
these structural features should thus be taken into account when re - engineering cycloadditions in computations and experiments .
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors | site - specific labeling of biomolecules is rapidly advancing due to the discovery of novel mutually orthogonal reactions .
quantum chemistry studies have also increased our understanding of their relative rates , although these have until now been based on highly simplified reactants . here
we examine a set of strain - promoted click - type cycloaddition reactions of n - propyl azide , 3-benzyl tetrazine and 3-benzyl-6-methyl tetrazine with cyclooctenes / ynes , in which we aim to address all relevant structural details of the reactants .
our calculations have included the obligatory handles used to attach the label and biomolecule as these can critically influence the stereochemistry and electron demand of the reaction .
we systematically computed orbital gaps , activation and distortion energies using density functional theory and determined experimental rates for validation .
our results challenge the current paradigm of the inverse electron demand for this class of reactions .
we found that the ubiquitous handles , when next to the triple bond of cyclooctynes , can switch the diels alder type ligations to normal electron demand , a class we term as spinedac reactions .
electron donating substituents on tetrazine can enhance normal demand but also increase distortion penalties .
the presence and isomeric configuration of handles thus determine the reaction speed and regioselectivity .
our findings can be directly utilized in engineering genuine cycloaddition click chemistries for biological labeling . |
as people are living longer due to advances in health care and lifestyles , spine surgeons will be faced with a greater number of surgical problems in the aged population and with that comes an expected increase in perioperative medical complications .
another substantial challenge is advanced osteoporosis , which is found very predictably in this population . with this poor bone mineralization
comes an array of complications associated with fixation failure . until osteoporosis can be better managed in the perioperative period , surgeons will need to aggressively pursue methods to enhance fixation in the aging spine .
pedicle screw fixation is still the most popular fixation method , but to date this fixation proves to be inadequate in many patients with osteoporosis .
previous studies have shown that not tapping prior to placing a pedicle screw will enhance screw fixation in the osteoporotic spine.1 it has also been shown that cement augmentation of screws in the spine will also enhance fixation.2
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13 both techniques can be useful in a given situation . however , the effect of tapping versus not tapping when using cement to augment screws has not been evaluated fully .
in addition , surgeons have choices to make regarding the trajectory of the pedicle screws they implant .
although many studies regarded this , very few have addressed implications of changing screw trajectory in the face of osteoporosis .
one study by santoni et al did , however , and espouses a more lateral trajectory in the face of osteoporosis in an attempt to put the screw closer to a cortical buttress.14 to the authors ' knowledge , this has not achieved widespread acceptance for unclear reasons . the purpose of this study is to evaluate the effects of some technical choices a surgeon may make when faced with the need to achieve pedicle screw fixation in the osteoporotic vertebra .
one choice faced is whether to pretap prior to placement of pedicle screws into an osteoporotic spinal segment .
another choice the surgeon faces is the preferred screw trajectory in an osteoporotic spinal segment .
we chose to investigate the mechanical ramifications of a straight - ahead trajectory versus a cephalad - oriented screw trajectory .
synthetic osteoporotic cancellous bone block models were utilized as previously published by choma et al.3 osteoporotic bone was simulated by a 0.09-g / ml density open - cell rigid foam block attached with cement to a short fiber - filled epoxy sheet ( sawbones , pacific research laboratories , vashon , washington , united states ) .
the epoxy sheet mechanically simulated the dense superior end plate of the vertebra , and the whole structure can be seen in fig .
section blocks 30 mm wide 60 mm deep were used for testing .
two groups were tested a nonaugmented ( na ) group and a group augmented with bioactive cement ( bioc ) .
this is a proprietary mixture of calcium sulfate and calcium phosphate provided by wright medical ( arlington , tennessee , united states ) . in each specimen of both groups a centrally located pilot hole , 3.0 mm in diameter by 40 mm deep ,
was drilled into the end of each block using a straight lenke - type probe , and then the blocks were threaded with a 5.5-mm - diameter tap ( stryker xia , kalamazoo , mi , united states ) to a depth of 0 , 20 , 30 , or 40 mm depending upon the configuration .
starter holes and screwdrivers were guided along tracks using custom jigs to ensure proper and repeatable orientation of screw placement .
bioc , if applicable to the group being tested , was then injected ( 1.5 to 2.0 ml ) for samples using a syringe and jamshidi needle ( carefusion , san diego , ca , united states ) before a screw was manually inserted until the base of the head rested against the foam .
the cement augmentation distribution for various tapping depths was characterized visually using backlit photography ( fig .
, we tested four samples per specified tap depth , and in the augmented group , we tested five samples for each tap depth .
more samples were tested in cement group as there was prestudy concern for more variability with the use of cement .
fixed - head pedicle screws were used in both subgroups ( na and bioc ) , which were 6.5 mm in diameter by 40 mm long ( stryker xia ) .
a rod 5.5 mm in diameter by 65 mm long ( stryker xia ) was then inserted into the screw head and secured by tightening the set screw to 12 n / m to complete the test specimen . each augmented sample cured overnight prior to testing , which was well beyond the manufacturer 's recommended minimum time .
synthetic vertebrae with the epoxy end plate .
augmented cases featuring different tap depths .
identical synthetic osteoporotic bone blocks were used to evaluate the effect of varying screw trajectory in the sagittal plane .
two screw trajectories were evaluated in this study : a straight - ahead ( 0 degrees ) screw , parallel to the superior end plate , and a cephalad - oriented screw ( 23 degrees cephalad ) , in which the tip approaches the superior end plate .
we chose 23 degrees based upon screw geometry so that a consistent entry point was maintained between orientation groups and because it allowed the screw tip to contact the end plate .
four groups were evaluated in this portion of the study : an na screw control group ( na-0 ) with the screw placed at 0 degrees relative to the end plate ; an na screw control group ( na-23 ) with the screw placed at 23 degrees relative to the end plate ; a group augmented with calcium phosphate ( cp-0 ) with the screw placed 0 degrees relative to the end plate ; and a group augmented with calcium phosphate ( cp-23 ) with the screw placed 23 degrees relative to the end plate . prior to screw placement , a centrally located pilot hole , 3.0 mm in diameter by 40 mm deep , was drilled into the end of each block using a straight lenke - type probe and then threaded with a 5.5-mm - diameter tap ( stryker xia ) to a depth of 30 mm .
starter holes and screwdrivers were guided along either 0- or 23-degree degree tracks to ensure proper and repeatable orientation of screw placement .
cement , if applicable , was then injected ( 1.5 to 2.0 ml ) for each sample using a syringe and jamshidi needle .
next , a pedicle screw was manually inserted until the base of the head rested against the foam . in the na group
, we tested four samples per specified screw trajectory ( 0 or 23 degrees ) , and in the augmented group , we tested five samples for each screw trajectory .
more samples were tested in cement group as there was prestudy concern for more variability with the use of cement . for both parts of this study , the foam block epoxy sheet assembly , which simulated vertebral bone , was securely mounted between the superior and inferior steel plates of the specimen - loading fixture .
the specimen 's rod was held securely in an angle vise and tilted 45 degrees relative to the test machine 's horizontal table in a sagittal plane orientation .
a 46.6-cm - long pushrod was attached to the universal joint of the test machine 's load cell ram and the axis of the pin in the yoke attaching it to the specimen - loading fixture .
the pushrod was used to transmit a force f along the pushrod 's axis ( vertical initially ) to the test specimen at a point 45 mm anterior of the rod 's z axis and 10 mm below the inferior surface of the superior steel end plate .
this created equal components of pullout force fx and transverse force fz in combination with a flexion bending moment m = fz 0.015
this configuration simulates a combined loading condition in a kyphotic thoracic spine with no external constraints on the relative motion in the sagittal plane between the screw and vertebra .
motion of the pedicle screw relative to the simulated vertebra was measured with a 0.01-mm resolution 3d infrared optical tracking system ( ndi optotrak certus , waterloo , ontario , canada ; fig .
quasi - static loading tests were performed using a servo - hydraulic test machine ( instron 8821s , 100 royall street , canton , massachusetts , united states ) . in quasi - static testing ,
compressive load was applied in displacement control at a rate of 0.40 mm / s until catastrophic failure of the specimen occurred .
failure initiation was defined as the first maximum magnitude of compression force f that resulted in nearly constant or declining load resistance for at least 1 mm of additional ram displacement .
differences in failure initiation force and corresponding screw displacements were statistically compared by using a student t ( two - tailed , unequal variance ) test .
significance was taken as p 0.01 .
instron setup with the optotrak sensors attached .
( reprinted from choma tj , frevert wf , carson wl , waters np , pfeiffer fm .
biomechanical analysis of pedicle screws in osteoporotic bone with bioactive cement augmentation using simulated in vivo multicomponent loading .
spine ( phila pa 1976 ) 2011;36(6):454462 , with permission from lippincott williams & wilkins.3 )
synthetic osteoporotic cancellous bone block models were utilized as previously published by choma et al.3 osteoporotic bone was simulated by a 0.09-g / ml density open - cell rigid foam block attached with cement to a short fiber - filled epoxy sheet ( sawbones , pacific research laboratories , vashon , washington , united states ) .
the epoxy sheet mechanically simulated the dense superior end plate of the vertebra , and the whole structure can be seen in fig .
section blocks 30 mm wide 60 mm deep were used for testing .
two groups were tested a nonaugmented ( na ) group and a group augmented with bioactive cement ( bioc ) .
this is a proprietary mixture of calcium sulfate and calcium phosphate provided by wright medical ( arlington , tennessee , united states ) . in each specimen of both groups a centrally located pilot hole , 3.0 mm in diameter by 40 mm deep ,
was drilled into the end of each block using a straight lenke - type probe , and then the blocks were threaded with a 5.5-mm - diameter tap ( stryker xia , kalamazoo , mi , united states ) to a depth of 0 , 20 , 30 , or 40 mm depending upon the configuration .
starter holes and screwdrivers were guided along tracks using custom jigs to ensure proper and repeatable orientation of screw placement .
bioc , if applicable to the group being tested , was then injected ( 1.5 to 2.0 ml ) for samples using a syringe and jamshidi needle ( carefusion , san diego , ca , united states ) before a screw was manually inserted until the base of the head rested against the foam .
the cement augmentation distribution for various tapping depths was characterized visually using backlit photography ( fig .
, we tested four samples per specified tap depth , and in the augmented group , we tested five samples for each tap depth .
more samples were tested in cement group as there was prestudy concern for more variability with the use of cement .
fixed - head pedicle screws were used in both subgroups ( na and bioc ) , which were 6.5 mm in diameter by 40 mm long ( stryker xia ) .
a rod 5.5 mm in diameter by 65 mm long ( stryker xia ) was then inserted into the screw head and secured by tightening the set screw to 12 n / m to complete the test specimen . each augmented sample cured overnight prior to testing , which was well beyond the manufacturer 's recommended minimum time .
synthetic vertebrae with the epoxy end plate .
augmented cases featuring different tap depths .
identical synthetic osteoporotic bone blocks were used to evaluate the effect of varying screw trajectory in the sagittal plane .
two screw trajectories were evaluated in this study : a straight - ahead ( 0 degrees ) screw , parallel to the superior end plate , and a cephalad - oriented screw ( 23 degrees cephalad ) , in which the tip approaches the superior end plate .
we chose 23 degrees based upon screw geometry so that a consistent entry point was maintained between orientation groups and because it allowed the screw tip to contact the end plate .
four groups were evaluated in this portion of the study : an na screw control group ( na-0 ) with the screw placed at 0 degrees relative to the end plate ; an na screw control group ( na-23 ) with the screw placed at 23 degrees relative to the end plate ; a group augmented with calcium phosphate ( cp-0 ) with the screw placed 0 degrees relative to the end plate ; and a group augmented with calcium phosphate ( cp-23 ) with the screw placed 23 degrees relative to the end plate . prior to screw placement , a centrally located pilot hole , 3.0 mm in diameter by 40 mm deep , was drilled into the end of each block using a straight lenke - type probe and then threaded with a 5.5-mm - diameter tap ( stryker xia ) to a depth of 30 mm .
starter holes and screwdrivers were guided along either 0- or 23-degree degree tracks to ensure proper and repeatable orientation of screw placement .
cement , if applicable , was then injected ( 1.5 to 2.0 ml ) for each sample using a syringe and jamshidi needle .
next , a pedicle screw was manually inserted until the base of the head rested against the foam . in the na group
, we tested four samples per specified screw trajectory ( 0 or 23 degrees ) , and in the augmented group , we tested five samples for each screw trajectory .
more samples were tested in cement group as there was prestudy concern for more variability with the use of cement . for both parts of this study , the foam block epoxy sheet assembly , which simulated vertebral bone , was securely mounted between the superior and inferior steel plates of the specimen - loading fixture .
the specimen 's rod was held securely in an angle vise and tilted 45 degrees relative to the test machine 's horizontal table in a sagittal plane orientation .
a 46.6-cm - long pushrod was attached to the universal joint of the test machine 's load cell ram and the axis of the pin in the yoke attaching it to the specimen - loading fixture .
the pushrod was used to transmit a force f along the pushrod 's axis ( vertical initially ) to the test specimen at a point 45 mm anterior of the rod 's z axis and 10 mm below the inferior surface of the superior steel end plate .
this created equal components of pullout force fx and transverse force fz in combination with a flexion bending moment m = fz 0.015 n / m at the vertebra center .
this configuration simulates a combined loading condition in a kyphotic thoracic spine with no external constraints on the relative motion in the sagittal plane between the screw and vertebra .
motion of the pedicle screw relative to the simulated vertebra was measured with a 0.01-mm resolution 3d infrared optical tracking system ( ndi optotrak certus , waterloo , ontario , canada ; fig .
quasi - static loading tests were performed using a servo - hydraulic test machine ( instron 8821s , 100 royall street , canton , massachusetts , united states ) . in quasi - static testing ,
compressive load was applied in displacement control at a rate of 0.40 mm / s until catastrophic failure of the specimen occurred .
failure initiation was defined as the first maximum magnitude of compression force f that resulted in nearly constant or declining load resistance for at least 1 mm of additional ram displacement .
differences in failure initiation force and corresponding screw displacements were statistically compared by using a student t ( two - tailed , unequal variance ) test .
( reprinted from choma tj , frevert wf , carson wl , waters np , pfeiffer fm .
biomechanical analysis of pedicle screws in osteoporotic bone with bioactive cement augmentation using simulated in vivo multicomponent loading .
spine ( phila pa 1976 ) 2011;36(6):454462 , with permission from lippincott williams & wilkins.3 )
tables 1 and 2 present the force to failure for screws placed into holes tapped to different depths ranging from 0 to 40 mm . in the absence of cement augmentation
each na sample shows a consistent decrease in force to failure as the tap depth increases . in comparing the untapped versus the tapped screw ,
the force to failure decreases 52% if the pilot hole is tapped to 40 mm prior to screw insertion ( 30.93 2.1 n versus 14.87 0.3 n , respectively ) .
this behavior is consistent with previous studies evaluating the effect of tapping prior to screw placement in the lumbar spine.11
12
alternatively , the samples with cement augmentation increased the force to failure as the tap depth increased . the average force to failure increased in each case that the tap depth increased ( tables 1 and 3 ) .
the average force to failure increased 54% when comparing untapped versus pretapping the entire screw length ( 40.35 1.6 n versus 61.95 1.8 n , respectively ) .
a graphical representation of the force to failure for na and augmented screws relative to varied tap depths is shown in fig .
abbreviation : bioc , bioactive cement . in comparing each case of the augmented versus na specimens ,
the p values are all less than 0.01 , taken as a statistically significant difference between the experimental augmented cases and na control cases .
abbreviations : bioc , calcium phosphate and calcium sulfate cement augmentation ; na , nonaugmented .
f
max ( n ) p values in student t test ( two - tailed , unequal variance ) , statistical significance : p < 0.01 .
pedicle screws oriented at 23 degrees relative to the end plate had a significantly higher failure force than their respective counterparts at 0 degrees in both the na and bioc groups ( p < 0.01 ; tables 5 and 6 ) . in the na group , simply changing the trajectory of the screw doubled the average force of failure from 20.95 n to 39.82 n. the cases with the largest change in failure strength were the na screw at 0 degrees versus the calcium phosphate screw at 23 degrees . in this comparison , there was a 3.5-fold increase in the average failure strength from 20.95 n to 73.17 n. again , this comparison 's p value was much less than 0.01 , making it statistically significant . in the bioc group ,
the average force to failure in the bioc group inserted at 0 degrees was 56.82 4.7
n , and the mean force to failure for the screws inserted at 23 degrees was 73.17 2.1
n ( fig . 5 ) . orienting the screw up to the vertebral end plate led to significantly higher force to failure values in both cases , with what appears to be an independent effect by augmenting the screw with cement .
abbreviations : bioc , calcium phosphate and calcium sulfate cement augmentation ; na , nonaugmented . student t test ( two - tailed , unequal variance , statistical significance : p < 0.01 ) .
tables 1 and 2 present the force to failure for screws placed into holes tapped to different depths ranging from 0 to 40 mm . in the absence of cement augmentation
each na sample shows a consistent decrease in force to failure as the tap depth increases . in comparing the untapped versus the tapped screw ,
the force to failure decreases 52% if the pilot hole is tapped to 40 mm prior to screw insertion ( 30.93 2.1 n versus 14.87 0.3 n , respectively ) .
this behavior is consistent with previous studies evaluating the effect of tapping prior to screw placement in the lumbar spine.11
12
alternatively , the samples with cement augmentation increased the force to failure as the tap depth increased . the average force to failure increased in each case that the tap depth increased ( tables 1 and 3 ) .
the average force to failure increased 54% when comparing untapped versus pretapping the entire screw length ( 40.35 1.6 n versus 61.95 1.8 n , respectively ) .
a graphical representation of the force to failure for na and augmented screws relative to varied tap depths is shown in fig .
abbreviation : bioc , bioactive cement . in comparing each case of the augmented versus na specimens ,
the p values are all less than 0.01 , taken as a statistically significant difference between the experimental augmented cases and na control cases .
abbreviations : bioc , calcium phosphate and calcium sulfate cement augmentation ; na , nonaugmented .
f
max ( n ) p values in student t test ( two - tailed , unequal variance ) , statistical significance : p < 0.01 .
pedicle screws oriented at 23 degrees relative to the end plate had a significantly higher failure force than their respective counterparts at 0 degrees in both the na and bioc groups ( p < 0.01 ; tables 5 and 6 ) . in the na group , simply changing the trajectory of the screw doubled the average force of failure from 20.95 n to 39.82 n. the cases with the largest change in failure strength were the na screw at 0 degrees versus the calcium phosphate screw at 23 degrees . in this comparison , there was a 3.5-fold increase in the average failure strength from 20.95 n to 73.17 n. again , this comparison 's p value was much less than 0.01 , making it statistically significant . in the bioc group ,
the average force to failure in the bioc group inserted at 0 degrees was 56.82 4.7 n , and the mean force to failure for the screws inserted at 23 degrees was 73.17 2.1
n ( fig . 5 ) . orienting the screw up to the vertebral end plate led to significantly higher force to failure values in both cases , with what appears to be an independent effect by augmenting the screw with cement
abbreviations : bioc , calcium phosphate and calcium sulfate cement augmentation ; na , nonaugmented .
student t test ( two - tailed , unequal variance , statistical significance : p < 0.01 ) .
pedicle screw fixation in the osteoporotic spine is expected to be a major challenge facing spinal surgeons for the foreseeable future .
fairly clear evidence including the results of this study supports the biomechanical superiority of not tapping prior to placing standard na pedicle screws in an osteoporotic spine . in our study , tapping prior to screw placement led to a 52% decrease in force to failure of the screw fixation .
tapping removes bone that could potentially be compressed by the placement of a screw and therefore not tapping and leaving this bone in place will likely lead to better fixation .
the choice of tapping versus not tapping when placing cement - augmented screws certainly seems less intuitive , which we felt justified further study .
our study used an osteoporotic model with multicomponent loading that was previously established by choma et al.3 the multicomponent loading is thought to more closely approximate how screws might fail in vivo , and therefore this combined pullout , toggle , and translational force was used in our study instead of a one - dimensional screw pullout model . in our study , we evaluated tapping versus not tapping prior to cement augmentation of a screw in a cancellous bone block model . tapping to 40
mm prior to augmentation and screw placement led to a 54% increase in force to failure when compared with a nontapped cement - augmented screw .
the results of our study suggest that tapping prior to placement of cement augmentation substantially improves loads to failure of screw fixation in an osteoporotic model .
it was also clear in our data that tapping longer instead of shorter prior to cement augmentation led to substantially greater stability .
we hypothesize that the tapping creates a larger cavity in which the bioactive cement can more uniformly distribute around the screw and the screw bone interface . tapping longer
limitations exist whenever a synthetic bone model is used and this should be recognized , but this previously established model provides a uniform reproducible simulation of osteoporotic bone.3 synthetic bone blocks helped to minimize the effect of geometric and material property variability associated with cadaveric vertebra .
worst - case scenario of minimal cortical contact of the screw in the pedicle , which is inherent in a cadaveric model due to the pedicle 's oval cross - sectional shape .
there should be some caution translating this research to an in vivo application as tapping prior to placing augmentation and screws may have consequences that have not been well studied .
one potential disadvantage of tapping first , prior to augmentation and screw placement , is that the tap creates a larger hole and could lead to a pedicle breach and therefore a conduit for cement extravasation .
this breach may not have been present if the tract was not tapped prior to cement placement . the obvious advantage to tapping first , beyond the enhanced fixation as suggested in our research ,
the tapping also more closely approximates the diameter of the screw and therefore should better allow the surgeon to palpate a breach or a potentially misdirected screw trajectory .
the other component of this study was the evaluation of screw trajectory and the effect of sagittal screw angulation with or without screw augmentation . in the na screws , changing the angulation from 0 ( straight ahead ) to 23
the hypothesis for such a dramatic difference is that this angulation of screws allows the screw tip to better approximate the simulated superior end plate . in vivo
this cephalad angulation of the screw could better approximate the screw tip to the relatively denser bone found adjacent to the superior end plate .
this might lead to fewer early instrumentation failures in the osteoporotic spine and hopefully translate to improved clinical results . further clinical study is clearly needed .
one certainly needs to be aware of anatomic constraints of placing screws with more cephalad orientation .
violating the end plate or pedicle with this cephalad trajectory could certainly have very undesirable consequences .
the results between the augmented screws with different trajectories also showed a substantial improvement in the group with greater cephalad angulation , but the difference between the two trajectories was less dramatic ( 29% increase in load to failure ) .
cement augmentation alone provides substantial stability , and the marginal increase in stability achieved with a cephalad oriented and augmented screw is likely not worth further investigation given the anatomic constraints and risks of angulating screws cephalad .
unfortunately , the number of patients with osteoporosis and patients with low bone mineral density continues to rise as our population is living longer .
standard pedicle screw fixation in this population is often inadequate and can lead to devastating complications.15
16
17
18 because of this , we need to investigate and exploit every potential advantage for stable fixation in the aging spine .
based on an established osteoporotic bone block model with multicomponent loading , it appears that there is a substantial improvement in stability when tapping and tapping long prior to bioactive cement augmentation of pedicle screws . in our study , pretapping the entire screw length prior to placement of augmented screws led to a 54% increase in load to failure when compared with a nontapped but still augmented screw .
this is a relatively straightforward adjustment in surgical technique when placing augmented screws into the osteoporotic spine and has the potential to further limit early fixation failure .
based on the same osteoporotic model , we conclude that it appears cephalad angulated pedicle screw placement provides substantially greater stability when compared with a standard straight - ahead trajectory with both augmented and na screw constructs .
the screws that are oriented cephalad would more closely approximate the dense bone of the superior end plate and therefore lead to greater fixation . | study design biomechanical study of pedicle screw fixation in osteoporotic bone .
objective to investigate whether it is better to tap or not tap osteoporotic bone prior to placing a cement - augmented pedicle screw .
methods initially , we evaluated load to failure of screws placed in cancellous bone blocks with or without prior tapping as well as after varying the depths of tapping prior to screw insertion .
then we evaluated load to failure of screws placed in bone block models with a straight - ahead screw trajectory as well as with screws having a 23-degree cephalad trajectory ( toward the end plate ) .
these techniques were tested with nonaugmented ( na ) screws as well as with bioactive cement ( bioc ) augmentation prior to screw insertion .
results in the na group , pretapping decreased fixation strength in a dose - dependent fashion . in the bioc group ,
the tapped screws had significantly greater loads to failure ( p < 0.01 ) . comparing only the screw orientation , the screws oriented at 23 degrees cephalad had a significantly higher failure force than their respective counterparts at 0 degrees ( p < 0.01 ) .
conclusions standard pedicle screw fixation is often inadequate in the osteoporotic spine , but this study suggests tapping prior to cement augmentation will substantially improve fixation when compared with not tapping .
angulating screws more cephalad also seems to enhance aging spine fixation . |
public health is an important subject which requires the application of the discipline of health economics to facilitatte the identification of cost effective interventions to improve public health . without this targeting public health investment
all countries have to make difficult choices about how to allocate scarce resources amongst competing uses .
everywhere there is continuous debate about the size of the public sector and the need to balance this against the funding of a vigorous private sector . within the public sector
difficult choices have to be made between and within essential services such education , defence and health care .
it is characterised by increasing demand due to the ageing of populations and technological change , rising expenditures and a debate about the relative roles of curative medicine and investments in public health .
an appealing analogy is that investment upstream on prevention would save subsequent expenditure downstream on care and cure .
but is prevention better than cure , and if so which interventions offer the greatest benefit to patients at least cost ? given the universal scarcity of resources , economic recession and austerity , answers to this crucial question remain elusive .
how can knowledge about the relative productivity of investing in curative and preventive care be improved ?
a discipline is a way of analysing the world which creates hypotheses that are subject to empirical testing .
it is a mode of thinking that facilitates the use of particular concepts and relationships that can be used to ask questions and collect data . over time
examples of social science disciplines particularly relevant to the production of health and health care are psychology , sociology and economics .
history , medicine and public health are three areas which can be called subjects . these topics can be explored using social science disciplines .
for instance fogel and engerman postulated that slavery in the usa was economically efficient ( if morally repugnant ) .
social medicine , community medicine and public health are three titles of discourse on the subject of health production .
many decades ago it was defined by holland as a subject that covers the organisation and evaluation of health care systems and the medical aspects of the administration of health care service . nowadays
the subject public health covers not only the production and distribution of health care but also the production and distribution of health .
the subject of public health involves use of a broad range of activities , only some of which are disciplines e.g. epidemiology , statistics , sociology , social policy , public administration , management , psychology , operations research and planning . of theses some are disciplines and
some are not e.g. epidemiology which involves the analysis of the causes and distribution of disease in populations rather than individuals is a discipline like the social sciences .
the vigour and success of a subject such as public health depends on the complementary use of a range of disciplines .
public health exhibits a peculiar deficiency : the relative neglect of the use of economics .
the poor exploitation of the discipline of economics by public health practitioners is epitomised by the marmot report .
this report was commissioned by the labour government and focused the identification of inequalities in health in the united kingdom and how these long established variations could be reduced .
the report concluded that investment in remedial policies was required in six areas : give every child the best start in life ; enable all children , young people and adults to maximise their capabilities and have control over their lives , create fair employment and good work for all ; ensure a healthy standard of living for all ; create and develop healthy and sustainable places and communities ; strengthen the role and impact of ill health preventions .
it begs answers to two crucial questions that can only be answered by economic evaluation of competing investments in public health .
firstly how are these policy objectives ranked ? secondly what trade - offs are there within and between each group
failure to address these issues leaves decision makers ill informed about how to prioritise investments in the production of health .
such ignorance is not bliss as it is likely to waste scarce economic resources and frustrate improvements in public health and reductions in health inequalities .
public health is a subject area of great policy importance but its development has been myopic .
returning to the issue of health inequalities , where the united kingdom and the united states have despite their relative affluence some of greatest health inequalities in the developed world , there continues to be political and social concern but a failure to develop evidence based remedial policies . in the uk
there have been a series of reports over decades : the black report , the acheson report , and more recently the marmot report .
these reports have offered great detail about differences in mortality and morbidity of rich and poor groups , exploiting cross section and cohort data each of these uk reports has described in great detail the differences in life expectation , mortality and morbidity of rich and poor social groups .
each report has attracted high levels of public interest and political agreement that such inequalities should be reduced . however a characteristic of this very pertinent work has been an absence of detail about which types of investment would be most efficient in reducing health inequalities .
for instance , with a child born today in deprived glasgow in scotland having a life expectation at least a decade less than a child born in affluent kensington and chelsea in london , the policy question is how can than inequality be reduced best at least cost ?
for instance is it best to target resources at young children and develop in their first three years of life their cognitive and non cognitive skills , thereby enhancing their life time earnings potential and capacity to invest in health activities ? if the answer to this question is yes , which policy interventions are most cost effective ?
investing in health production in this way implies relative neglect of older children and adults , leaving them to their fate and premature death and excess morbidity during their lifetimes .
where is the evidence that this is an efficient policy ? in both the public health and the economics literature on the reduction of health inequalities there appears to be a consensus that investment in interventions in the early years , particularly pre - school , are likely to be more efficient .
investment later , for instance in teenagers , is unlikely to be good value for money as their cognitive and non cognitive skills may be difficult to improve compared to those in early life .
the public health - social epidemiology approach to inequality if it neglects economics will continue to myopic , providing a diagnosis but little insight into the efficient treatment of the problem . how can economics and public health be married or better integrated to produce more efficient and comprehensive public policy .
this task is not easy as the literature is often poor in terms of trial design and reporting .
for instance katikireddi and colleagues have analysed a recent english policy document which espouses investment in those interventions that are effective .
they show how current politicians offer the rhetoric of funding only those investments that are demonstrably effective .
however despite their good intent , katikireddi and his colleagues show that the quantity and quality of evidence of effectiveness is limited and some government policies lack an evidence base of effectiveness they appraise evidence for a range of policies : early years , physical activity , food , community interventions , and inequalities and show the limits of the evidence base .
the result of poor evaluation of effectiveness of public health interventions is that policies tend to be based on faith rather than evidence .
does the banning of smoking in public places such as restaurants and bars reduce tobacco consumption ?
a study comparing smoke free legislation in scotland with england prior to its banning smoking in public places has shown that it had no effect on tobacco consumption in scotland .
the policy may have reduced passive smoking mortality and morbidity , for which there is no comparable data , and improved environmental cleanliness but its effect on tobacco consumption appears to be zero .
evidence of effectiveness is a necessary but not a sufficient criterion for investment in the production of health in the curative or the public health sectors .
what is effective may not be cost effective but what is cost effective is always effective .
assume that intervention a produces 50 quality adjusted life years ( qalys or years of good quality life ) and intervention b produces 250 qalys . the myopic medical paradigm , and
the patient or beneficiary from such investment would choose intervention b as it produces the best outcome i.e. 200 more qalys than investing in intervention a. however , this conclusion is premature as it ignores the cost of the alternatives .
all investment decisions involve an opportunity cost i.e. a decision to spend on one option deprives the beneficiaries of another option of benefits .
if we assume that intervention a costs 500 and intervention b costs 5000 , this means that a produces each qaly at the cost of 10 whilst intervention b produces each qaly at a cost of 20 . if the programme budget is 100000 , intervention a which is least effective produces 10000 qalys whilst intervention b ( the more effective ) produces only 5000 qalys .
thus when investing in health care , curative and public health , evidence is needed of effectiveness and cost effectiveness of competing interventions .
they could have usefully developed their argument and noted more thoroughly that the cost effectiveness evidence base is even poorer .
why is the quality of effectiveness and cost effectiveness research in public health so poor ?
it seems that the lessons disseminated by the cochrane collaboration ( www.cochrane.org ) have failed to affect practice in public health .
the problem , as chalmers has emphasised in his criticism of research in curative medicine , is not lack of resources but the poor quality of research work .
the literature exhibits a tendency to select multiple end points for appraisal when single end points might be preferred .
how long should trials continue e.g. when measuring tobacco quit rates success within the year may be cancelled out later by smoking resumption ? costing interventions for essential cost effectiveness studies is essential but difficult .
however health care systems generally have poor unit cost data as audit and performance management systems tend to be focused on macro expenditure control i.e. staying in budget .
what costs should be included in an evaluation ? should it focus on the costs to the local health service ? or should health service costs and costs to the patient and their carers
the english national institute for health and clinical excellence ( nice ) is required to carry out technology and public health appraisals focused only on the costs to the nhs of the competing interventions and ignores broader social cost issues .
another complex aspect of economic evaluation which is particularly acute in public health is the discounting costs and benefits .
if offered 100 now or 100 in a year s time , most would opt for 100 now i.e. they prefer benefits now to benefits in the future . if offered the choice of paying a debt now or paying it off in the future , most would prefer to delay payment and pay in the future .
when carrying out economic evaluation what choice of discount rates ` should be used for costs and benefits ?
should the rates be the same for costs and benefits ? currently the english nice uses different discount rates for cost and benefits : six per cent for costs and one and a half per cent for benefits .
the combined effects of poor evaluation of the effectiveness and the reluctance of public health practitioners to incorporate economic evaluation into their work has created a paucity of evidence to inform investment in public health .
as ever , and in medicine , the volume of literature is substantial and growing but sadly its quality is poor .this problem is epitomised by the work of nice which although charged with the task of evaluating public health policies is finding it difficult to make progress due to the poor quality of effectiveness and economic data being reported in the literature .
public health is a subject area of great policy importance but its development has been myopic .
returning to the issue of health inequalities , where the united kingdom and the united states have despite their relative affluence some of greatest health inequalities in the developed world , there continues to be political and social concern but a failure to develop evidence based remedial policies . in the uk
there have been a series of reports over decades : the black report , the acheson report , and more recently the marmot report .
these reports have offered great detail about differences in mortality and morbidity of rich and poor groups , exploiting cross section and cohort data each of these uk reports has described in great detail the differences in life expectation , mortality and morbidity of rich and poor social groups .
each report has attracted high levels of public interest and political agreement that such inequalities should be reduced . however a characteristic of this very pertinent work has been an absence of detail about which types of investment would be most efficient in reducing health inequalities .
for instance , with a child born today in deprived glasgow in scotland having a life expectation at least a decade less than a child born in affluent kensington and chelsea in london , the policy question is how can than inequality be reduced best at least cost ?
for instance is it best to target resources at young children and develop in their first three years of life their cognitive and non cognitive skills , thereby enhancing their life time earnings potential and capacity to invest in health activities ? if the answer to this question is yes , which policy interventions are most cost effective ?
investing in health production in this way implies relative neglect of older children and adults , leaving them to their fate and premature death and excess morbidity during their lifetimes .
where is the evidence that this is an efficient policy ? in both the public health and the economics literature on the reduction of health inequalities there appears to be a consensus that investment in interventions in the early years , particularly pre - school , are likely to be more efficient .
investment later , for instance in teenagers , is unlikely to be good value for money as their cognitive and non cognitive skills may be difficult to improve compared to those in early life .
the public health - social epidemiology approach to inequality if it neglects economics will continue to myopic , providing a diagnosis but little insight into the efficient treatment of the problem . how can economics and public health be married or better integrated to produce more efficient and comprehensive public policy .
this task is not easy as the literature is often poor in terms of trial design and reporting .
for instance katikireddi and colleagues have analysed a recent english policy document which espouses investment in those interventions that are effective .
they show how current politicians offer the rhetoric of funding only those investments that are demonstrably effective .
however despite their good intent , katikireddi and his colleagues show that the quantity and quality of evidence of effectiveness is limited and some government policies lack an evidence base of effectiveness they appraise evidence for a range of policies : early years , physical activity , food , community interventions , and inequalities and show the limits of the evidence base .
the result of poor evaluation of effectiveness of public health interventions is that policies tend to be based on faith rather than evidence .
does the banning of smoking in public places such as restaurants and bars reduce tobacco consumption ?
a study comparing smoke free legislation in scotland with england prior to its banning smoking in public places has shown that it had no effect on tobacco consumption in scotland .
the policy may have reduced passive smoking mortality and morbidity , for which there is no comparable data , and improved environmental cleanliness but its effect on tobacco consumption appears to be zero .
evidence of effectiveness is a necessary but not a sufficient criterion for investment in the production of health in the curative or the public health sectors .
what is effective may not be cost effective but what is cost effective is always effective .
assume that intervention a produces 50 quality adjusted life years ( qalys or years of good quality life ) and intervention b produces 250 qalys . the myopic medical paradigm , and the patient or beneficiary from such investment would choose intervention b as it produces the best outcome i.e. 200 more qalys than investing in intervention a. however , this conclusion is premature as it ignores the cost of the alternatives .
all investment decisions involve an opportunity cost i.e. a decision to spend on one option deprives the beneficiaries of another option of benefits .
if we assume that intervention a costs 500 and intervention b costs 5000 , this means that a produces each qaly at the cost of 10 whilst intervention b produces each qaly at a cost of 20 .
if the programme budget is 100000 , intervention a which is least effective produces 10000 qalys whilst intervention b ( the more effective ) produces only 5000 qalys .
thus when investing in health care , curative and public health , evidence is needed of effectiveness and cost effectiveness of competing interventions .
they could have usefully developed their argument and noted more thoroughly that the cost effectiveness evidence base is even poorer .
why is the quality of effectiveness and cost effectiveness research in public health so poor ?
it seems that the lessons disseminated by the cochrane collaboration ( www.cochrane.org ) have failed to affect practice in public health .
the problem , as chalmers has emphasised in his criticism of research in curative medicine , is not lack of resources but the poor quality of research work . however , public health research is not easy to design and implement .
the literature exhibits a tendency to select multiple end points for appraisal when single end points might be preferred .
how long should trials continue e.g. when measuring tobacco quit rates success within the year may be cancelled out later by smoking resumption ?
however health care systems generally have poor unit cost data as audit and performance management systems tend to be focused on macro expenditure control i.e. staying in budget .
what costs should be included in an evaluation ? should it focus on the costs to the local health service ? or should health service costs and costs to the patient and their carers
the english national institute for health and clinical excellence ( nice ) is required to carry out technology and public health appraisals focused only on the costs to the nhs of the competing interventions and ignores broader social cost issues .
another complex aspect of economic evaluation which is particularly acute in public health is the discounting costs and benefits .
if offered 100 now or 100 in a year s time , most would opt for 100 now i.e. they prefer benefits now to benefits in the future .
if offered the choice of paying a debt now or paying it off in the future , most would prefer to delay payment and pay in the future .
when carrying out economic evaluation what choice of discount rates ` should be used for costs and benefits ?
should the rates be the same for costs and benefits ? currently the english nice uses different discount rates for cost and benefits : six per cent for costs and one and a half per cent for benefits .
the combined effects of poor evaluation of the effectiveness and the reluctance of public health practitioners to incorporate economic evaluation into their work has created a paucity of evidence to inform investment in public health .
as ever , and in medicine , the volume of literature is substantial and growing but sadly its quality is poor .this problem is epitomised by the work of nice which although charged with the task of evaluating public health policies is finding it difficult to make progress due to the poor quality of effectiveness and economic data being reported in the literature .
the topic of public health is not making sufficient impact on resource allocation in all health care systems .
the important issues it raises , as epitomised by the analysis of health inequalities , tend to create considerable public interest but little impact on the use of resources .
this is a product of both the failure of the public health industry to conduct and report well designed trails of the effectiveness of the policies they espouse and their failure to include cost components in their studies .
the absence of cost data and the absence of economic evaluation make prioritisation of competing investments impossible . in the absence of economic data resources
its mitigation requires that the practitioners of the arts and science of public health embraces the techniques of economic evaluation urgently and comprehensively . | with resources always scarce , limited resources have to be targeted at those interventions , prevention and cure , that give the greatest population health gain at least cost .
mere identification of what works in prevention is inadequate unless this evidence is supplemented with economic analysis that identifies what is cost effective .
public health without the use of economics is incomplete . |
it has been established for a long time that psychological interventions can markedly alter patients ' thinking patterns , beliefs , attitudes , emotional states , and behaviors .
the neural mechanisms mediating such alterations were poorly understood before the advent of functional neuroimaging techniques ( eg , single - photon emission computed tomography [ spect ] , positron emission tomography [ pet ] , and functional magnetic resonance imaging [ fmri ] ) . since the turn of the new millenium
, a number of functional neuroimaging studies have been conducted to elucidate this important issue .
some of these studies have explored the neural impact of various forms of psychotherapy in individuals with major depressive disorder ( mdd ) .
other neuroimaging studies have investigated the effects of psychological interventions for anxiety disorders ( obsessive - compulsive disorder [ ocd ] , panic disorder [ pd ] , post - traumatic stress disorder [ ptsd ] , social phobia , and spider phobia ) .
these studies are reviewed in the present article , and the putative neural mechanisms of change in psychotherapy are discussed . emphasis is put on brain imaging investigations of psychotherapy for major depressive disorder ( mdd ) .
to date , a few neuroimaging studies have been carried out during the resting state to measure the impact of psychotherapy in people with mdd . in one of these studies ,
after this initial scan , patients had six 1-hour weekly sessions of interpersonal psychotherapy ( ipt ) .
ipt is a brief form of psychotherapy that helps depressed individuals learn how to deal more effectively with others , to reduce conflict , and gain support from family and friends .
the decrease in depressive symptoms was associated with increased regional cerebral blood flow ( rcbf ) in the posterior cingulate cortex and right basal ganglia .
brody and colleagues have also explored the impact of ipt on regional cerebral metabolic activity in individuals with mdd .
twenty - four participants with mdd and 16 normal control volunteers ( who received no treatment ) underwent fdg- ( fluorodeoxyglucose- ) pet scanning before and after 12 weeks .
the initial fdg - pet scan revealed that mdd participants had higher metabolism than control volunteers in the prefrontal cortex ( pfc ) , caudate , and thalamus , and lower metabolism in the anterior inferior temporal lobe .
this is consistent with neuroimaging studies of mdd showing regional metabolic abnormalities in the pfc , anterior cingulate gyrus , and temporal lobe .
following ipt , mdd participants had metabolic changes in the direction of normalization in these brain regions .
symptomatic improvement was accompanied by significant increases in the left temporal lobe and anterior insula , and significant decreases in the right middle frontal gyrus ( including both the ventrolateral pfc [ vlpfc ] and the dorsolateral pfc [ dlpfc ] ) , right dorsal caudate , and left middle anterior cingulate cortex ( acc ) .
moreover , improvement in cognitive function positively correlated with changes in dlpfc metabolism , whereas reductions of ventral and dorsal pfc metabolism were associated with decreases in anxiety / somatization and psychomotor retardation symptoms .
other neuroimaging studies have examined the effects of cognitive behavioral therapy ( cbt ) in people with mdd .
cbt seeks to train patients to identify and change negative beliefs and negative interpretations related to the past , present , and future . in one of these investigations , goldapple
et al used fdg - pet to measure the brain changes induced by cbt in 17 unmedicated individuals with mdd .
participants were scanned before and after a 15- to 20-session cbt treatment . during treatment , participants learned a number of behavioral and cognitive strategies aiming to combat dysphoric mood and diminish automatic reactivity to negative thoughts and attitudes .
specifically they were taught cognitive monitoring to dismantle ostensibly complex chains of thinking and feeling into separate elements ; they were also requested to increase the frequency of pleasant events in their lives , to record their thinking using thought records , and to test their interpretations and beliefs between sessions .
this improvement was accompanied by increases in the parahippocampal gyrus and dorsal acc ( brodmann area [ ba ] 24 ) , and decreases in dorsal ( ba 9 and 46 ) , ventral ( ba 47 and 11 ) , and medial ( ba 9 , 10 and 11 ) pfc . in another 18fdg - pet investigation , kennedy and
coworkers scanned 12 individuals with mdd before and after 16 weeks of treatment with cbt .
response to cbt ( n=7 ) was associated with increased glucose metabolism in the right inferior occipital cortex , as well as reduced glucose metabolism in the lateral orbitofrontal cortex ( ba 11 and 47 ) and left dorsomedial pfc ( ba 8) .
therefore , the identification of the neural correlates of symptom reduction is a primary aim of the neuroimaging studies of psychotherapy . in this context
, symptom provocation can allow researchers to compare brain responses to trigger scenarios or stimuli before and after treatment , and thus assess the impact of psychotherapy on brain activity .
using fmri and a symptom provocation paradigm ,
dichter et al examined the effects of behavioral activation therapy for depression ( batd)a form of therapy designed to enhance engagement with positive stimuli and reduce avoidance behaviors on the neural correlates of cognitive control in emotional contexts .
fifteen people with mdd were scanned before and after therapy while they performed a task requiring cognitive control in both sad and neutral contexts . before batd , the participants recruited prefrontal cortical areas ( right orbital frontal cortex [ ba 47 ] , right frontal pole [ ba 10 ] , and paracingulate gyrus [ ba 9 ] ) to a greater extent to cognitive control stimuli presented in sad contexts than in neutral contexts .
following batd , decreased activation in response to cognitive control stimuli presented within a sad context was noted in these prefrontal structures . of note , the magnitude of pretreatment activation in the part of the paracingulate gyrus cluster responsive to treatment predicted the magnitude of depressive symptom change after batd .
the effect of a long - term , psychodynamic intervention has been recently assessed in recurrently depressed unmedicated individuals ( n=16 ) .
descriptions containing personal core sentences previously extracted from an attachment interview alternated with presentations of attachment - related scenes with neutral descriptions . compared with control participants
, mdd participants displayed a greater activation in the subgenual cingulate cortex [ ba 25 ] , medial pfc [ ba 8 and 9 ] , and left anterior hippocampus / amygdala before treatment , and a reduction in these brain regions after long - term psychodynamic therapy .
a limbic - cortical - striatal - pallidal - thalamic circuit has been proposed to play a pivotal role in the pathogenesis and maintenance of the mdd .
this circuit has connections to several cortical areas including the medial pfc , the dorsomedial / dorsal anterolateral pfc , the mid and posterior cingulate cortex , the anterior superior temporal gyrus , and the entorhinal and posterior parahippocampal cortices .
several studies exploring the brain metabolic correlates of mdd have reported , during resting state , metabolic abnormalities in these structures , including the dorsolateral prefrontal areas ( known to be involved in cognitive control and working memory ) and ( para ) limbic regions ( presumably implicated in ruminative thoughts and negative emotional states ) .
it seems likely that distinct psychotherapies , such as ipt and cbt , exert differing effects on the brain at cellular and molecular levels .
unfortunately , we know very little regarding this basic issue . as for the global level
, reduced glucose metabolism in the orbitofrontal areas ( ventromedial and ventrolateral ) following cbt has been interpreted as correlates of the learned reduction of ruminations and maladaptive associative memories .
these cortical regions which are anatomically connected to the amygdala , hypothalamus , and brain stem are thought to be implicated in the integration of experiential stimuli with emotional salience .
furthermore , the orbital frontal cortex has been shown to be involved with emotional processing biases in depressed individuals .
response to cbt was also found to be associated with a reduction of glucose metabolic activity in the medial pfc .
this cortical area , which seems to be implicated in self - referential processing of emotional stimuli , is activated during a wide variety of emotional tasks , including attention to subjective feeling , recollection of emotionally charged personal life events , and processing of emotion - related meanings .
the fmri activation studies conducted by dichter et al and buchheim et al indicate that batd and long - term , psychodynamic therapy can also modulate the activity of brain regions and circuits implicated in various aspects of emotion processing .
the reduced activation of the anterior hippocampus / amygdala complex detected after psychodynamic therapy , in response to personally relevant material , is particularly interesting given that this cerebral structure displays enhanced reactivity in mdd . along the same lines ,
a reduction in the reactivity of the subgenual cingulate cortex was also detected in the buchheim et al study .
now this portion of the cingulate cortex appears to be critically involved in mood dysregulation and its resolution .
it is important to recognize that the functional neuroimaging studies of psychotherapy for mdd do not always yield similar findings .
several potentially biasing factors may lead to contrasting results and render problematic a direct comparison between these studies .
for example , mdd may have many different causes that are difficult to differentiate clinically .
this may lead to a variability in symptoms and regional brain abnormalities across depressed individuals .
the differences in rationale , technique , and efficacy of the various psychotherapeutic modalities investigated constitute another factor .
furthermore , inconsistencies in results can be produced by a more or less rigid adherence to a given framework , varying numbers of sessions , distinct milieus ( eg , individual vs group therapy ) , and the use of single vs multiple therapists .
other methodological factors that vary between neuroimaging studies of psychotherapy include the phenomena measured ( eg ,
metabolic activity vs hemodynamic activity ) , the sensitivity and spatial / temporal resolutions of the neuroimaging techniques used , and the methods for examining regional brain activity ( eg , voxel - based techniques , region - of - interest - based approaches ) .
the sample size , the type of control participants ( eg , healthy , waitlist ) , and the point of the second scan within the treatment course , may also lead to divergent results .
a number of functional neuroimaging studies have been conducted , during resting state , to explore the effects of psychological interventions for anxiety disorders .
these researchers used fdg - pet to measure regional glucose metabolism in individuals with ocd before and after 10 weeks of structured exposure and the four- step cognitive behavioral treatment method .
the goal of this treatment method is to teach people with ocd to respond to the intrusive thoughts and urges in a new and more adaptive way .
the first step involves teaching patients to relabel the intrusive thoughts and urges as symptoms of the brain disorder known as ocd . in the second step ,
patients are encouraged to reattribute the disturbing and persistent nature of the symptoms to false messages arising out of a dysfunctional brain .
the primary aim of the first two steps is to produce an alteration in perspective regarding ocd symptoms , which results in patients appreciating the fact that they have a critically important choice to make concerning their behavioral responses in the moments after symptoms intrude into consciousness . in the third step
, patients learn to change behavioral responses while the uncomfortable intrusive thoughts and urges are still present . in the fourth step
, patients come to revalue the intrusive thoughts and urges as much less important , and the fear and anxiety associated with them vanish gradually .
one aspect of this training that is especially crucial is mindfulness ( or mindful awareness ) , ie , the ability to observe one 's own mental phenomena with the calm clarity of an impartial spectator .
this ability allows the patient to create a distance between his / her experience of the self and his / her experience of the ocd symptoms .
it also increases his / her capacity to choose how to respond to intrusive thoughts and urges .
the results of the pet scans revealed significant bilateral decreases in caudate glucose metabolic rates that were greater in treatment responders than those seen in poor responders .
in addition , correlations of brain activity , in the right hemisphere , between the orbitofrontal gyrus and the head of the caudate nucleus , and the orbital gyrus and the thalamus , diminished significantly after effective treatment .
the hyperactivity of the caudate before treatment , and the reduction of its activity after intervention , are consistent with the alleged role of this structure in the pathophysiology of ocd .
the impact of cbt has also been investigated with fdg - pet in individuals with pd .
the therapy was a 6-week standard group treatment program for pd , consisting of education and corrective information , cognitive restructuring , in vivo exposure , and problem solving , as well as training in diaphragmatic breathing and relaxation .
the severity of panic disorder was measured with the panic disorder severity scale ( pdss ) .
furthermore , fdg glucose utilization decreases were measured in the right inferior temporal gyrus , and superior and inferior frontal gyri , whereas glucose utilization increases were detected ( mostly in the left hemisphere ) in the inferior frontal gyrus , middle temporal gyrus , and insula . in a similar study , decreased glucose utilization was found in the right hippocampus , left anterior cingulate , left cerebellum , and pons , whereas increased glucose utilization was detected bilaterally in the medial pfc in pd participants who showed improvement after cbt .
other neuroimaging investigations have used a symptom provocation paradigm to measure the effects of psychological interventions for anxiety disorders .
for instance , lindauer et al utilized technetium spect to examine the impact of brief eclectic psychotherapy ( bep ) in individuals with ptsd ( these individuals were randomly assigned to the treatment or a waiting list ) and traumatized control participants .
bep includes a focal psychodynamic approach and incorporates several techniques used in cbt protocols ( eg , cognitive restructuring , imaginal exposure ) .
greater activation was measured in the right insula and right dlpfc in the ptsd group compared with the control group .
after effective psychotherapy , lower activation was found in the right dlpfc relative to the ptsd patients on the waiting list . according to lindauer and coworkers ,
the decreased dlpfc activation is related to the fact that working memory is no longer occupied by traumatic memories after effective psychotherapy .
furmark et al have used a symptom provocation paradigm and oxygen-15-pet to measure the effects of cbt on regional cerebral blood flow ( rcbf ) in social phobia .
previously untreated patients with this disorder were scanned during an anxiogenic public speaking task before and after 9 weeks of treatment or waiting time .
symptoms improved significantly following cbt , but remained unchanged in the waiting list control group . in treatment responders , clinical improvement was associated with a reduced rcbf response to public speaking in the amygdala , hippocampus , and the periamygdaloid , rhinal , and parahippocampal cortices . since the amygdaloid - hippocampal complex has been hypothesized to form an alarm system that is activated by threatening events , furmark et al proposed that a reduction of neural activity in this structure and neighboring cortical areas might be a mechanism by which cbt exerts its anxiolytic effect .
the results of a recent fmri study suggest that functional neuroimaging can predict psychotherapy success in individuals with social phobia . in this investigation ,
fmri was carried out during responses to social signals of threat ( fearful / angry faces ) in patients with social phobia before and after 12 weeks of cbt .
whole brain voxel - wise analyses revealed that therapeutic success was predicted by increased pre treatment activation to threatening faces in higher - order visual regions ( superior and middle temporal gyrus ) , and cognitive and emotion processing areas ( dorsal acc , dorsomedial pfc ) .
these findings are consistent with cognitive models associating reduction in threat processing bias with clinical recovery .
so far , a few fmri studies have been conducted to identify changes in brain activation following cbt in spider phobics . in one of these studies , we used fmri to measure brain responses to the viewing of film excerpts depicting spiders , 1 week before cbt and 1 week after cbt .
responders to cbt were defined as participants who were able to touch , without reporting fear reactions , an entire series of pictures depicting spiders , tv screen spiders , and real spiders .
the fmri results showed that in spider phobics before cbt , the transient state of fear triggered by the phobogenic stimuli was associated with significant activation of the right lpfc , the parahippocampal gyrus , and visual associative cortical areas .
in our view , the activation of the lpfc reflected the use of metacognitive strategies aimed at self - regulating the fear triggered by the spider film excerpts , whereas the parahippocampal activation reflected an automatic reactivation of the contextual fear memory that led to the development of avoidance behavior and the maintenance of spider phobia . after successful completion of cbt , no significant activation was found in the lpfc and the parahippocampal gyrus .
the neuroimaging studies reviewed in this article suggest that alterations in thought patterns , beliefs , feelings , and behaviors occurring during psychotherapeutic interventions can lead to a normalization of functional brain activity at a global level .
these interventions seem to exert potent modulating effects on the brain regions and circuits mediating the symptoms of mdd and anxiety disorders .
for example , the reduction in the medial pfc activity following psychodynamic therapy might suggest that a function of this brain region the extinction of learned associations may no longer be required when the patient is no longer ruminating , rather than the increased activity at baseline representing a source of the pathology .
similarly , increased metabolism in a given brain region may reflect a downstream effect of decreased inhibition in a separate cerebral structure that is more proximate to the functional abnormality .
there is now evidence that psychotherapeutic interventions can modulate different types of neural processes . with respect to this issue , lehto and
colleagues have used spect and the ( 123i)-cit radioligand to compare , in depressive outpatients , serotonin transporter ( sert ) levels before and after 12 months of psychodynamic psychotherapy .
such an increase might have contributed to an enhancement of serotonergic activity in the previously depressed participants .
, a diffusion tensor imaging ( dti ) study was recently conducted to investigate this possibility .
outpatients diagnosed with mdd underwent dti before and after a 4-week course of guided imagery psychotherapy .
fractional anisotropy ( fa)which is thought to reflect microstructural properties of white matter such as myelination , axon caliber , and fiber density was measured in depressed patients and healthy controls , before and after treatment , using whole brain voxel - wise analysis .
clinical improvement was associated with higher fa in the right thalamus . at an early stage of the intervention ,
higher fa was found in a part of the frontal lobe associated with emotion regulation .
as previously mentioned , mdd may have multiple distinct etiologies which are difficult to distinguish clinically .
patient subgrouping based on neurobiological information acquired via neuroimaging may help us understand why some depressed patients improve with specific psychotherapies and others do not .
these researchers utilized multivariate analysis and discovered that the expression of individual brain patterns of activity separated , almost perfectly , a group of schizophrenic patients from a comparison group .
but it does not appear far - fetched to think that in the not - too - distant future , the refinement of functional and molecular neuroimaging data acquisition and analysis techniques will help clinicians to improve patient outcomes by providing useful information related to the selection of optimal treatment and the evaluation of psychotherapy effects . | it has been long established that psychological interventions can markedly alter patients ' thinking patterns , beliefs , attitudes , emotional states , and behaviors .
little was known about the neural mechanisms mediating such alterations before the advent of functional neuroimaging techniques . since the turn of the new millenium
, several functional neuroimaging studies have been conducted to tackle this important issue .
some of these studies have explored the neural impact of various forms of psychotherapy in individuals with major depressive disorder .
other neuroimaging studies have investigated the effects of psychological interventions for anxiety disorders .
i review these studies in the present article , and discuss the putative neural mechanisms of change in psychotherapy .
the findings of these studies suggest that mental and behavioral changes occurring during psychotherapeutic interventions can lead to a normalization of functional brain activity at a global level . |
a 47-year - old man was admitted to hospital with worsening malaise , fatigue , drenching night sweats , anorexia and a 15 kg weight loss .
four months previously , he developed dyspnea , orthopnea , exertional chest heaviness and a single episode of hemoptysis , for which he had been empirically treated with two 10-day courses of oral antibiotics ( cefuroxime , then clarithromycin ) .
transient swelling , erythema and tenderness over his shins ensued , followed by numbness of his fingers and the left side of his body .
a dental procedure ( unknown ) had been performed 12 months before symptom onset . on examination
his temperature was 36.8c , heart rate 80 beats / min , blood pressure 95/50 mmhg , respiratory rate 18 breaths / min and oxygen saturation 99% on 3 l nasal prongs .
cardiovascular examination revealed a soft s2 , an s3 , a grade ii / vi aortic systolic ejection murmur and a grade iii / vi diastolic decrescendo murmur at the left lower sternal border , without signs of congestive heart failure .
fundoscopic examination revealed changes consistent with hypertension , but did not demonstrate retinal hemorrhages or roth s spots .
there was a tender area of induration on the palm of his left hand and the dorsum of his left foot ( figure 1a ) , but no erythema nodosum .
abnormal laboratory investigations included a white blood cell count of 14.110/l ( normal values 410/l to 1110/l ) , neutrophils
1110/l ( normal values 210/l to 810/l ) , a normocytic , normochromic anemia with a hemoglobin level of 117
g / l ) and elevated inflammatory markers ( c - reactive protein level 64 mg
/ l [ normal level 0 mg / l to 8 mg / l ] and erythrocyte sedimentation rate 28 mm ( normal value 0 mm to 10 mm ) .
three temporally distinct sets of blood cultures were obtained , and empirical therapy with intravenous vancomycin and ceftriaxone was initiated .
transesophageal echocardiogram demonstrated a 1.7 cm vegetation on a previously unrecognized bicuspid aortic valve , with severe aortic insufficiency ( figure 1b ) .
magnetic resonance angiography of the brain revealed a focus of restricted diffusion in the corpus callosum , concerning for an infarct . by 48 h , all blood cultures yielded a gram - negative bacillus ( oxidase positive , catalase negative and urease negative ) identified as cardiobacterium hominis ( figure 1c ) .
the identification was confirmed by matrix - assisted laser desorption ionization time - of - flight ( vitek ms ) and the vitek nh identification card ( biomrieux canada inc , canada ) . at the time of aortic valve replacement ,
both native aortic leaflets were markedly thickened and a perivalvular abscess was observed at the aortic valve root , which subsequently grew c hominis .
initial etest ( biomrieux canada inc ) results , using mueller - hinton agar supplemented with 5% defibrinated sheep blood ( mhb ) , demonstrated resistance to ceftriaxone and cefotaxime ( figure 1d ) .
the nitrocefin - based test ( nitrocefin sr112 , oxoid microbiology products , usa ) for beta - lactamase production was negative .
subsequently , broth microdilution ( bmd ) in cation - adjusted mueller - hinton broth supplemented with 5% lysed horse blood ( camhb - lhb ) as well as etest susceptibilities using brucella blood agar ( 1 ) demonstrated susceptibility to all agents tested ( table 1 ) .
endovascular infection with c hominis , a member of the hacek group of microorganisms ( haemophilus species , actinobacillus actinomycetemcomitans , cardiobacterium hominis , eikenella corrodens and kingella species ) , is usually insidious in onset , with a prolonged subacute course characterized by leukocytosis , anemia , splenomegaly , embolic phenomena , congestive heart failure and weight loss ( 2 ) .
dental work ( as well as routine oral hygiene / quality of dentition ) and bicuspid aortic valve are both well - documented risk factors for developing infective endocarditis due to hacek microorganisms ( 2 ) .
although c hominis is of relatively low virulence , endovascular infection complicates 95% of all cases of bacteremia , with the aortic valve being most commonly affected ( 3,4 ) .
peripheral and central nervous system emboli occur frequently in c hominis endocarditis , noted in 51% and 21% of cases , respectively , especially when the aortic valve is involved ( 3,5,6 ) .
prognosis is generally favourable , with a 93% cure rate for both native and prosthetic valve infection ( 3 ) .
a third - generation cephalosporin is the drug of choice for infection with hacek organisms ( 8) . in our patient , initial minimum inhibitory concentration determinations using etest methodology on mhb agar demonstrated resistance to third - generation cephalosporins ( nitrocephin negative ) .
this complicated early management of the patient because it appeared to preclude the use of first - line therapy for c hominis endocarditis .
repeat testing with clinical and laboratory standards institute ( clsi)-approved reference methods ( 1 ) demonstrated susceptibility to third - generation cephalosporins .
major error with other recent c hominis isolates ( n=5 ) in our laboratory ( data not shown ) .
penicillin resistance due to beta - lactamase production has been documented in c hominis ( 9,10 ) , but cephalosporin resistance has only been described in a single case report ( 9 ) based on disc diffusion testing .
guidelines for susceptibility testing of fastidious organisms , including the hacek group , are relatively new ( 1 ) . for cardiobacterium species ,
the recommended testing method is bmd in cambh - lhb ( as for pneumococcus and other fastidious organisms ) .
although not endorsed by clsi , many laboratories use etest or other gradient strip methodologies for ease of use and accessibility . in the present case , use of a non - clsi - approved antibiotic gradient strip susceptibility testing methodology resulted in a major error . the risk of a
the likely cause of the major error was the use of an antibiotic gradient susceptibility testing method with media ( mhb ) not adequately validated in microbiological studies .
mhb agar has previously been used in agar dilution and etest methodologies for other hacek - group organisms ( 1113 ) .
the etest application guide recommends using either brucella blood agar or mueller - hinton agar with 1% hemoglobin and 1% isovitalex ( bd , usa ) for hacek organisms ( 14 ) ; however , the cited literature provides no data on the use of mueller - hinton agar for susceptibility testing of c hominis .
a subpopulation of c hominis with isolated resistance to cephalosporins due to specific penicillin - binding protein mutations ( as has been described in streptococcus pneumoniae ) was also considered to be unlikely given that the intermediate zones did not grow when subcultured to another agar plate and the repeated susceptibility testing using bmd did not show any evidence of resistance . returning to the case ,
the patient received six weeks of intravenous ceftriaxone following his aortic valve replacement , rather than a standard four - week course ( 8) , because of his septic cerebral embolus .
repeat echocardiography demonstrated complete resolution of his perivalvular abscess and a properly functioning prosthetic aortic valve .
his neurological symptoms continued to improve , although he still had residual left - sided numbness at three - month follow - up .
this case highlights both the typical clinical presentation of endovascular infection with c hominis and the potential issues that may arise when performing susceptibility testing for fastidious organisms in the clinical microbiology laboratory . | cardiobacterium hominis is a fastidious organism that can rarely cause subacute bacterial endocarditis .
this report describes a case involving a 47-year - old man with subacute endocarditis due to c hominis ; the isolate was initally found to be resistant to third - generation cephalosporins , but was later found to be susceptible using an alternative method .
the authors discuss the likely cause of this error and emphasize the importance of adherence to fully validated methods for the determination of antibiotic susceptibilities . |
diabetes prevention is critical for women with gestational diabetes mellitus ( gdm ) , which affects 714% of pregnancies ( 5 ) , as women with gdm are seven times more likely to develop type 2 diabetes than parous women without gdm ( 6 ) .
postpartum weight retention increases diabetes risk ( 7 ) , and postpartum weight management for women with gdm is recommended ( 8) . yet
evidence to support health system adoption of postpartum diabetes prevention programs for women with gdm is lacking .
in addition to traditional clinical trials enrolling carefully selected volunteers with a history of gdm under ideal conditions ( 4 ) , pragmatic trials evaluating the effectiveness of diabetes prevention programs in real - world clinical settings among diverse populations are needed to increase generalizability and inform health system adoption ( 9 ) .
the gestational diabetes effects on moms ( gem ) cluster randomized controlled trial compared an existing postpartum diabetes prevention program of mailed recommendations delivered by the kaiser permanente northern california ( kpnc ) perinatal center ( 10 ) ( usual care ) to usual care plus a diabetes prevention program ( dpp)derived lifestyle intervention ( 1 ) .
the dpp - derived intervention was delivered on behalf of the kpnc perinatal center and offered as an optional routine care program ( 11 ) .
the goal of the intervention was to help women with gdm 1 ) reach pregravid weight if their pregravid bmi was < 25.0 kg / m or 2 ) lose 5% of pregravid weight if their pregravid bmi was 25.0 kg / m ( 11 ) .
primary outcomes were the proportion of women who reached postpartum weight goals and weight change from pregravid to postpartum across the 12-month postpartum follow - up period .
secondary outcomes included changes from pregnancy to postpartum in daily total energy intake , percent of calories from fat , physical activity , hypertension , and depression .
the kaiser foundation research institute human subjects committee approved the trial and waived the requirement for informed consent for the intervention .
women provided verbal consent for the survey component of the trial after all medical facilities were randomized ( 11 ) .
the setting was kpnc , an integrated health care delivery system with 44 medical facilities managing 33,000 births annually .
kpnc membership closely approximates the surrounding population ( 12 ) and is dynamic with respect to enrollment .
randomization was blocked on facility size ( i.e. , the expected annual number of women with gdm , in three strata : < 25 , 2574 , and 75 ) .
a restricted randomization scheme was used to ensure acceptable between - condition balance ( i.e. , maximum between - condition relative difference ) in expected racial / ethnic distributions and the number of women contacted by an ongoing unrelated observational study , both overall and within facility size stratum ( 11,13 ) .
all investigators , data collectors , and health care providers were blinded to condition assignment .
potentially eligible women included pregnant women 18 years old diagnosed with gdm ( n = 2,480 ) according to the carpenter and coustan criteria between march 2011 and march 2012 , as recommended by the american college of obstetricians and gynecologists ( 14 ) during this period and implemented across all 44 kpnc medical facilities .
women were excluded if they did not have any telephone contact with the kpnc perinatal center during pregnancy ( n = 142 ) , were missing data on pregravid bmi ( n = 40 ) , or had a neonatal loss ( n = 18 ) , leaving 2,280 women : 1,087 in the intervention condition and 1,193 in usual care ( fig .
a , 12 women were not health plan members and did not have clinic - measured weight , 5 of them provided self - reported weight ; b , 9 women were not health plan members and did not have clinic - measured weight , 4 of them provided self - reported weight ; c , 37 women were not health plan members and did not have clinic - measured weight , 26 of them provided self - reported weight ; d , 36 women were not health plan members and did not have clinic - measured weight , 25 of them provided self - reported weight ; e , 84 women were not health plan members and did not have clinic - measured weight , 61 of them provided self - reported weight ; f , 101 women were not health plan members and did not have clinic - measured weight , 66 of them provided self - reported weight only .
all women with gdm at kpnc are offered supplemental care from the kpnc perinatal center consisting of telephone - based case management for glucose control during pregnancy ( 10 ) . at 6 weeks postpartum
, the kpnc perinatal center sends a letter encouraging women to be screened for diabetes and provides printed materials that emphasize a healthy bmi , participation in 30 min of physical activity daily , and healthy eating .
if screening results indicate prediabetes , women are mailed materials about lifestyle prevention strategies and educational classes .
in addition to usual care , women attending medical facilities assigned to the intervention were mailed , within 2 weeks of the gdm diagnosis , a tailored letter detailing a personalized goal for gestational weight gain with healthy eating and physical activity tips ( 11 ) .
after delivery , women were offered a print / telephone - based lifestyle program modeled after the dpp intervention ( 11 ) .
the program was presented as optional routine care and delivered by gem coaches on behalf of the kpnc perinatal center . during the core of the postpartum intervention ( 6 weeks to 6 months ) , the primary target was to help women achieve the postpartum weight goals : 1 ) reaching pregravid weight if their pregravid bmi was < 25.0 kg / m or 2 ) losing 5% of pregravid weight if their pregravid bmi was 25.0 kg / m ( 11 ) .
women were mailed a 13-session guidebook to review via telephone with a lifestyle coach , a registered dietitian employed for the trial and based at the kpnc perinatal center .
women were encouraged to set weekly goals for daily fat and caloric intake and to work up to or continue participating in 150 min of moderate- to vigorous - intensity physical activity per week ( 11 ) .
behavior change techniques were individualized to women s preferences , resources , and cultural context using motivational interviewing and theoretical constructs derived from social cognitive theory ( 15 ) and the transtheoretical model ( 16 ) .
all telephone sessions were audiotaped ; a random 10% were coded using a brief checklist of intervention components to assess fidelity to the protocol .
fidelity was high ( mean proportion of intervention components present = 95% , range 60100% ) . during the maintenance phase ( 712 months postpartum )
the direct cost of the intervention included coaches work hours , calculated using the may 2012 median annual wage for registered dietitians ( 17 ) plus fringe benefits ( 29% of annual wages ) , and the cost of printing and mailing materials .
all 2,280 eligible women in the 44 medical facilities were invited to complete study surveys ( 11 ) ; 1,783 ( 78.2% ) completed the baseline survey during pregnancy after the gdm diagnosis ( mean gestational age at completion 29.4 weeks [ sd 6.1 ] ) .
survey responders did not differ from nonresponders except for being less likely to be of non - hispanic white origin ( 11 ) .
responders to the baseline survey were asked to complete surveys at 6 weeks , 6 months , and 12 months postpartum ( 11 ) , with 91.6 , 87.9 , and 84.0% retention rates , respectively .
pregravid bmi was calculated from clinic - measured pregravid weight obtained through the electronic health record ( ehr ; 86.0% ) , weight ascertained at the first prenatal clinic visit before 10 weeks ' gestation ( 10.5% , from the ehr ) , or self - reported pregravid weight on the gem pregnancy survey ( 3.5% ) .
clinic - measured postpartum weight was obtained through the ehr for each time point , regardless of participation in the intervention or the gem surveys .
the block food frequency questionnaire ( 18 ) and the pregnancy physical activity questionnaire ( 19 ) were used at baseline and 6 months postpartum to assess usual daily total energy intake and weekly physical activity over the past 3 months .
hypertension was defined as present during pregnancy and at each postpartum time point by searching the ehr for any abnormal blood pressure measurements ( systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg ) or for use of antihypertensive medications .
depression was defined as present during pregnancy and at each postpartum time point by searching the ehr for diagnoses of depression , use of antidepressant medications , or depression score 10 on patient health questionnaire 9 ( phq-9 ) ( 20 ) or score 10 on phq-8 ( 21 ) from surveys at each time point .
age , race / ethnicity , parity , and laboratory test results were also obtained from the ehr .
prediabetes and diabetes were defined according to american diabetes association glyc - emic thresholds ( 22 ) ( see supplementary methods ) .
the sample size of 2,280 , of which 97.4% had at least one clinic - measured weight during the 12-month postpartum follow - up period , allowed for robust estimation of the average effect of the intervention on postpartum weight goals assessed through clinic - measured weight across the 12-month postpartum follow - up period .
we focused post hoc minimum detectable effect estimation at each point in time accounting for the cluster randomization .
of the sample , 93.4 , 71.9 , and 62.3% had clinic - measured weight at 6 weeks , 6 months , and 12 months postpartum , respectively , which led to a minimum detectable absolute difference in the proportion meeting postpartum weight goals ranging from 5.6 to 10.2% , 6.2 to 10.6% , and 6.6 to 11.0% , respectively , across the range in expected proportion meeting goals in the usual care condition ( 1525% , based on our pilot trial [ 24% ] and across the range in expected intraclass correlation [ 0.010.05 ] ) ( see supplementary methods ) .
we note the additional power and precision gained in the analysis of treatment effects by incorporating the repeated measures of meeting postpartum weight goals .
analyses used marginal regression models to estimate population average intervention effects , using logistic regression with estimation via generalized estimating equations in analyses of meeting the weight goals and other dichotomous outcomes , and linear mixed regression in analyses of weight change and changes in other continuous outcomes after adjusting for baseline values .
regression models accounted for the within - medical facility correlation between patients and within - person correlation among repeated measurements for valid estimation of treatment effects and associated standard errors .
variations in treatment condition differences over time were examined via the introduction of appropriate cross - product terms . for all analyses ,
if women had a subsequent pregnancy , they were censored at the estimated time of conception .
primary outcome analysis for the effect of the intervention included only clinic - measured postpartum weights .
sensitivity analyses to address potential bias from missing clinic - measured weight included clinic - measured or self - reported postpartum weight collected during postpartum surveys . additional sensitivity analyses included clinic - measured or imputed postpartum weight ( 23 ) .
two exploratory analyses assessed intervention effectiveness across number of sessions completed and using an instrumental variable to estimate the complier average causal effect , a measure of the effectiveness of the intervention among those who comply with assigned treatment , with the randomization indicator used as the instrumental variable ( 24 ) . for this analysis , all or none
a key applicable assumption is that the outcomes among noncompliers are not affected by randomization condition assignment .
exploratory outcome analysis of prediabetes or diabetes incidence used cox proportional hazards regression to estimate hazard ratios associated with the intervention , controlling for pregnancy fasting and 3-h glucose values and pregravid bmi .
the setting was kpnc , an integrated health care delivery system with 44 medical facilities managing 33,000 births annually .
kpnc membership closely approximates the surrounding population ( 12 ) and is dynamic with respect to enrollment .
randomization was blocked on facility size ( i.e. , the expected annual number of women with gdm , in three strata : < 25 , 2574 , and 75 ) .
a restricted randomization scheme was used to ensure acceptable between - condition balance ( i.e. , maximum between - condition relative difference ) in expected racial / ethnic distributions and the number of women contacted by an ongoing unrelated observational study , both overall and within facility size stratum ( 11,13 ) .
all investigators , data collectors , and health care providers were blinded to condition assignment .
potentially eligible women included pregnant women 18 years old diagnosed with gdm ( n = 2,480 ) according to the carpenter and coustan criteria between march 2011 and march 2012 , as recommended by the american college of obstetricians and gynecologists ( 14 ) during this period and implemented across all 44 kpnc medical facilities .
women were excluded if they did not have any telephone contact with the kpnc perinatal center during pregnancy ( n = 142 ) , were missing data on pregravid bmi ( n = 40 ) , or had a neonatal loss ( n = 18 ) , leaving 2,280 women : 1,087 in the intervention condition and 1,193 in usual care ( fig .
a , 12 women were not health plan members and did not have clinic - measured weight , 5 of them provided self - reported weight ; b , 9 women were not health plan members and did not have clinic - measured weight , 4 of them provided self - reported weight ; c , 37 women were not health plan members and did not have clinic - measured weight , 26 of them provided self - reported weight ; d , 36 women were not health plan members and did not have clinic - measured weight , 25 of them provided self - reported weight ; e , 84 women were not health plan members and did not have clinic - measured weight , 61 of them provided self - reported weight ; f , 101 women were not health plan members and did not have clinic - measured weight , 66 of them provided self - reported weight only .
all women with gdm at kpnc are offered supplemental care from the kpnc perinatal center consisting of telephone - based case management for glucose control during pregnancy ( 10 ) . at 6 weeks postpartum
, the kpnc perinatal center sends a letter encouraging women to be screened for diabetes and provides printed materials that emphasize a healthy bmi , participation in 30 min of physical activity daily , and healthy eating .
if screening results indicate prediabetes , women are mailed materials about lifestyle prevention strategies and educational classes .
in addition to usual care , women attending medical facilities assigned to the intervention were mailed , within 2 weeks of the gdm diagnosis , a tailored letter detailing a personalized goal for gestational weight gain with healthy eating and physical activity tips ( 11 ) .
after delivery , women were offered a print / telephone - based lifestyle program modeled after the dpp intervention ( 11 ) .
the program was presented as optional routine care and delivered by gem coaches on behalf of the kpnc perinatal center . during the core of the postpartum intervention ( 6 weeks to 6 months )
, the primary target was to help women achieve the postpartum weight goals : 1 ) reaching pregravid weight if their pregravid bmi was < 25.0 kg / m or 2 ) losing 5% of pregravid weight if their pregravid bmi was 25.0 kg / m ( 11 ) .
women were mailed a 13-session guidebook to review via telephone with a lifestyle coach , a registered dietitian employed for the trial and based at the kpnc perinatal center .
women were encouraged to set weekly goals for daily fat and caloric intake and to work up to or continue participating in 150 min of moderate- to vigorous - intensity physical activity per week ( 11 ) .
behavior change techniques were individualized to women s preferences , resources , and cultural context using motivational interviewing and theoretical constructs derived from social cognitive theory ( 15 ) and the transtheoretical model ( 16 ) .
all telephone sessions were audiotaped ; a random 10% were coded using a brief checklist of intervention components to assess fidelity to the protocol .
fidelity was high ( mean proportion of intervention components present = 95% , range 60100% ) . during the maintenance phase ( 712 months postpartum ) ,
the direct cost of the intervention included coaches work hours , calculated using the may 2012 median annual wage for registered dietitians ( 17 ) plus fringe benefits ( 29% of annual wages ) , and the cost of printing and mailing materials .
all 2,280 eligible women in the 44 medical facilities were invited to complete study surveys ( 11 ) ; 1,783 ( 78.2% ) completed the baseline survey during pregnancy after the gdm diagnosis ( mean gestational age at completion 29.4 weeks [ sd 6.1 ] ) .
survey responders did not differ from nonresponders except for being less likely to be of non - hispanic white origin ( 11 ) .
responders to the baseline survey were asked to complete surveys at 6 weeks , 6 months , and 12 months postpartum ( 11 ) , with 91.6 , 87.9 , and 84.0% retention rates , respectively .
pregravid bmi was calculated from clinic - measured pregravid weight obtained through the electronic health record ( ehr ; 86.0% ) , weight ascertained at the first prenatal clinic visit before 10 weeks ' gestation ( 10.5% , from the ehr ) , or self - reported pregravid weight on the gem pregnancy survey ( 3.5% ) .
clinic - measured postpartum weight was obtained through the ehr for each time point , regardless of participation in the intervention or the gem surveys .
the block food frequency questionnaire ( 18 ) and the pregnancy physical activity questionnaire ( 19 ) were used at baseline and 6 months postpartum to assess usual daily total energy intake and weekly physical activity over the past 3 months .
hypertension was defined as present during pregnancy and at each postpartum time point by searching the ehr for any abnormal blood pressure measurements ( systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg ) or for use of antihypertensive medications .
depression was defined as present during pregnancy and at each postpartum time point by searching the ehr for diagnoses of depression , use of antidepressant medications , or depression score 10 on patient health questionnaire 9 ( phq-9 ) ( 20 ) or score 10 on phq-8 ( 21 ) from surveys at each time point .
age , race / ethnicity , parity , and laboratory test results were also obtained from the ehr .
prediabetes and diabetes were defined according to american diabetes association glyc - emic thresholds ( 22 ) ( see supplementary methods ) .
the sample size of 2,280 , of which 97.4% had at least one clinic - measured weight during the 12-month postpartum follow - up period , allowed for robust estimation of the average effect of the intervention on postpartum weight goals assessed through clinic - measured weight across the 12-month postpartum follow - up period .
we focused post hoc minimum detectable effect estimation at each point in time accounting for the cluster randomization .
of the sample , 93.4 , 71.9 , and 62.3% had clinic - measured weight at 6 weeks , 6 months , and 12 months postpartum , respectively , which led to a minimum detectable absolute difference in the proportion meeting postpartum weight goals ranging from 5.6 to 10.2% , 6.2 to 10.6% , and 6.6 to 11.0% , respectively , across the range in expected proportion meeting goals in the usual care condition ( 1525% , based on our pilot trial [ 24% ] and across the range in expected intraclass correlation [ 0.010.05 ] ) ( see supplementary methods ) .
we note the additional power and precision gained in the analysis of treatment effects by incorporating the repeated measures of meeting postpartum weight goals .
analyses used marginal regression models to estimate population average intervention effects , using logistic regression with estimation via generalized estimating equations in analyses of meeting the weight goals and other dichotomous outcomes , and linear mixed regression in analyses of weight change and changes in other continuous outcomes after adjusting for baseline values .
regression models accounted for the within - medical facility correlation between patients and within - person correlation among repeated measurements for valid estimation of treatment effects and associated standard errors .
variations in treatment condition differences over time were examined via the introduction of appropriate cross - product terms . for all analyses ,
if women had a subsequent pregnancy , they were censored at the estimated time of conception .
primary outcome analysis for the effect of the intervention included only clinic - measured postpartum weights .
sensitivity analyses to address potential bias from missing clinic - measured weight included clinic - measured or self - reported postpartum weight collected during postpartum surveys . additional sensitivity analyses included clinic - measured or imputed postpartum weight ( 23 ) .
two exploratory analyses assessed intervention effectiveness across number of sessions completed and using an instrumental variable to estimate the complier average causal effect , a measure of the effectiveness of the intervention among those who comply with assigned treatment , with the randomization indicator used as the instrumental variable ( 24 ) . for this analysis , all or none compliance was defined as completing one or more telephone sessions ( 25 ) .
a key applicable assumption is that the outcomes among noncompliers are not affected by randomization condition assignment .
exploratory outcome analysis of prediabetes or diabetes incidence used cox proportional hazards regression to estimate hazard ratios associated with the intervention , controlling for pregnancy fasting and 3-h glucose values and pregravid bmi .
the intervention and usual care conditions were comparable on prerandomization characteristics such as medical facility size ( fig . 1 ) and racial / ethnic and bmi distributions ; however , they differed by fasting and 3-h glucose ( table 1 ) . among the 1,087 women in the intervention
, 50.3% completed one or more telephone sessions ; specifically , 18.8% completed 13 sessions , 16.2% completed 412 sessions , and 15.3% completed all 13 sessions .
baseline characteristics by treatment condition : the gem trial hypertension was defined as systolic blood pressure 140 mmhg , diastolic blood pressure 90 mmhg , or use of antihypertensive medications .
depression was defined as score 10 on phq-8 or phq-9 , physician diagnosis , or use of antidepressant medications .
ogtt , oral glucose tolerance test . * p < 0.05 for the comparison with the intervention condition by two - tailed student t test .
639 women in the intervention and 668 women in usual care with valid diet data at baseline and 6 months postpartum ( e.g. , those with daily total energy intake 600 and 4,000 kcal at both time points ) .
522 women in the intervention and 560 women in usual care with valid physical activity data at baseline and 6 months postpartum ( e.g. , those with 20 h / week of total physical activity and 9 h / week of moderate physical activity at both time points ) . clinic - measured postpartum weight was obtained at least once during the 12-month postpartum follow - up for 97.4% of women ( n = 2,221 ) . in the primary analysis that included only clinic - measured postpartum weight , on average , over the 12-month postpartum period ,
women in the medical facilities assigned to the intervention had a statistically significant 28% higher odds ( odds ratio [ or ] 1.28 [ 95% ci 1.10 , 1.47 ] ) of meeting postpartum weight goals than women in the medical facilities assigned to usual care ( table 2 ) .
the proportion meeting weight goals was significantly higher in the intervention facilities than usual care facilities at 6 weeks ( 25.5 vs. 22.4% ; 1.17 [ 1.01 , 1.36 ] ) and 6 months postpartum ( 30.6 vs. 23.9% ; 1.45 [ 1.14 , 1.83 ] ) .
at 12 months ( 6 months after the intervention ended ) , the condition difference was reduced ( 33.0 vs. 28.0% ; 1.25 [ 0.96 , 1.62 ] ) and no longer statistically significant .
the absolute difference between conditions was 3.1% at 6 weeks , 6.7% at 6 months , and 5.0% at 12 months postpartum . in analyses examining weight change from pregravid to postpartum ( table 2 ) ,
women in the intervention facilities retained less weight than women in usual care facilities at 6 months postpartum ( mean 0.39 kg [ sd 5.5 ] vs. 0.95 kg [ 5.5 ] ; adjusted mean condition difference 0.64 kg [ 95% ci 1.13 , 0.14 ] ) ; the condition difference was attenuated at 12 months postpartum and no longer statistically significant ( 0.43 kg [ 0.98 , 0.11 ] ) ( table 2 ) .
sensitivity analyses that included self - reported or imputed weight if the clinic - measured weight was missing yielded similar results ( tables 2 and 3 ) .
the intervention was equally effective across racial / ethnic groups ( p value for condition race / ethnicity interactions > 0.25 ) .
the intervention was also equally effective across bmi strata ( supplementary tables 1 and 2 ) .
proportion of women meeting the postpartum weight goals with ors estimating differences between conditions and mean changes in weight from pregravid to postpartum with mean differences between conditions : the gem trial * p value for condition differences ; p values for treatment follow - up time point interaction > 0.05 . adjusted for pregravid weight .
mean changes in physical activity and diet from pregnancy to 6 months postpartum with mean differences between conditions : the gem trial * adjusted for baseline values .
in exploratory analyses , the instrumental variable estimates of the absolute differences between conditions among compliers ( defined as completing one or more telephone sessions ) were almost double the absolute differences observed in the primary analysis : estimates for proportions meeting weight goals were 6.2% at 6 weeks , 13.3% at 6 months , and 9.9% at 12 months ; estimates for differences in mean weight change were 0.62 kg at 6 weeks , 1.27 kg at 6 months , and 0.86 kg at 12 months postpartum . in additional exploratory analyses , women who completed all 13 sessions had significantly higher odds of meeting the postpartum weight goals than women in usual care at 6 months ( 3.97 [ 2.75 , 5.72 ] ) and 12 months postpartum ( 2.16 [ 1.52 , 3.07 ] ) ( fig .
2b ) , women who completed 412 sessions retained less weight than women in usual care at 6 months postpartum ( mean 0.13 kg [ sd 5.7 ] vs. 0.94 kg [ 5.5 ] , adjusted mean condition difference 1.1 kg [ 95% ci 1.8 , 0.41 ] ) and at 12 months postpartum ( 0.06 kg [ 5.6 ] vs. 0.50 kg [ 5.4 ] ; 1.0 kg [ 1.9 , 0.13 ] ) .
women who completed all 13 sessions lost more weight than women in usual care at 6 months ( mean 1.89 kg [ sd 5.5 ] vs. 0.94 kg [ 5.5 ] , adjusted mean condition difference 2.3 kg [ 95% ci 3.1 , 1.5 ] ) and 12 months postpartum ( 1.19 kg [ 6.1 ] vs. 0.50 kg [ 5.4 ] ; 1.6 kg [ 2.4 , 0.68 ] ) . or ( 95% ci ) estimating the condition difference in meeting the postpartum weight goals ( a ) and adjusted mean condition difference ( 95% ci ) ( b ) for weight change from pregravid by number of intervention sessions completed and time since delivery : the gem trial . at 6 months postpartum ,
the number of women by number of completed telephone sessions were as follows : 338 for 0 sessions , 151 for 13 sessions , 171 for 412 sessions , and 104 for 13 sessions .
at 12 months , the number of women by number of completed telephone sessions were as follows : 301 for 0 sessions , 134 for 13 sessions , 112 for 412 sessions , and 129 for 13 sessions .
at 6 months postpartum , women in both conditions increased minutes per week spent in vigorous - intensity activity ; however , women in the intervention facilities showed a significantly greater increase ( adjusted mean condition difference 15.4 min / week [ 95% ci 4.9 , 25.8 ] ) ( table 3 ) .
no condition differences were observed for time spent in moderate - intensity activity or total volume of activity ( i.e. , met minutes per week ) .
women in intervention facilities and usual care facilities reported similar reductions in daily total energy intake and percent of calories from fat , with no condition differences ( table 3 ) .
similar results for diet and physical activity were observed in sensitivity analyses that included imputed values if survey data were missing ( table 3 ) .
no condition differences were observed for hypertension or depression ( supplementary table 3 ) . after excluding women with possible pregestational diabetes ( 21 in the intervention and 29 in usual care ) , 873 ( 83.5% ) women in the intervention and 974 ( 85.4% ) in usual care completed postpartum diabetes screening .
fewer women in the intervention facilities developed prediabetes or diabetes than in usual care facilities ( 310 [ 29.7% ] and 37 [ 3.5% ] in intervention facilities and 370 [ 32.4% ] and 50 [ 4.4% ] in usual care facilities , respectively ) .
however , the risk estimate for prediabetes / diabetes combined did not reach statistical significance ( 33.2 vs. 36.8% ; hazard ratio 0.90 [ 95% ci 0.78 , 1.04 ] ) ( supplementary fig .
the mean number of call attempts to reach women and complete telephone sessions was 2.2 ( sd 1.8 ) .
the first session lasted a mean of 28.2 min ( 8.6 ) ; subsequent sessions lasted a mean of 18.8 min ( 7.4 ) .
direct intervention costs per person among all 1,087 women in the intervention were $ 78.00 ( $ 19.00 for printing and mailing and $ 59.00 for telephone sessions ) ; costs increased to $ 121.00 ( $ 19.00 for printing and mailing and $ 102.00 for telephone sessions ) if the cost of telephone sessions was calculated among women who completed one or more telephone sessions . during the 12 months postpartum
, there were no significant differences between intervention and usual care in the proportion of women who had an emergency room visit for sprains / strains ( n = 9 [ 0.8% ] vs. n = 16 [ 1.3% ] ; p = 0.24 ) or fractures ( n = 2 [ 0.2% ] vs. 6 [ 0.5% ] ; p = 0.29 ) .
the proportion of women in the intervention and usual care who were hospitalized or had an emergency room visit was 2.1 vs. 1.8% ( p = 0.64 ) and 7.8 vs. 10.1% ( p = 0.05 ) , respectively .
no condition differences were observed in underlying medical diagnosis except that fewer women had emergency room visits due to gallbladder disorders in the intervention than usual care ( n = 3 [ 0.3% ] vs. n = 15
clinic - measured postpartum weight was obtained at least once during the 12-month postpartum follow - up for 97.4% of women ( n = 2,221 ) . in the primary analysis that included only clinic - measured postpartum weight , on average , over the 12-month postpartum period ,
women in the medical facilities assigned to the intervention had a statistically significant 28% higher odds ( odds ratio [ or ] 1.28 [ 95% ci 1.10 , 1.47 ] ) of meeting postpartum weight goals than women in the medical facilities assigned to usual care ( table 2 ) .
the proportion meeting weight goals was significantly higher in the intervention facilities than usual care facilities at 6 weeks ( 25.5 vs. 22.4% ; 1.17 [ 1.01 , 1.36 ] ) and 6 months postpartum ( 30.6 vs. 23.9% ; 1.45 [ 1.14 , 1.83 ] ) .
at 12 months ( 6 months after the intervention ended ) , the condition difference was reduced ( 33.0 vs. 28.0% ; 1.25 [ 0.96 , 1.62 ] ) and no longer statistically significant .
the absolute difference between conditions was 3.1% at 6 weeks , 6.7% at 6 months , and 5.0% at 12 months postpartum . in analyses examining weight change from pregravid to postpartum ( table 2 ) ,
women in the intervention facilities retained less weight than women in usual care facilities at 6 months postpartum ( mean 0.39 kg [ sd 5.5 ] vs. 0.95 kg [ 5.5 ] ; adjusted mean condition difference 0.64 kg [ 95% ci 1.13 , 0.14 ] ) ; the condition difference was attenuated at 12 months postpartum and no longer statistically significant ( 0.43 kg [ 0.98 , 0.11 ] ) ( table 2 ) .
sensitivity analyses that included self - reported or imputed weight if the clinic - measured weight was missing yielded similar results ( tables 2 and 3 ) .
the intervention was equally effective across racial / ethnic groups ( p value for condition race / ethnicity interactions > 0.25 ) .
the intervention was also equally effective across bmi strata ( supplementary tables 1 and 2 ) .
proportion of women meeting the postpartum weight goals with ors estimating differences between conditions and mean changes in weight from pregravid to postpartum with mean differences between conditions : the gem trial * p value for condition differences ; p values for treatment follow - up time point interaction > 0.05 . adjusted for pregravid weight .
mean changes in physical activity and diet from pregnancy to 6 months postpartum with mean differences between conditions : the gem trial * adjusted for baseline values .
in exploratory analyses , the instrumental variable estimates of the absolute differences between conditions among compliers ( defined as completing one or more telephone sessions ) were almost double the absolute differences observed in the primary analysis : estimates for proportions meeting weight goals were 6.2% at 6 weeks , 13.3% at 6 months , and 9.9% at 12 months ; estimates for differences in mean weight change were 0.62 kg at 6 weeks , 1.27 kg at 6 months , and 0.86 kg at 12 months postpartum . in additional exploratory analyses , women who completed all 13 sessions had significantly higher odds of meeting the postpartum weight goals than women in usual care at 6 months ( 3.97 [ 2.75 , 5.72 ] ) and 12 months postpartum ( 2.16 [ 1.52 , 3.07 ] ) ( fig .
2b ) , women who completed 412 sessions retained less weight than women in usual care at 6 months postpartum ( mean 0.13 kg [ sd 5.7 ] vs. 0.94 kg [ 5.5 ] , adjusted mean condition difference 1.1 kg [ 95% ci 1.8 , 0.41 ] ) and at 12 months postpartum ( 0.06 kg [ 5.6 ] vs. 0.50 kg [ 5.4 ] ; 1.0 kg [ 1.9 , 0.13 ] ) .
women who completed all 13 sessions lost more weight than women in usual care at 6 months ( mean 1.89 kg [ sd 5.5 ] vs. 0.94 kg [ 5.5 ] , adjusted mean condition difference 2.3 kg [ 95% ci 3.1 , 1.5 ] ) and 12 months postpartum ( 1.19 kg [ 6.1 ] vs. 0.50 kg [ 5.4 ] ; 1.6 kg [ 2.4 , 0.68 ] ) . or ( 95% ci ) estimating the condition difference in meeting the postpartum weight goals ( a ) and adjusted mean condition difference ( 95% ci ) ( b ) for weight change from pregravid by number of intervention sessions completed and time since delivery : the gem trial . at 6 months postpartum , the number of women by number of completed telephone sessions were as follows : 338 for 0 sessions , 151 for 13 sessions , 171 for 412 sessions , and 104 for 13 sessions . at 12 months ,
the number of women by number of completed telephone sessions were as follows : 301 for 0 sessions , 134 for 13 sessions , 112 for 412 sessions , and 129 for 13 sessions .
at 6 months postpartum , women in both conditions increased minutes per week spent in vigorous - intensity activity ; however , women in the intervention facilities showed a significantly greater increase ( adjusted mean condition difference 15.4 min / week [ 95% ci 4.9 , 25.8 ] ) ( table 3 ) .
no condition differences were observed for time spent in moderate - intensity activity or total volume of activity ( i.e. , met minutes per week ) .
women in intervention facilities and usual care facilities reported similar reductions in daily total energy intake and percent of calories from fat , with no condition differences ( table 3 ) .
similar results for diet and physical activity were observed in sensitivity analyses that included imputed values if survey data were missing ( table 3 ) .
after excluding women with possible pregestational diabetes ( 21 in the intervention and 29 in usual care ) , 873 ( 83.5% ) women in the intervention and 974 ( 85.4% ) in usual care completed postpartum diabetes screening .
fewer women in the intervention facilities developed prediabetes or diabetes than in usual care facilities ( 310 [ 29.7% ] and 37 [ 3.5% ] in intervention facilities and 370 [ 32.4% ] and 50 [ 4.4% ] in usual care facilities , respectively ) .
however , the risk estimate for prediabetes / diabetes combined did not reach statistical significance ( 33.2 vs. 36.8% ; hazard ratio 0.90 [ 95% ci 0.78 , 1.04 ] ) ( supplementary fig .
the mean number of call attempts to reach women and complete telephone sessions was 2.2 ( sd 1.8 ) .
the first session lasted a mean of 28.2 min ( 8.6 ) ; subsequent sessions lasted a mean of 18.8 min ( 7.4 ) .
direct intervention costs per person among all 1,087 women in the intervention were $ 78.00 ( $ 19.00 for printing and mailing and $ 59.00 for telephone sessions ) ; costs increased to $ 121.00 ( $ 19.00 for printing and mailing and $ 102.00 for telephone sessions ) if the cost of telephone sessions was calculated among women who completed one or more telephone sessions .
during the 12 months postpartum , there were no significant differences between intervention and usual care in the proportion of women who had an emergency room visit for sprains / strains ( n = 9 [ 0.8% ] vs. n = 16 [ 1.3% ] ; p = 0.24 ) or fractures
( n = 2 [ 0.2% ] vs. 6 [ 0.5% ] ; p = 0.29 ) . the proportion of women in the intervention and usual care who were hospitalized or had an emergency room visit was 2.1 vs. 1.8% ( p = 0.64 ) and 7.8 vs. 10.1% ( p = 0.05 ) , respectively .
no condition differences were observed in underlying medical diagnosis except that fewer women had emergency room visits due to gallbladder disorders in the intervention than usual care ( n = 3 [ 0.3% ] vs. n = 15 [ 1.2% ] ; p = 0.008 ) .
this cluster randomized trial demonstrated that a health system based print / telephone lifestyle intervention derived from the dpp modestly reduced postpartum weight retention and increased vigorous - intensity physical activity among women with gdm .
postpartum weight retention and weight gain are associated with increased risk for diabetes ( 7 ) , recurrent gdm ( 26 ) , and abnormal cardiometabolic profile ( 27 ) .
the reductions in postpartum weight retention and the suggestion of reductions in prediabetes / diabetes incidence , for which gem was not powered , might be explained by the significant increase in vigorous - intensity activity among women in the intervention facilities , since no condition differences were observed for changes in daily total energy intake and percent of calories from fat .
it has been reported that vigorous - intensity activity is associated with decreased risk of diabetes among women with a history of gdm ( 28 ) and greater sports participation is associated with reduced postpartum weight retention ( 27 ) .
condition differences in weight observed in gem were similar to that observed in other cluster randomized trials . in the tianjin gestational diabetes prevention program cluster randomized trial ( 29 ) , women in a lifestyle intervention weighed 1 kg less than women in the control condition at 12 months postpartum .
the gem condition difference was also similar to that observed at 12 months in the da qing cluster randomized trial ( 3 ) of adults with prediabetes , in which lifestyle intervention led to 40 and 60% reductions in diabetes risk at 6 years ( 3 ) and 20 years ( 30 ) , respectively , suggesting potential long - term beneficial effects of modest weight loss . in gem ,
condition differences in the proportion meeting postpartum weight goals were statistically significant , on average , over the entire 12-month postpartum period , and at 6 weeks and 6 months postpartum .
although condition differences at the 12-month postpartum time point were not statistically significant , they were similar in magnitude to those at 6 months postpartum , when the intervention ended .
it is noteworthy that intervention effects on postpartum weight retention among women with gdm are greater in trials randomized at the individual level ( 31,32 ) than cluster randomized trials ( 29 ) , since only a select group of motivated volunteers participate in the former .
although in cluster randomized trials such as gem only a portion of eligible women participate , it should be noted that the effect sizes reported are for all women in the facilities assigned to the intervention .
instrumental variable analysis suggested that the intervention effect would be at least twofold greater if all women had completed one or more sessions , suggesting a potential for great impact if we can improve patient engagement in such lifestyle interventions .
to illustrate , in the pilot study that preceded gem , with randomization at the individual level , more women in the intervention met the postpartum weight goals , with a 16% absolute difference between conditions by 12 months postpartum ( 31 ) .
a trial among 75 women with gdm found women in a web - based lifestyle intervention were closer to their pregravid weight than control subjects at 12 months postpartum ( 0.7 vs. + 4.0 kg ) ( 32 )
. limitations of the gem trial include missing clinic - measured weight for 26.4% of women at 6 months and 32.7% at 12 months postpartum .
these missing data are due to the pragmatic nature of the gem trial ; participants did not volunteer for a study with 12 months of longitudinal follow - up on body weight measurements .
we obtained women s weights from the ehr , although this required women to visit their medical facility .
still , 97.4% of women contributed to the analysis of measured weight across the entire 12-month postpartum period .
furthermore , results based on clinic - measured postpartum weight were comparable to those of sensitivity analyses
. observed differences in postpartum weight retention remained significant in a sensitivity analysis that included clinic - measured weight or self - reported weight ( with only 13.7 and 18.6% still missing at 6 and 12 months , respectively ) and a sensitivity analysis that imputed missing clinic - measured weight . given the robustness of our results ,
strengths of this cluster randomized trial include the ability to randomize all medical facilities in the kpnc region ; the large number of facilities and women , and their racial / ethnic diversity ; intent - to - treat analyses including all facilities and women ; blinding of investigators , data collectors , and health care providers ; and follow - up extending 6 months beyond the end of the intervention .
a unique strength was that the primary analysis was based on clinic - measured weight obtained via ehr , regardless of women s participation in the surveys or intervention .
gem features several aspects of pragmatic trials , which prioritize generalizability at the potential expense of effect size given implicit sample heterogeneity and lower intervention adherence ( 33 ) .
in contrast to traditional clinical trials , gem had minimal exclusion criteria and the intervention was delivered at the health system level as optional routine care to an unselected population ( i.e. , regardless of motivation , perinatal complications , or comorbidities ) , which likely contributed to the 50.3% intervention uptake .
although this uptake is high for a pragmatic trial ( 34,35)and considerably higher than , for example , the recently reported uptake of 13% in the health system based veterans health administration move ! lifestyle change program , the largest such program in the u.s .
( 36)uptake likely impacted the condition differences observed and shows how challenging it is to engage patients in prevention programs . in gem ,
although prespecified categories for the number of completed sessions were used , women were not randomized to these categories , and those who completed more sessions may have lost more weight due to increased motivation for a healthy lifestyle . nevertheless , these dose - response analyses ( 37 ) suggest that to increase the effectiveness of lifestyle interventions , strategies are needed to increase and sustain patient engagement . if health systems were to adopt postpartum interventions for women with gdm , clinician referrals and other strategies to increase patient engagement might increase uptake and effectiveness ( 38 ) . finally , the cost per woman of the gem intervention was relatively modest , and the printed / telephone modality is easily translatable to other settings . in conclusion
, this cluster randomized trial of diabetes prevention strategies delivered at the health system level to women with gdm demonstrated that a dpp - derived , print / telephone - based lifestyle intervention was superior to usual care in reducing postpartum weight retention , a risk factor for diabetes , and increasing physical activity .
although condition differences were modest , differences of similar magnitude have been shown to reduce long - term diabetes incidence in at - risk adults ( 30 ) .
these findings , from a trial embedded in real - world practice , may encourage health systems to adopt dpp - derived postpartum interventions to help women with gdm manage their weight and increase physical activity , thereby potentially preventing or delaying the onset of diabetes . | objectiveto compare the effectiveness of diabetes prevention strategies addressing postpartum weight retention for women with gestational diabetes mellitus ( gdm ) delivered at the health system level : mailed recommendations ( usual care ) versus usual care plus a diabetes prevention program ( dpp)derived lifestyle intervention.research design and methodsthis study was a cluster randomized controlled trial of 44 medical facilities ( including 2,280 women with gdm ) randomized to intervention or usual care .
the intervention included mailed gestational weight gain recommendations plus 13 telephone sessions between 6 weeks and 6 months postpartum .
primary outcomes included the following : proportion meeting the postpartum goals of 1 ) reaching pregravid weight if pregravid bmi < 25.0 kg / m2 or 2 ) losing 5% of pregravid weight if bmi 25.0 kg / m2 ; and pregravid to postpartum weight change.resultson average , over the 12-month postpartum period , women in the intervention had significantly higher odds of meeting weight goals than women in usual care ( odds ratio [ or ] 1.28 [ 95% ci 1.10 , 1.47 ] ) .
the proportion meeting weight goals was significantly higher in the intervention than usual care at 6 weeks ( 25.5 vs. 22.4% ; or 1.17 [ 1.01 , 1.36 ] ) and 6 months ( 30.6 vs. 23.9% ; or 1.45 [ 1.14 , 1.83 ] ) .
condition differences were reduced at 12 months ( 33.0 vs. 28.0% ; or 1.25 [ 0.96 , 1.62 ] ) .
at 6 months , women in the intervention retained significantly less weight than women in usual care ( mean 0.39 kg [ sd 5.5 ] vs. 0.95 kg [ 5.5 ] ; mean condition difference 0.64 kg [ 95% ci 1.13 , 0.14 ] ) and had greater increases in vigorous - intensity physical activity ( mean condition difference 15.4 min / week [ 4.9 , 25.8]).conclusionsa dpp - derived lifestyle intervention modestly reduced postpartum weight retention and increased vigorous - intensity physical activity . |
thrombotic thrombocytopenic purpura ( ttp ) is a microangiopathic hemolytic disorder that is induced by marked reduction in the level of von willebrand factorcleaving protease , adamts13 .
it is characterized by development of microthrombi in small blood vessels leading to consumptive thrombocytopenia and hemolytic anemia . although ttp is commonly associated with neurologic and renal dysfunction 1 , 2 , cardiac involvement is rarely seen .
unfortunately , most cases with ttp with cardiac involvement are diagnosed on autopsy where a massive myocardial necrosis is usually evident 4 . in this report , we present a case of ttpinduced myocardial infarction treated successfully with plasma exchange .
a 59yearold man with history of hypertension , coronary artery disease ( cad ) , and medications noncompliance presented to the emergency room with a 2day history of chest pain , lethargy , and headache .
the chest pain was retrosternal , pressurelike , increasing in severity , radiating to the left arm , and associated with diaphoresis .
the patient was supposed to be on low dose aspirin , metoprolol tartrate , and atorvastatin .
laboratory studies showed elevated troponin t at 0.33 ng / ml , low platelet count at 9,000/l , low hemoglobin ( 6.4 gm / dl ) , and elevated creatinine of 3.3 mg / dl .
the patient prothrombin time , international normalized ratio , and partial thromboplastin time were within normal limits .
ekg .
due to the severity of chest pain , he was started on nitroglycerin infusion and transferred to the cardiac intensive care unit .
further workup revealed elevated lactate dehydrogenase at 857 units / l , undetectable haptoglobin level and elevated indirect bilirubin level at 2.0 mg / dl .
peripheral blood smear revealed increased number of schistocytes ( 5 per high power field ; fig .
ttp was diagnosed and adamts13 level was requested . a oncedaily plasma exchange was initiated .
after 6 days of daily plasma exchange , platelet count increased to 195,000/l , hemoglobin level stabilized at 10.1 gm / dl , creatinine level decreased to 1.45 mg / dl , troponin t level decreased to 0.09 ng / ml , lactate dehydrogenase level decreased to 246 units / l , and his chest pain improved with complete resolution of hemolysis and marked improvement of energy level .
3 ) . peripheral blood smear showing increased number of schistocytes ( arrows ) .
this is the second ekg which demonstrates improvement of the st segment depression as compared to fig .
prior to discharge , the patient underwent cardiac catheterization and revealed no evidence of occlusive cad .
three weeks later , a repeat echocardiogram showed an improved left ventricular ejection fraction to 6570% .
in this report , we present a case with typical acute coronary syndrome ( acs ) features including leftsided chest pain , elevated troponin t level , and risk factors for atherosclerosis such as hypertension and prior history of cad .
ttp can rarely present with acs due to development of microthrombi in the small vessels supplying the myocardium , which makes it difficult to distinguish from atherosclerotic acs , particularly in patients with multiple risk factors , as in our case .
the presence of anemia , thrombocytopenia , and evidence of hemolysis should raise the suspicion for ttpinduced acs .
in addition , cardiac involvement in ttp patients can manifest as mi , tachyarrhythmias , conduction disturbances , congestive heart failure ( chf ) , cardiogenic shock , myocarditis , myocardial necrosis , or sudden cardiac death 4 , 5 , 6 , 7 , 8 .
in fact , most ttp patients with cardiac involvement are diagnosed postmortem at the time of autopsy , where thrombosis of the small vessels supplying the myocardium is seen in as high as in 77% of patients 9 , 10 , 11 , 12 .
the presence of microthrombi in the microcirculation of the myocardium and the presence of anemiarelated high cardiac output may contribute to cardiac dysfunction , leading to symptomatic chf 11 . in a study conducted by balasubramaniyam et al .
, the 20012010 nationwide inpatient sample database was used to identify patients aged 18 years with the diagnosis of ttp and acute mi . in this study ,
multiple variables were examined including age , gender , tobacco use , hypertension , diabetes mellitus , dysplipidemia , and prior history of cad or stroke .
conducted a study on 14 patients with ttp who underwent coronary angiography due to mi .
our patient had angiography after six sessions of plasma exchange , and did not demonstrate significant cad . unlike our patient
since cardiac involvement in ttp carries a higher mortality rate , prompt recognition of cardiac involvement is vital 5 , 15 .
a study conducted by benhamou et al . showed that a troponin i level higher than 0.25 g / l in patients with ttp predicts more refractory disease and is associated with a threefold increase in the risk of death , regardless of cardiac symptoms .
the authors of this study concluded that troponin level should be used as a prognostic indicator in patients with ttp 16 . due to its rarity
however , plasma exchange improves symptoms in the majority of these patients and should be initiated as soon as possible . in our patient ,
cardiac , neurologic , hematologic , and renal manifestations of ttp improved with plasma exchange .
invasive procedure such as coronary angiography have low yield in these patients and may even exacerbate ttp . as a result , it should be deferred until organ functions improve and hemolytic markers normalize .
17 , 18 , 19 , 20 . based on our case , it is reasonable to initiate plasma exchange transfusion for such patients without delay in addition to any supportive care measures .
imaging of the coronary arteries and other invasive procedures can be performed following clinical stabilization of the patient and improvement in laboratory parameters which will make such procedures safer .
| key clinical messagein patients presenting with thrombotic thrombocytopenia purpura and nonst elevation myocardial infarction , prompt initiation of plasma exchange takes precedence over other invasive diagnostic procedures for coronary artery disease .
such procedures should be delayed until clinical condition and laboratory parameters have been stabilized . |
bedwetting or nocturnal enuresis ( ne ) is the most common childhood urologic complaint and the most common issue reported at school examinations .
ne affects 10% to 15% of those aged 5 years , 5% of those aged 10 years , and 1% of those aged 15 years .
it can have a significant effect on the child or young person 's behavior and emotional well - being .
it may cause a child to be marginalized by family and friends and may instigate anger , punishments , and rejection in caregivers , culminating in a loss of the child 's self - confidence .
such psychoemotional effects have been found to improve after treatment of ne . according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) ,
ne is defined as bedwetting with a severity of at least twice per week in children over 5 years of age when not provoked by congenital or acquired defects of the central nervous system or by the direct physiological effect of a substance ( such as a diuretic ) .
it has been divided into primary ne ( having never experienced a lengthy spell of dry nights ) and secondary ne ( having a history of being dry for at least 6 months ) .
the occurrence of ne seems to be determined by 3 main factors that direct treatment implications : 1 ) nocturnal polyuria , which suggests a need for treatment with desmopressin ; 2 ) nocturnal bladder reservoir function , which indicates a need for a bladder - training program coupled with anticholinergic treatment ( e.g. , oxybutynin ) ; and 3 ) the arousal response to bladder fullness during sleep , which suggests that treatment with an enuresis alarm may be a good treatment option .
however , one should be cautious in extrapolating the findings of these studies to other communities given the possible differences in socioeconomic conditions that affect the clinical outcome .
because local saudi data on the usefulness of these treatment strategies for ne are scanty , we conducted this study to determine and compare the clinical efficacy of an enuresis alarm , desmopressin , and the combination of both regimens in the treatment of saudi children with primary monosymptomatic nocturnal enuresis ( pmne ) .
this multicenter , prospective , randomized , comparative study was conducted between october 2009 and march 2013 .
patients were consecutively enrolled from patients referred to the urology and pediatric clinics at salman bin abdul - aziz university hospital , al - kharj ; royal commission hospital , jubail ; and king khalid hospitals , al - kharj and tabouk , kingdom of saudi arabia , for treatment of enuresis .
the size of the research sample was determined by using the graphpad prism sample size calculator ( graphpad software , la jolla , ca , usa ) . on the basis of previous studies [ 9 - 15 ] of the trial parameters ( reduction in wetting frequencies , response to treatment , relapse rates , etc . ) , a clinically important difference of 30% in the proportion of these parameters was considered acceptable .
we considered a dropout rate of 10% . to obtain a significance level of 0.05 and a power of 80%
all patients underwent thorough history - taking including a lower urinary tract symptoms ( luts ) questionnaire , physical examination , urinalysis , midstream urine culture , and measurement of serum creatinine and serum electrolytes .
the severity of ne was categorized as mild ( 2 - 3 times / wk ) , moderate ( 4 - 6 times / wk ) , and severe ( 7 times / wk ) .
patients 6 to 14 years of age , having wetting episodes at least twice per week over 3 consecutive months , and whose parents agreed to allocation of treatment were eligible for enrollment . patients with identifiable luts other than ne and those with other physical disorders , such as diabetes
; with abnormal urine analysis results ; with renal , urological , cardiovascular , or neurological diseases ; or previously treated with an enuresis alarm or desmopressin were excluded from study . out of 167 patients who met the inclusion criteria , 31 chose not to participate .
the remaining 136 patients were randomly divided into three groups through a computer randomization program and received treatment for 12 weeks : group i , the enuresis alarm group ( ea group , n=45 ) ; group ii , the desmopressin group ( d group , n=46 ) ; and group iii , combined enuresis alarm and desmopressin group ( ea / d group , n=45 ) ( fig .
1 ) . to keep the groups closely balanced , we used randomization in blocks of three and each center had its own list . in patients using the alarm device ,
the children were instructed to test the alarm before sleep , with the sound ( or vibration ) in mind , and the families were instructed to awake the child immediately after onset of the alarm and to send him to the bathroom to finish voiding . in patients assigned to desmopressin , an oral lyophilisate pharmaceutical form of desmopressin ( melt formulation ) was used .
the patients were advised to place the tablet under the tongue and to allow it to dissolve completely without water .
if no considerable response was achieved after 2 weeks ( > 1 wet night / wk ) , the doses were increased to 240 g .
all patients were advised to urinate just before going to bed and to restrict their fluid intake ( not to drink more than sufficient to satisfy thirst ) from 1 hour before bedtime .
they were also instructed to avoid drinking liquids with a diuretic effect at night , to keep regular sleep hours , and to keep a record of night wetting episodes .
in addition , the children used a star chart for recording the number of dry and wet nights and compliance with treatment .
the protocol and all study procedures were conducted in conformity with the ethical guidelines of the declaration of helsinki of 1975 .
the study protocol was approved by the ethics committee of the participating hospitals , and parents of all patients enrolled in this study provided written informed consent .
follow - up visits were scheduled on a weekly basis during the treatment period and 3 months after treatment discontinuation . at each follow - up visit , blood pressure ,
serum sodium was measured at the first follow - up visit and at the end of treatment .
treatment efficacy was determined by comparison of wet nights in the pretreatment period with the numbers during the last week of treatment .
treatment outcomes were categorized as either success or failure on the basis of the 1-week nocturnal records at the end of treatment .
successful outcomes included the following responses , as defined by the standardization committee of the international children 's continence society ( iccs ) : full response , no wet nights ; response , 90% reduction in the number of wet nights ; partial response , 50% to 89% reduction in the number of wet nights .
treatment failure was defined as nonresponse ( < 50% reduction in the number of wet nights ) .
a relapse was defined as more than one wetting episode per month after full response . among those assigned to therapy
, treatment efficacy analyses were performed by using the intention - to - treat population , defined as all enrolled patients who received at least one dose of any study medication .
statistical analyses were performed by using paired or independent t - tests when appropriate for continuous variables and chi - square tests for categorical variables .
the treatment outcomes ( response , noncompliance , and relapse rates ) were compared by using chi - square tests .
the differences in the mean number of wetting episodes per week before treatment , at the end of treatment , and at the end of follow - up were assessed by using one - way analysis of variance ( anova ) .
within - group comparisons over these 3 time points were performed by paired t - test .
this multicenter , prospective , randomized , comparative study was conducted between october 2009 and march 2013 .
patients were consecutively enrolled from patients referred to the urology and pediatric clinics at salman bin abdul - aziz university hospital , al - kharj ; royal commission hospital , jubail ; and king khalid hospitals , al - kharj and tabouk , kingdom of saudi arabia , for treatment of enuresis .
the size of the research sample was determined by using the graphpad prism sample size calculator ( graphpad software , la jolla , ca , usa ) . on the basis of previous studies [ 9 - 15 ] of the trial parameters ( reduction in wetting frequencies , response to treatment , relapse rates , etc . ) , a clinically important difference of 30% in the proportion of these parameters was considered acceptable .
we considered a dropout rate of 10% . to obtain a significance level of 0.05 and a power of 80%
all patients underwent thorough history - taking including a lower urinary tract symptoms ( luts ) questionnaire , physical examination , urinalysis , midstream urine culture , and measurement of serum creatinine and serum electrolytes .
the severity of ne was categorized as mild ( 2 - 3 times / wk ) , moderate ( 4 - 6 times / wk ) , and severe ( 7 times / wk ) .
patients 6 to 14 years of age , having wetting episodes at least twice per week over 3 consecutive months , and whose parents agreed to allocation of treatment were eligible for enrollment . patients with identifiable luts other than ne and those with other physical disorders , such as diabetes ; with abnormal urine analysis results ; with renal , urological , cardiovascular , or neurological diseases ; or previously treated with an enuresis alarm or desmopressin were excluded from study .
out of 167 patients who met the inclusion criteria , 31 chose not to participate .
the remaining 136 patients were randomly divided into three groups through a computer randomization program and received treatment for 12 weeks : group i , the enuresis alarm group ( ea group , n=45 ) ; group ii , the desmopressin group ( d group , n=46 ) ; and group iii , combined enuresis alarm and desmopressin group ( ea / d group , n=45 ) ( fig .
1 ) . to keep the groups closely balanced , we used randomization in blocks of three and each center had its own list . in patients using the alarm device ,
the children were instructed to test the alarm before sleep , with the sound ( or vibration ) in mind , and the families were instructed to awake the child immediately after onset of the alarm and to send him to the bathroom to finish voiding . in patients assigned to desmopressin , an oral lyophilisate pharmaceutical form of desmopressin ( melt formulation ) was used .
the patients were advised to place the tablet under the tongue and to allow it to dissolve completely without water .
if no considerable response was achieved after 2 weeks ( > 1 wet night / wk ) , the doses were increased to 240 g .
all patients were advised to urinate just before going to bed and to restrict their fluid intake ( not to drink more than sufficient to satisfy thirst ) from 1 hour before bedtime .
they were also instructed to avoid drinking liquids with a diuretic effect at night , to keep regular sleep hours , and to keep a record of night wetting episodes .
in addition , the children used a star chart for recording the number of dry and wet nights and compliance with treatment .
the protocol and all study procedures were conducted in conformity with the ethical guidelines of the declaration of helsinki of 1975 .
the study protocol was approved by the ethics committee of the participating hospitals , and parents of all patients enrolled in this study provided written informed consent .
follow - up visits were scheduled on a weekly basis during the treatment period and 3 months after treatment discontinuation . at each follow - up visit , blood pressure ,
serum sodium was measured at the first follow - up visit and at the end of treatment .
treatment efficacy was determined by comparison of wet nights in the pretreatment period with the numbers during the last week of treatment .
treatment outcomes were categorized as either success or failure on the basis of the 1-week nocturnal records at the end of treatment .
successful outcomes included the following responses , as defined by the standardization committee of the international children 's continence society ( iccs ) : full response , no wet nights ; response , 90% reduction in the number of wet nights ; partial response , 50% to 89% reduction in the number of wet nights .
treatment failure was defined as nonresponse ( < 50% reduction in the number of wet nights ) .
a relapse was defined as more than one wetting episode per month after full response .
among those assigned to therapy , treatment efficacy analyses were performed by using the intention - to - treat population , defined as all enrolled patients who received at least one dose of any study medication .
statistical analyses were performed by using paired or independent t - tests when appropriate for continuous variables and chi - square tests for categorical variables .
the treatment outcomes ( response , noncompliance , and relapse rates ) were compared by using chi - square tests .
the differences in the mean number of wetting episodes per week before treatment , at the end of treatment , and at the end of follow - up were assessed by using one - way analysis of variance ( anova ) .
within - group comparisons over these 3 time points were performed by paired t - test .
16 ( sas institute inc . , cary , nc , usa ) , medcalc ver . 12 ( medcalc software , ostend , belgium ) , and graphpad prism
1 , a total of 136 patients were enrolled in this study and 115 patients completed the treatment .
the 21 patients who did not complete the study included 9 patients from group i , 4 patients from group ii , and 8 patients from group iii .
group i consisted of 32 boys and 13 girls ( mean age , 8.212.05 years ) , group ii consisted of 30 boys and 16 girls ( mean age , 7.972.10 years ) , and group iii consisted of 33 boys and 12 girls ( mean age , 8.011.96 years ) .
the mean number of wet nights / wk was 4.561.71 for group i , 5.041.58 for group ii , and 4.731.66 for group iii .
there were no statistically significant differences between the 3 groups in terms of age , sex , mean wetting frequencies , or severity of ne ( p>0.05 ) .
the baseline characteristics of the studied patients are summarized in table 1 . at the end of treatment ,
wetting frequencies were significantly reduced in all groups , by 54.8% , 65.1% , and 78.9% in the groups ea , d , and ea / d , respectively ( table 2 ) .
rebound occurred in all groups after discontinuation of treatment ; however , the reduction in wetting frequencies remained significant until the end of follow - up ( fig .
2 ) . the wetting frequencies were analyzed week by week during treatment and follow - up periods ( fig .
3 ) . groups d and ea / d patients showed immediate improvement and registered statistically significant reductions in wetting frequencies from week 1 .
group ea patients showed gradual improvement and statistically significant reduction was observed only from week 4 . at the end of treatment ,
the wetting frequencies were significantly higher in the group ea / d than in the groups ea and d ( p<0.0001 and p=0.018 , respectively ) .
after discontinuation of treatment , the group d showed an immediate rebound of wetting and frequencies became higher than in the groups ea and ea / d until the end of follow - up .
in the groups ea / d and ea , significant rebound was observed at follow - up weeks 3 and 10 , respectively ; there was no significant difference in wetting frequencies between the groups ea and ea / d from follow - up week 1 until the end of follow - up . counting defaulters due to noncompliance issues as a form of failure ( intent - to - treat analysis ) , the full response and partial response rates were 13.3% and 37.8% in the group ea , 26.1% and 43.5% in the group d , and 40.0% and 33.3% in the group ea / d , respectively .
anova showed that the full response to treatment was significantly higher in the group ea / d than in the group ea ( p=0.025 ) , whereas other group pairs were not significantly different .
the relapse rates were higher in the group d ( 66.6% ) than in the group ea ( 16.6% ) or the group ea / d ( 33.3% ) . a significant difference was observed only between the groups ea and d ( p=0.019 ) .
noncompliance rates were higher in the groups ea ( 20.0% ) and ea / d ( 17.8% ) than in the group d ( 8.7% ) , but the difference was not statistically significant ( p=0.230 , fisher exact test ) .
this study was the first to evaluate and compare the efficacy of an enuresis alarm , desmopressin treatment , or a combination of both regimens for the treatment of enuretic saudi children .
our results agreed with previous global patterns demonstrating that an enuresis alarm , desmopressin , and a combination of both are effective treatment modalities for saudi children with pmne .
the average wetting frequencies of participants in the 3 groups were significantly reduced during treatment and 3 months after treatment when compared with pretreatment values .
the percentage of patients who responded to treatment was 51.1% , 69.6% , and 73.3% in the groups ea , d , and ea / d , respectively , and response was sustained after treatment withdrawal in 83.4% , 33.4% , and 66.7.0% of full responders in the 3 groups , respectively .
although several modalities ( behavioral and pharmacological ) are available for the treatment of primary ne , none has proven complete success , most likely as a result of incomplete understanding of the pathophysiology of ne .
alarm therapy is an effective treatment strategy for primary ne owing to its efficacy , low relapse rates , and absence of side effects .
it is presumed to cure primary ne as a result of conditioning effects on arousal or by increasing nocturnal bladder reservoir function .
the most important disadvantage of alarm treatment is the late onset of its impact ; it starts to result in effects in a couple of weeks .
this might decrease the motivation of the child and family . in our study , the alarm device exerted a significant effect after 4 weeks . also , the rates of noncompliance were higher in the groups treated with an alarm .
however , some factors could have an effect on the response to alarm treatment , particularly those of a psychological nature , such as marital status conflict , lack of motivation , and parental penalty .
the physiological aspects related to a poor response to alarm treatment embody the problem of awakening with the sound of the device .
desmopressin has been available as intranasal spray and tablet formulations for the treatment of primary ne for many years ; the convenient , sublingual oral lyophilisate ( melt ) formulation is a more recent development .
desmopressin oral melt is the preferable formulation over tablet and requires no water intake and is associated with high compliance among children aged 5 to 11 years .
overall , desmopressin is a safe drug with few side effects and low risks even when used for many years .
the only serious potential side effect that has been reported in children with ne who were treated with desmopressin is water intoxication with hyponatremia and convulsions .
consequently , the enuresis indication has been removed for the nasal spray in several countries .
regardless of the formulation used , there are recognizable and preventable risk factors for hyponatremia .
an unsuitably high fluid intake before desmopressin administration could be a key risk issue . in our study ,
the oral melt tablet was used 1 hour before bedtime and evening fluids were allowed as desired with no side effects detected .
the efficacy of an alarm device and desmopressin in the treatment of primary ne was evaluated and compared in several previous studies .
similar to these studies , we found that desmopressin elucidated a more rapid onset of action than the alarm , but desmopressin was associated with a significantly higher relapse rate .
the combined therapy was significantly better than alarm therapy , but not desmopressin therapy , in achieving an immediate short - term response ; however , its effect was no better than the alarm in the long term .
three months after treatment discontinuation , there was no significant difference between the 2 groups concerning relapse rates .
however , in the present study , no statistically significant difference was noted between the 3 groups in noncompliance rates .
a british study investigated 71 enuretic children and compared the use of combined desmopressin and alarm with alarm monotherapy and found that 76% of children receiving combined therapy became dry compared with 46% of those using the alarm alone , with similar relapse rates , 15% and 19% , respectively . in a placebo - controlled study , leebeek - groenewegen et al .
compared the efficacy of combined enuresis alarm and desmopressin with alarm monotherapy in the treatment of 93 enuretic children and found a significant reduction in wetting frequencies with combined therapy compared with alarm monotherapy .
however , no significant difference was seen between the 2 groups in the relapse rates after treatment discontinuation .
reported their crossover trial of combined desmopressin and alarm versus placebo and alarm and showed a significant reduction in wet nights with combined therapy during the 2 weeks of observation compared with placebo and alarm .
however , the 2-week treatment period was inadequate because alarm therapy takes as long as 5 to 8 weeks before success can be determined . in the randomized comparative study of fai - ngo ng et al .
, 105 chinese enuretic children received an enuresis alarm , desmopressin , or combined enuresis alarm and desmopressin therapy for 12 weeks and were followed for another 12 weeks . patients with 1 wet night
/ wk in the last 4 weeks were defined as full responders , patients with > 1 wet night / wk but a > 50% reduction in the wetting frequency were defined as partial responders , and patients with a < 50% reduction in wetting frequency were defined as nonresponders .
those authors determined that the total , full , and partial response rates were 42.9% , 22.9% , and 20% for alarm therapy ; 52.5% , 24% , and 10.5% for desmopressin therapy ; and 78.1% , 62.5% , and 15.6% for combined therapy , respectively .
they attributed the lower effects especially with alarm and desmopressin monotherapy to the overcrowded living community of the chinese population , which affects the motivation and compliance of families and children .
also , the possibility of high bladder dysfunction in chinese children with primary ne may be a contributing factor .
they assessed the relapse rate as the percentage of total responders ( complete plus partial ) in each group when treatment was stopped and found no significant differences between the three groups regarding relapse rates . in our study , the relapse rates were significantly higher in the group d than in the group ea ; all ea patients who fully responded to treatment remained dry until the end of follow - up .
one study reported that relapse rates with desmopressin in adults depend on the initial tailored dose ; however , this needs to be confirmed with further studies on children .
moreover , the calculated results are greatly affected by the methods used for outcome measurement .
this point may also explain the heterogeneity in the proportion of full and partial responders for each treatment modality in most of the studies .
in the present study , the proportion of full to partial response rates seemed to be lower than in the previous report because we used the iccs definitions to evaluate the response to treatment and only patients with no wet nights were considered full responders .
one limitation of the present study was the lack of an untreated control group , because the possibility of spontaneous resolution could not be excluded . in our departments
we are not permitted , for ethical purposes , to exclude or to delay treatment of patients , especially if the treatment modalities used have been shown to be effective in previous studies . however , the main focus of the present study was to compare the different treatment strategies for pmne and to evaluate whether the combination of enuresis alarm and desmopressin has a beneficial effect over either treatment alone .
another limitation was the short follow - up period , because 3 months of follow - up is insufficient for valid recommendation .
therefore , these results should be clinically applied with caution . controlled studies with larger study populations and longer follow - up
should be conducted to facilitate the standardization of the appropriate method for treatment of pmne and to determine if the results in this study are maintained over time .
desmopressin , enuresis alarm , and combined therapy are effective in the treatment of saudi children with pmne .
combined therapy showed better immediate response rates than did the alarm but no significant difference in relapse rates .
we suggest that adding desmopressin to therapy with an enuresis alarm is of benefit only in achieving a rapid response if children or families seek rapid improvement . | purposewe evaluated and compared the effectiveness of an enuresis alarm , desmopressin medication , and their combination in the treatment of saudi children with primary monosymptomatic nocturnal enuresis ( pmne).materials and methodsa total of 136 children with pmne were randomly assigned to receive an enuresis alarm alone ( ea group , n=45 ) , desmopressin alone ( d group , n=46 ) , or a combination of both ( ea / d group , n=45 ) .
patients were followed weekly during treatment and for 12 weeks after treatment withdrawal.resultsduring treatment , wetting frequencies were significantly reduced in all groups and remained significantly lower than pretreatment values until the end of follow - up . in the d and ea / d groups , an immediate reduction in wetting frequencies was observed , whereas a longer time was required to reach a significant reduction in the ea group .
the full and partial response rates were 13.3% and 37.8% in the ea group , 26.1% and 43.5% in the d group , and 40.0% and 33.3% in the ea / d group .
a significant difference was observed only between the ea and ea / d groups ( p=0.025 ) .
relapse rates were higher in the d group ( 66.6% ) than in the ea ( 16.6% ) and ea / d ( 33.3% ) groups .
a significant difference was observed between the d and ea groups only ( p=0.019).conclusionsdesmopressin , an enuresis alarm , and combined therapy are effective in the treatment of saudi children with pmne .
desmopressin produced an immediate effect but relapses were common .
the enuresis alarm provided gradual effects that persisted posttreatment .
the combined therapy was superior to the alarm in achieving an immediate response ; however , its effect was not better than that of the alarm long term . |
thalassemia epitomizes disorders of hemoglobin synthesis , characterized by reduced synthesis of either the alpha - globin or -globin chains of the hemoglobin molecule .
-thalassemia encompasses a group of monogenic diseases that have in common defective synthesis of -globin resulting from a variety of molecular defects that reduce or abolish the normal synthesis of the -globin chains of hemoglobin .
the defects involved are extremely heterogeneous and give rise to a large phenotypic spectrum , with patients that are almost asymptomatic to those requiring regular blood transfusions to sustain life .
being an autosomal recessive disorder characterized by microcytosis and hemolytic anemia , it accounts for thousands of childhood deaths per year , primarily in regions of the world in which malaria used to be endemic .
thorough clinical , radiographic and laboratory assessment is crucial in diagnosing thalassemia along with any other hematological disorder .
two cases of -thalassemia showing distinct features are presented and the importance of detailed examination of the cases has been highlighted .
a 19-year - old male patient presented with the complaint of malaligned teeth in his upper and lower jaws since last 6 - 7 years he wanted to get it corrected . past dental history was non - contributory ; however , patient gave a history of blood transfusions every 15 days for the past 18 years .
history also revealed that the patient was hospitalized for the same about 15 days ago . on examination
, the patient seemed to be conscious , cooperative and well - oriented , but with poor built .
lower palpebral conjunctiva showed pallor , but no signs of jaundice , pigmentation , clubbing of nails , skin eruptions or any other deformities , cyanosis or edema were noted .
intraoral examination showed malaligned teeth in both upper and lower jaws , chiefly in the anterior region [ figure 1 ] .
radiographic examination was non - contributory as intraoral periapical radiograph [ figure 2 ] and lateral cephalogram [ figure 3 ] did not disclose any abnormality , except for slight variations in the diploic space along with flaring of maxillary anterior teeth , suggestive of malocclusion .
based on the patient 's history and clinical presentation , a provisional diagnosis of thalassemia was given .
laboratory investigations made known that the patient had normocytic normochromic anemia . to substantiate with the provisional diagnosis , a hemoglobin electrophoresis was run on paragon gel at a ph of 8.6 , which showed an absence of -globin of hemoglobin . based on the clinical features , past medical history in association with hematological finding ,
patient presenting typical chipmunk facies along with malaligned teeth in upper and lower jaws intraoral periapical radiograph of case 1 with no significant findings lateral cephalogram of case 1 showing slight variations in the diploic space an 8-year - old male child was brought by his mother with a complaint of malaligned teeth in upper front region of jaw .
past dental history was non - influential ; however a history of blood transfusions since past 7 years was given by patient 's mother .
on clinical examination , slight malocclusion was noted in upper anterior teeth associated with deep bite [ figure 4 ] .
radiographic examination was non - related to the medical history as intraoral periapical radiograph [ figure 5 ] and lateral cephalogram did not show any significant changes [ figure 6 ] .
nevertheless , hemoglobin electrophoresis test showed absence of -globin of hemoglobin , as was observed in the first case .
though the clinical features and radiographic features did not contribute much , with the help of past medical history and laboratory investigation , a diagnosis of -thalassemia minor was given .
patient presenting with slight malocclusion in upper anterior teeth intraoral periapical radiograph of case 2 with no significant findings lateral cephalogram of case 2 with no significant findings
a 19-year - old male patient presented with the complaint of malaligned teeth in his upper and lower jaws since last 6 - 7 years he wanted to get it corrected . past dental history was non - contributory ; however , patient gave a history of blood transfusions every 15 days for the past 18 years .
history also revealed that the patient was hospitalized for the same about 15 days ago . on examination
, the patient seemed to be conscious , cooperative and well - oriented , but with poor built .
lower palpebral conjunctiva showed pallor , but no signs of jaundice , pigmentation , clubbing of nails , skin eruptions or any other deformities , cyanosis or edema were noted .
intraoral examination showed malaligned teeth in both upper and lower jaws , chiefly in the anterior region [ figure 1 ] .
radiographic examination was non - contributory as intraoral periapical radiograph [ figure 2 ] and lateral cephalogram [ figure 3 ] did not disclose any abnormality , except for slight variations in the diploic space along with flaring of maxillary anterior teeth , suggestive of malocclusion .
based on the patient 's history and clinical presentation , a provisional diagnosis of thalassemia was given .
laboratory investigations made known that the patient had normocytic normochromic anemia . to substantiate with the provisional diagnosis , a hemoglobin electrophoresis was run on paragon gel at a ph of 8.6 , which showed an absence of -globin of hemoglobin . based on the clinical features , past medical history in association with hematological finding ,
patient presenting typical chipmunk facies along with malaligned teeth in upper and lower jaws intraoral periapical radiograph of case 1 with no significant findings lateral cephalogram of case 1 showing slight variations in the diploic space
an 8-year - old male child was brought by his mother with a complaint of malaligned teeth in upper front region of jaw . past dental history
was non - influential ; however a history of blood transfusions since past 7 years was given by patient 's mother . on clinical examination , slight malocclusion was noted in upper anterior teeth associated with deep bite [ figure 4 ] .
radiographic examination was non - related to the medical history as intraoral periapical radiograph [ figure 5 ] and lateral cephalogram did not show any significant changes [ figure 6 ] .
nevertheless , hemoglobin electrophoresis test showed absence of -globin of hemoglobin , as was observed in the first case . though the clinical features and radiographic features did not contribute much , with the help of past medical history and laboratory investigation , a diagnosis of -thalassemia minor was given .
patient presenting with slight malocclusion in upper anterior teeth intraoral periapical radiograph of case 2 with no significant findings lateral cephalogram of case 2 with no significant findings
thalassa , meaning sea , as the original cases were reported from the region of the mediterranean sea . cooley and lee , in 1925 , described thalassemia as a severe anemia that is associated with bone changes and splenomegaly .
only later it was established that thalassemia was attributable to a partial autosomal dominant gene , for which the homozygous state was termed thalassemia major and the heterozygous state was termed thalassemia minor .
thalassemia minor occurs only when one defective gene for -globin molecule is inherited and the patient presents with no significant clinical manifestations , as noted in case 2 .
conversely , when two defective genes for the -globin molecules are inherited , the patient is affected with thalassemia major , also known as cooley 's anemia or mediterranean fever and presents with clinical manifestations as observed in case 1 .
furthermore , the disorders less severe than major forms but more severe than minor forms are described as thalassemia intermedia , which has been observed in 10% of patients with the homozygous trait .
thalassemia has been estimated as the most common inherited disorder that affects humans . according to recent reports , approximately out of 14 people only one
thalassemia , found in almost every ethnic group and geographic location , occurs more commonly in the mediterranean , african , indian and south - east asian populations . in developing countries like india
, hemoglobinopathies are seen to be increasing in an alarming rate due to lack of proper health - care . among the thalassemia patients in the world ,
about 10% belong to indian sub - continent , with 3 - 4% of them being carriers .
thalassemia syndromes are known to constitute a larger group of inherited hematologic disorders termed hemoglobinopathies .
therefore a defect in the structure of the globin chain can cause sickle cell anemia ; thalassemia is caused by a defect in the rate of synthesis of one or more globin chain .
the genetic defect in thalassemia results in reduced rate of synthesis of one of the globin chains that make up hemoglobin , which in turn can cause the formation of abnormal hemoglobin molecules , thus causing anemia , the characteristic presenting sign of thalassemia .
thalassemia major is generally detected during the 1 year of life because severe microcytic hypochromic anemia develops as fetal hemoglobin synthesis ceases after 3 - 4 months of age ; however in our case , normocytic normochromic anemia was noted .
the consequently produced exceptionally fragile red blood cells can survive for only few days in the peripheral circulation , which leads to critically increased rate of hematopoiesis ( up to 30 times the normal ) , in an attempt to maintain adequate oxygenation .
this results in massive bone marrow hyperplasia , in association with hepatosplenomegaly and lymphadenopathy due to extramedullary hematopoiesis , which could not be appreciated in the present case .
bone marrow hyperplasia could instigate marked but painless enlargement of the mandible and maxilla , resulting in characteristic chipmunk
facies , as was eminent in the first case , along with an evidence of frontal bossing .
if the patient is untreated , tissue hypoxia worsens and bacterial infections due to pneumococcal organisms often develop .
sooner or later , high - output cardiac failure occurs resulting in death of patients by 1 year of age as a result of infection or cardiac problems .
blood transfusion is the customary treatment for thalassemia major , administered every 2 - 3 weeks so as to simulate the normal hematologic state .
however , repeated blood transfusions unfortunately lead to iron overload due to constant infusion of exogenous red blood cells , causing hemochromatosis , ultimately leading to death .
heart , liver and endocrine glands are particularly affected by this toxic iron accumulation . to combat this problem , deferoxamine ( desferrioxamine )
bone marrow transplantation has also been used with considerable success for individuals who are relatively young , have little organ damage and have a human leukocyte antigen - matched donor . for patients with abnormal facial appearance caused by thalassemia
prevention of thalassemia is looked - for , either by screening for carriers of the genetic trait or by prenatal diagnosis .
it is an asymptomatic disease with mild anemia or none and very few red cell abnormalities are seen . in the present case ,
similar to thalassemia major , molecular genetics includes rare gene deletion in / , defects in transcription , processing or translation of -globin messenger ribonucleic acid ( mrna ) .
both patients are being treated by periodic blood transfusions , with an improvement in the general health being noted and the patients are under regular follow - up .
each of the thalassemia syndromes occurs due to specific gene mutation that is responsible for the decreased production of a specific globin .
this mutation could be in the form of deletions , rearrangement of the loci or point mutations leading to impaired transcription , processing , or translation of globin mrna , causing defective globin production .
variations in the severity of the clinical presentation may be a reflection of the specific alteration in the genetic code , for the reason that over 200 different mutations have been documented for this condition .
quite a few studies have been carried out to understand the genetic conformation of thalassemia .
thalassemias produce a deficiency of alpha or -globin , unlike sickle cell disease which produces a specific mutant form of -globin .
-globin chains are encoded by a single gene on chromosome 11 , while alpha globin chains are enclosed by two closely linked genes on chromosome 16 .
thus in a normal person with two copies of each chromosome , there are two loci encoding the -chain and four loci encoding the alpha chain .
deletion of one of the loci has a high prevalence in people of african or asian descent , making them more likely to develop alpha thalassemia , while -thalassemia is common in africans , greeks and italians .
to conclude , thalassemia constitutes a group of genetic disorders , which requires prompt diagnosis based on medical history , clinical examination , radiographic assessment , appropriate laboratory investigations in association with genetic studies .
irrespective of clinical features and thorough medical history laboratory investigations are must to be carried out in order to detect any blood disorder . | thalassemia is a rare , complex disease , representing a group of disorders of hemoglobin synthesis that are characterized by reduced synthesis of either the alpha - globin or -globin chains of the hemoglobin molecule .
defective synthesis of -globin resulting from a variety of molecular defects causes -thalassemia .
thalassemia is an autosomal recessive disorder , which requires prompt diagnosis and an appropriate treatment .
thorough clinical , radiographic and laboratory assessment helps in diagnosing thalassemia and any other hematological disorder . here
, two cases of -thalassemia showing distinct features are presented and the importance of detailed work - up of the cases has been highlighted . |
in the previous issue of critical care , protti and colleagues presented a series of patients with severe hyperlactatemia secondary to biguanide intoxication .
traditionally , hyperlactatemia in critically ill patients - and particularly those in shock - was normally interpreted as a marker of secondary anaerobic metabolism due to inadequate oxygen supply inducing cellular distress .
this view has recently been challenged with the demonstration that , during shock states , lactate production is , at least in part , linked to an increased aerobic glycolysis through 2 stimulation .
we recently demonstrated in a rat model that this mechanism occurs not only during sepsis ( high or normal blood flow ) , but also during hemorrhagic shock ( low blood flow ) . in clinical practice , there are clearly certain situations where hyperlactatemia is predominantly a reflection of tissue hypoperfusion with subsequent anaerobic metabolism .
cardiogenic shock , as demonstrated previously , is associated with hyperlactatemia with a very high lactate / pyruvate ratio . in theory ,
nevertheless , hemorrhagic shock , when prolonged , becomes an inflammatory shock and may therefore behave as septic shock .
the problem encountered with sepsis is more complex , although at least two situations are usually accompanied with hypoxia - associated hyperlactatemia .
the first situation is septic shock with catecholamine - resistant cardiocirculatory failure , especially in situations of low cardiac output .
the second circumstance is septic shock pre - emptively observed prior to volumetric expansion , as illustrated in the study of rivers and colleagues in which hyperlactatemia was associated with signs of poor oxygen delivery .
this leads to a decrease in the atp / adp ratio and an increase in the nadh / nad ratio .
a decrease in the atp / adp ratio induces both an accumulation of pyruvate , which can not be utilized by way of phosphofructokinase stimulation , and a decrease in pyruvate utilization by inhibiting pyruvate carboxylase , which converts pyruvate into oxaloacetate .
an increased nadh / nad ratio also increases pyruvate by inhibiting pyruvate dehydrogenase , and hence its conversion into acetylcoenzyme a. consequently , the increase in lactate production in an anaerobic setting is the result of an accumulation of pyruvate that is converted into lactate , which stems from alterations in the redox potential .
this conversion allows for the regeneration of some nad , enabling the production of atp by anaerobic glycolysis - although the process is clearly less efficient from an energy standpoint ( two atp molecules produced versus 36 ) .
it is important to consider that the modification of the redox potential induced by an increase in the nadh / nad ratio activates the transformation of pyruvate into lactate , and consequently increases the lactate / pyruvate ratio .
all in all , anaerobic energy metabolism is characterized by hyperlactatemia associated with an elevated lactate / pyruvate ratio , greater glucose utilization and low energy production .
recent observations have suggested that metformin , similarly to phenformin , may also inhibit mitochondrial respiration in tissues other than the liver . in the previous issue of critical care , using indirect measurement of oxygen consumption , protti and colleagues found that body oxygen consumption was markedly depressed despite a normal cardiac index evoking an inhibition of mitochondrial respiration .
unfortunately , arterial lactate / pyruvate and acetoacetate/-hydroxybutyrate ratios , as reflections of cytoplasmic and mitochondrial redox states , were unavailable .
interestingly , there was a clear correlation between drug clearance , correction of lactic acidosis and normalization of oxygen consumption .
clearly , the inhibition of mitochondrial respiration is not the unique mechanism involved in biguanide - induced lactic acidosis , since pure inhibition of mitochondrial function during cyanide poisoning is associated with death in the absence of antidote , and , similarly , since lactic acidosis associated with the use of nucleoside analogue reverse transcriptase inhibitors is due to an impairment of mitochondrial oxidative phosphorylation and is also associated with high mortality despite prompt therapy . to conclude , when looking at the literature , pure hypoxic causes of lactic acidosis are relatively rare in clinical practice . in the case of biguanide - induced lactic acidosis , the fact that the inhibition of mitochondrial respiration is reversible should encourage the early use of dialysis in order to accelerate drug elimination
| biguanide poisoning is associated with lactic acidosis .
the exact mechanism of biguanide - induced lactic acidosis is not well understood . in the previous issue of critical care , protti and
colleagues demonstrated that biguanide - induced lactic acidosis may be due in part to a reversible inhibition of mitochondrial respiration .
thus , in the absence of an antidote , increased drug elimination through dialysis is logical . |
in 2004 , two eblv-2 cases were identified in daubenton 's bats ( table 1 ) .
a diagnosis of eblv-2 infection was confirmed on brain samples with a fluorescent - antibody test , the mouse inoculation test , and a rapid taqman assay ( 8) .
attempts to culture eblv-2 from organs in both cases failed because of cytotoxicity of the samples , which destroyed the cell monolayer .
sample dilution reduced the cytotoxic effects of the sample on the cell monolayer ( used for virus isolation ) and enabled the development of small foci of infection ( bat 603/04 ) .
heminested rt - pcr detected virus rna in brain , tongue , thyroid gland , and bladder after the first round of amplification , and in salivary gland , heart , lung , intestine , and stomach after the second round of amplification .
we suspect that inappropriate storage of bat 696/04 in a freezer with repeated freezing and thawing before submission resulted in inactivation of virus in this sample .
heminested rt - pcr detected virus rna in samples of brain and stomach after the first round of pcr , and in samples of tongue , intestine , liver , and kidney after the second round of amplification .
an eblv-2-specific real - time pcr was developed to measure virus genome to quantify the potential viral rna load within organs .
analysis was only attempted on those organs with sufficient rna within the sample ( figure ) .
primers eblvna ( 5-cctggcagatgatgggac-3 ) and eblvnb ( 5-gccttttatcttggatcact-3 ) are located within the nucleoprotein gene and amplify a 221-bp target . an amplified product from a previous case ( 5 )
was purified by using the rneasy kit ( qiagen , valencia , ca , usa ) and quantified with a nanodrop wd-1000 spectrophotometer ( nanodrop technologies , inc . , wilmington , de , usa ) .
this procedure enabled the absolute number of copies of the amplicon to be calculated by its approximate molecular weight and avogadro 's number , as previously described ( 9 ) .
results of quantitative polymerase chain reaction ( pcr ) of viral genome copies within organ samples taken from 2 daubenton 's bats infected with european bat lyssavirus type 2 ( eblv-2 ) .
a ) standard curve of duplicate dilutions of known quantities of eblv-2 amplicon . a 20% ( vol / vol ) sample ( 10-fold dilution series ) separated by electrophoresis on a 1% agarose gel
b ) real - time pcr amplification of eblv-2 genomic rna from organ samples from a bat ( 696/04 ) in lancashire , united kingdom infected with eblv-2 .
dr , fluorescence change generated by a baseline - corrected algorithm calculated from every amplification cycle . a 20% ( vol / vol ) sample of each amplification separated by electrophoresis on a 1% agarose gel is shown in the inset .
br , brain ; to , tongue ; li , liver ; ki , kidney ; in , intestine ; st , stomach ; - , negative sample ( no template ) ; + , positive sample from a previous human case ( 5 ) .
rna was isolated from each organ with trizol ( invitrogen , carlsbad , ca , usa ) and quantified .
dilutions were made to either 0.25 g/l ( bat 603/04 ) or 1 g/l ( bat 696/04 ) to standardize the quantity of rna used for reverse transcription .
primer eblvna was used for cdna synthesis from the genomic ( negative ) sense strand as previously described ( 10 ) .
all pcrs were performed by using sybr green jumpstart taq readymix ( sigma , saint louis , mo , usa ) and an mx3000p real - time thermal cycler ( stratagene , la jolla , ca , usa ) .
a dilution series of the control amplicon was amplified simultaneously with the organ samples to create a standard curve for comparison of the threshold value ( ct ) with target copy number ( figure , panel a ) .
a representative plot of amplification curves from organ samples taken from bat 696/04 is shown in the figure , panel b , with 10 l of product separated by electrophoresis on a 1% agarose gel included for comparison .
the quantitative results for viral rna load for both bats are shown in table 2 . in both cases ,
eblv-2 was also found in the bladder of bat 603/04 but not in the kidney of bat 696/04 from which the bladder was not recovered because of carcass decomposition .
the detection and quantification of eblv-2 rna in bat organs by real - time pcr show the potential distribution of this virus .
the choice of organ tested in both cases was severely limited by degradation of the carcass before investigation .
furthermore , live virus could not be recovered from many organs because of cytotoxicity of the samples and virus degradation caused by repeated freezing and thawing .
since the brain is the main site of eblv-2 replication , this finding suggests that the virus displays a similar neurotropism to classical rabies virus .
rabies virus , especially in the late stages of disease , disseminates from the brain to other innervated sites within the host ( 11 ) . for eblv-2
, the tongue was consistently found to contain detectable levels of viral rna in this study and a previous study ( 7 ) .
genomic rna was also found in the stomach and intestines of 3 bats investigated ( this study and ) .
all of these organs are highly innervated tissues , although virus rna in the stomach could result from swallowing virus .
dissemination of rabies virus to the salivary glands and subsequent virus shedding enables transmission through biting .
detection of eblv-2 rna in the tongue of infected bats leads us to conclude that transmission of eblv-2 may occur through biting .
however , since eblv-2 genome was detected in a bladder sample , we can not exclude the possibility of virus release from urine . in future cases , where possible
, organs such as the salivary glands and lungs should be examined to provide further evidence for the route of virus transmission between bats . | organ distribution of european bat lyssavirus type 2 viral rna in its reservoir host , myotis daubentonii ( daubenton 's bat ) , was measured with a novel quantitative reverse transcription polymerase chain reaction assay .
high levels of genomic rna were found in the brain and were also detectable in the tongue , bladder , and stomach . |
antiviral therapy for hepatitis c virus ( hcv)-related chronic hepatitis results in a post - treatment sustained viral response ( svr ) in 50 - 90% of patients ( 1 ) .
virus genotype was reported to be the most important predictor of svr ( 2 ) .
in particular , genotype 1 patients are considered to be ' difficult - to - treat ' , whereas genotype 2 patients are considered to be ' easy - to - treat ' ( 2 ) .
other genotype - related factors / cofactors , potentially predictive of svr , are levels of viremia , virus interference on the genetic background of the host ( 3 ) , and the host features ( genetic background and metabolic interference ) ( 4 ) . based on viral kinetics , a decisional algorithm , which identified the ' stopping rules ' of therapy ,
patients with an early virological response ( evr ) i.e. after 12 weeks of treatment , have a high probability of an svr and are advised to continue treatment , whereas those who fail to respond after 12 weeks of treatment are asked to discontinue treatment ( 6 ) .
rapid virological response ( rvr ) , which corresponds to undetectable hcv - rna after 4 weeks of treatment ( 7 ) , has been shown to be a robust positive predictor of svr , and patients infected with hcv genotype 2 or 3 , who achieve rvr , are potential candidates for a short ( i.e. 6 months ) course of therapy ( 8 , 9 ) . while the lack of rvr does not necessarily result in a stopping rule , achieving rvr may serve to motivate patients and has implications , as to the duration of treatment in individual cases .
the rvr has a significantly higher predictive value than evr , and a series of studies , several of which are still ongoing , evaluated whether therapy could be optimized by adjusting the decisional therapeutic algorithm , based on the rvr ( 10 ) .
this retrospective study , conducted in a series of consecutive patients undergoing antiviral therapy with pegylated interferon ( peg - ifn)-2a or -2b and ribavirin , for hcv - related chronic hepatitis , was designed to evaluate the strength of rvr , as a predictor of svr , compared to other well - recognized factors / cofactors of response to antiviral therapy , as the primary end - point .
a secondary end - point of this study was to also analyze the possible biochemical , metabolic and/or virological interferences on rvr .
we retrospectively studied 315 consecutive outpatients affected by hcv - related chronic hepatitis from january 2009 to september 2011 , recruited from three tertiary centers of the second university of naples , who were undergoing antiviral therapy with peg - ifn- and ribavirin , according to nih guidelines ( 11 ) epidemiological and clinical characteristics are reported in table 1 .
inclusion criteria were : 1 ) elevated alanine transaminase ( alt ) levels during the last 6 months ; 2 ) hcv antibodies , and 3 ) no history of alcohol abuse .
exclusion criteria were : 1 ) overt infection with other hepatitis viruses ( i.e. hbsag+ ) ; 3 ) alcohol abuse ( > 20 mg / day in women and > 30 mg / day in men , in the 5 years before enrollment ) evaluated according to reid et al .
( 12 ) ; 4 ) history of active drug abuse ; and 5 ) hiv - positive test .
abbreviations : alt , alanine transaminase ; bmi , body mass index ; evr , early virologic response ; pegifn , pegylated interferon ; rvr , rapid virologic response ; svr , sustained virologic response .
virological , epidemiological , biochemical and ultrasound data were recorded upon admission to the centers .
body mass index ( bmi ) was calculated at the time of liver biopsy . when possible , the apparent disease duration was determined by considering exposure to major risk factors , as infection onset .
diabetes mellitus was identified according to the american diabetes association criteria , namely fasting glucose > 126 mg / dl , on two separate occasions , or a positive oral glucose tolerance test , on two separate occasions ( 13 ) .
total cholesterol , triglycerides , gamma - glutamyl transferase ( ggt ) , alt and ferritin were measured after a 12-hour fast .
metabolic syndrome was diagnosed according to national cholesterol education program - adult treatment panel iii criteria ( 14 ) .
markers of hbv infection were tested by a commercially available enzyme - linked immunosorbent assay ( abbott laboratories , chicago , il , usa ) .
the study was approved by the ethics committee of the second university of naples and patients gave their informed consent .
hepatic percutaneous biopsy was performed with a surecut 17 g needle , via the intercostal route , and was echo - assisted .
liver specimens were used for histological examination if they were at least 1.5 cm long and contained more than five portal spaces .
specimens were fixed in formalin , embedded in paraffin , and stained with hematoxylin - eosin .
biopsies were evaluated with the ishak score ( 15 ) , and biopsies with steatosis were also scored , according to brunt s criteria ( 16 ) . all rna preparation and
the rna was extracted according to chomczynski and sacchi ( 17 ) , and c - dna was derived .
we identified hcv rna using a nested pcr , with primers that expanded the highly conserved 5 non - coding genomic region .
carry - over pcr contamination was avoided by applying the measures suggested by kwok and higuchi ( 18 ) . to classify hcv genotypes , we hybridized serum pcr products to type- and subtype - specific probes 1a , 1b , 2a , 2b and 3a .
the probes had to fulfill two criteria : no more than two mismatches , compared with the corresponding published sequences of the same subtype , and they had to differ by three or more mismatches , compared with published sequences of other types and subtypes .
the only exception is probe 2b , which had only two mismatches , compared with the corresponding sequence of type 3a ( 19 ) . when appropriate , clinical and laboratory data were compared with the student s t test or the mann - whitney test .
we used multivariate analysis ( logistic regression model ) to calculate associations among dependent and independent variables .
virological , epidemiological , biochemical and ultrasound data were recorded upon admission to the centers .
body mass index ( bmi ) was calculated at the time of liver biopsy . when possible , the apparent disease duration was determined by considering exposure to major risk factors , as infection onset .
diabetes mellitus was identified according to the american diabetes association criteria , namely fasting glucose > 126 mg / dl , on two separate occasions , or a positive oral glucose tolerance test , on two separate occasions ( 13 ) .
total cholesterol , triglycerides , gamma - glutamyl transferase ( ggt ) , alt and ferritin were measured after a 12-hour fast .
metabolic syndrome was diagnosed according to national cholesterol education program - adult treatment panel iii criteria ( 14 ) .
markers of hbv infection were tested by a commercially available enzyme - linked immunosorbent assay ( abbott laboratories , chicago , il , usa ) .
the study was approved by the ethics committee of the second university of naples and patients gave their informed consent .
hepatic percutaneous biopsy was performed with a surecut 17 g needle , via the intercostal route , and was echo - assisted .
liver specimens were used for histological examination if they were at least 1.5 cm long and contained more than five portal spaces .
specimens were fixed in formalin , embedded in paraffin , and stained with hematoxylin - eosin .
biopsies were evaluated with the ishak score ( 15 ) , and biopsies with steatosis were also scored , according to brunt s criteria ( 16 ) .
all rna preparation and hcv rna determination steps were carried out under rnase - free conditions .
the rna was extracted according to chomczynski and sacchi ( 17 ) , and c - dna was derived .
we identified hcv rna using a nested pcr , with primers that expanded the highly conserved 5 non - coding genomic region .
carry - over pcr contamination was avoided by applying the measures suggested by kwok and higuchi ( 18 ) .
to classify hcv genotypes , we hybridized serum pcr products to type- and subtype - specific probes 1a , 1b , 2a , 2b and 3a .
the probes had to fulfill two criteria : no more than two mismatches , compared with the corresponding published sequences of the same subtype , and they had to differ by three or more mismatches , compared with published sequences of other types and subtypes .
the only exception is probe 2b , which had only two mismatches , compared with the corresponding sequence of type 3a ( 19 ) .
when appropriate , clinical and laboratory data were compared with the student s t test or the mann - whitney test .
we used multivariate analysis ( logistic regression model ) to calculate associations among dependent and independent variables .
independent predictive factors of svr were identified using univariate analysis in all patients ( table 2 ) .
multivariate analysis was carried out in patients divided according to genotype ( table 3 ) .
univariate analysis showed that staging ( p = 0.001 ) , bmi ( p = 0.037 ) , diabetes ( p = 0.000 ) , genotype ( p = 0.000 ) , rvr ( p = 0.000 ) , and viral load ( p = 0.013 ) were significantly associated to svr ( table 2 ) , whereas at multivariate analysis , only genotype ( p = 0.007 ) and rvr ( p = 0.000 ) were significantly associated to svr ( table 3 ) .
as shown in table 3 , in genotype-1-infected patients , staging ( p = 0.018 ) , alt ( p = 0.046 ) , metabolic syndrome ( p = 0.041 ) , diabetes ( p = 0.009 ) , rvr ( p = 0.000 ) and evr ( p = 0.000 ) were significantly associated to svr , at univariate analysis , while only rvr and evr ( p = 0.000 ) were independently associated to svr , at multivariate analysis . in genotype 2
patients , only rvr was related to svr , at both univariate and multivariate analyses . abbreviations : alt , alanine transaminase ; bmi , body mass index ; evr , early virologic response ; homa , homeostasis model assessment ; ms , metabolic syndrome ; peg - ifn , pegylated interferon ; rvr , rapid virologic response .
abbreviations : alt , alanine transaminase ; bmi , body mass index ; evr , early virologic response ; ms , metabolic syndrome ; rvr , rapid virologic response .
tables 4 and 5 show the results of univariate and multivariate analyses of factors predictive of rvr in all patients and in patients divided according to genotype , respectively . diabetes ( p = 0.003 ) ,
genotype 2 ( p = 0.000 ) and hcv - rna ( p = 0.016 ) were significantly related to rvr at univariate analysis , whereas at multivariate analysis , only genotype 2 ( p = 0.000 ) was an independent predictor of rvr , in all patients .
on the other hand , no laboratory or clinical factors were predictive of rvr in genotype 1b patients , at either univariate or multivariate analysis .
in genotype 2 patients , staging ( p = 0.029 ) and diabetes ( p = 0.001 ) were the only significant predictors of rvr , at univariate analyses , whereas no factor was independently related to rvr , at multivariate analysis .
finally , we evaluated the predictive positive value of evr and rvr in all patients and in patients according to virus genotype .
the predictive positive value ( ppv ) of svr was significantly higher in rvr patients , in both genotype groups .
in particular , the ppvs of rvr and evr for svr were 88% and 65% , in all patients , respectively .
moreover , ppvs of rvr and evr for svr in genotype 1b patients were 82% and 62% , respectively and , in genotype 1 patients , 93% and 80% , respectively
. abbreviation : alt , alanine transaminase ; bmi , body mass index ; homa , homeostasis model assessment ; ms , metabolic syndrome ; peg - ifn , pegylated interferon .
in recent years , various attempts have been made to maximize the therapeutic response to antiviral therapy with peg - ifn and ribavirin in patients with hcv - related chronic hepatitis .
besides dosing adjustments and patient adherence to interferon and ribavirin , tailoring the treatment regimen to the individuality of patients could greatly improve the response to therapy , avoiding relapse and minimizing adverse events . a recent study , in which therapy was optimized according to the patient 's baseline characteristics and response to therapy , revealed that rvr is a predictor of svr , and that duration of treatment could be modulated in relation to this parameter ( 20 ) .
factors associated with svr are either pretreatment or fixed factors ( genotype , hcv - rna levels , histology , race , steatosis , adherence to therapy , etc . ) or dynamic factors ( rvr and evr ) .
a svr probably depends on multiple factors , and hence , can differ among individuals ( 21 , 22 ) . this large multicenter retrospective , consecutive study evaluated the predictive value of rvr for svr , in both difficult - to - treat and easy - to - treat patients .
we also assessed the predictive role of a number of parameters , as to rvr .
it is noteworthy that our data were obtained in clinical practice and in patients undergoing antiviral therapy with peg - ifn and ribavirin , according to nih guidelines ( 11 ) , treated with the standard of care for patients with hcv - related chronic hepatitis .
we found that rvr was the strongest predictor of svr in all of our patients infected with hepatitis c virus , although there were substantial differences between the two genotypes .
in fact , we found that rvr was the only independent factor associated with svr in overall ( i.e. easy - to - treat and difficult - to - treat ) patients , whereas in difficult - to - treat patients , both rvr and evr were independently related to svr .
this result suggests that patients who obtain rvr are likely to have an svr , irrespective of sex , age , fibrosis and comorbidity .
furthermore , in patients with genotype 1 , evr was related to svr also , and this finding has implications for treatment duration .
it is well known that exposure to ifn and ribavirin , when hcv - rna is negative , increases the possibility of maintaining the response and of obtaining an svr .
therefore , an extension of treatment may be beneficial in patients who achieve an evr ( 23 , 24 )
. we also found that the patients metabolic status may affect the likelihood of achieving an rvr .
in fact , diabetes was independently related to rvr in all patients , and in patients with genotype 2 , in particular .
therefore , all genotype 2 patients without comorbidity achieved an rvr . on the other hand , in patients with genotype 1 , no variable was directly related to rvr at univariate and multivariate analyses .
we hypothesize that genotype 1 , per se , influences the metabolic status of patients ( 25 ) . in conclusion
, our study suggests that the occurrence of rvr provides information about treatment outcome in hcv - infected patients .
nowadays , triple therapy with protease inhibitors ( 26 ) and results from interferon - free trials ( 27 ) represent a real option , as a rescue therapy , for those patients who fail to clear the virus after 4 weeks of treatment with peg - ifn and ribavirin . | background : standard [ i.e. pegylated interferon ( peg - ifn ) + ribavirin ] treatment of hepatitis c virus ( hcv)-related chronic hepatitis is associated with a sustained virological response ( svr ) in 50 - 90% of patients .
a rapid virological response ( rvr ) ( i.e. negative hcv - rna after 4 weeks of treatment ) predicts svr in almost 90% of patients.objectives:the main aim of this study was to assess the strength of rvr , as a predictive factor of antiviral treatment response.patients and methods : using univariate and multivariate analysis , we retrospectively evaluated biochemical , metabolic , genetic and viral variables that might affect both rvr and svr to peg - ifn plus ribavirin , in 315 consecutive outpatients affected by hcv - related chronic hepatitis.results:at univariate analysis , staging , body mass index , rvr , genotype and viral load were significantly related to svr ( p < 0.001 ) . at multivariate analysis ,
rvr and genotype remained significant ( p < 0.00001 ) .
the rvr had a predictive value of 83% . at univariate and multivariate analyses , diabetes ( p = 0.003 ) ,
genotype 2 ( p = 0.000 ) and hcv - rna values ( p = 0.016 ) were independent predictors of rvr , even though at multivariate analyses , only genotype 2 was significantly related to rvr .
when we stratified patients , according to genotype , no laboratory or clinical factors were predictive of rvr in genotype 1 patients at either univariate or multivariate analysis . in genotype 2 patients , staging ( p = 0.029 ) and diabetes ( p = 0.001 ) were the only significant predictors of rvr at univariate analyses , whereas no factor was independently related to rvr , at multivariate analysis.conclusions:the rvr is the strongest factor of svr and infection with hcv genotype 2 is significantly associated with rvr . neither biochemical and/or metabolic factors seem to exert influence on rvr . |
non - small cell lung carcinoma ( nsclc ) accounts for 80 - 85% of all lung carcinomas , and adenocarcinoma is the predominant histologic type .
the prognosis of these patients remains poor , with an overall 5-year survival rate of less than 15% despite the advanced therapeutic options available .
recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinomas , one associated with smoking and activation of the k - ras oncogene and the other not associated with smoking and activation of epidermal growth factor receptor ( egfr ) .
introduction of targeted therapy with epidermal growth factor receptor - tyrosine kinase inhibitors ( egfr - tki ) has revolutionized the treatment of adenocarcinoma .
patients with these tumors survive significantly longer with egfr - tki therapy than with conventional chemotherapy .
the aim of this study was to subtype poorly differentiated nsclc based on the expression of thyroid transcription factor-1 ( ttf-1 ) and p-63 and to evaluate egfr expression in adenocarcinoma .
somatic mutations within the tyrosine kinase catalytic domain of egfr lead to conformational changes that promote permanent active status and are found in approximately 20% of lung adenocarcinomas .
they are considered to be the most reliable predictors of response to egfr - tkis .
a large number of studies on egfr expression status in lung carcinoma are available in the western literature .
paraffin blocks of all the cases of poorly differentiated nsclc reported between 2006 and 2011 were retrieved from the archives of the pathology department of sri ramachandra medical college and were included in the study .
five - micron - thick paraffin sections were cut and immunostained for ttf-1 and p-63 and the tumors were subtyped .
immunohistochemical staining for ttf-1 was performed using the biogenex monoclonal ttf-1 mouse antibody ; clone ( bgx-397a ) diluted in phosphate - buffered saline ( pbs ) . staining for p-63
was performed using biogenex monoclonal p-63 mouse antibody ; clone ( 4a4 ) diluted in pbs .
subtyping of nsclc into adenocarcinoma and squamous cell carcinoma was based on the morphology and immunohistochemistry ( ihc ) .
subtyping of nsclc was based on the algorithm followed by the international association for the study of lung cancer / american thoracic society / european respiratory society ( iatc / ats / ers ) international multidisciplinary team .
immunohistochemical staining for total egfr protein was performed using the biogenex monoclonal egfr rabbit antibody ; clone ( ep38y ) pre - diluted in pbs on both control and test sections according to the manufacturer 's instructions .
slides were scored based on the cytoplasmic and/or membrane staining intensity as follows : 0 - no staining or faint staining intensity in < 10% of tumor cells ; 1 + = faint staining in > 10% of tumor cells ; 2 + = moderate staining ; 3 + = strong staining .
ninety cases of carcinoma lung were diagnosed during the 5-year period from 2006 to 2011 . of these ,
non - small cell carcinoma accounts to 93% ( 84 cases ) , followed by small cell carcinoma ( 3% ) , carcinoid tumor ( 1% ) , mucoepidermoid carcinoma ( 1% ) and pleomorphic carcinoma ( 1% ) [ table 1 ] [ chart 1 ] .
of the 84 cases of nsclc , 53 cases ( 63% ) were males and 31 cases ( 37% ) were females .
fifty - five percent of the nsclc were adenocarcinoma[figure 1 ] with a peak incidence between 61 and 70 years of age [ charts 2 and 3 ] .
of the 84 cases of nsclc , 46 cases ( 55% ) were immunohistochemically proven primary adenocarcinoma of the lung .
this was followed by squamous cell carcinoma ( 24% ) , adenosquamous carcinoma ( 4% ) and non - small cell carcinoma - not otherwise specified ( nsclc - nos ) ( 18% ) [ table 2 ] .
subtyping of nsclc was based on the algorithm followed by the iatc / ats / ers international multidisciplinary team [ table 3 ] .
ttf-1 was expressed in 55% of the adenocarcinoma cases[figure 2 ] and p-63 was positive in 24% of squamous cell carcinoma[figures 4 and 5 ] ; both markers were positive in different tumor cells in 4% of the cases and were considered adenosquamous carcinoma .
eighteen percent of the tumors were negative for both markers and were hence considered as nsclc - nos with a possibility of metastasis [ chart 4 ] .
semiquantitative scoring of adenocarcinoma for egfr positivity showed 3 + positivity in 85% of the cases[figure 3 ] , 2 + positivity in 10% of the cases and 1 + positivity in 5% of the cases .
egfr was positive in 89% of adenocarcinoma and negative in 11% of the cases [ charts 5 and 6 ] .
distribution of all types of lung carcinoma prevalence of lung carcinoma microphotograph of adenocarcinoma ( hematoxylin and eosin , 200 ) demographic profile based on gender distribution age - wise distribution of non - small cell lung carcinoma subtyping of non - small cell lung carcinoma based on immunohistochemistry subtyping of nsclc based on ihc markers microphotograph of adenocarcinoma ( thyroid transcription factor-1 , 200 ) microphotograph of adenocarcinoma ( epidermal growth factor receptor , 200 ) microphotograph of squamous cell carcinoma ( hematoxylin and eosin , 100 ) microphotograph of squamous cell carcinoma ( p-63 , 100 ) subtyping of non - small cell lung carcinoma immunohistochemical scoring of epidermal growth factor receptor in adenocarcinoma distribution of epidermal growth factor receptor in adenocarcinoma
nsclc accounts for 80 - 85% of all lung carcinomas , and adenocarcinoma is the predominant histologic type with a male predominance ( m : f ratio : 1.7:1 ) .
however , an increase in the incidence of adenocarcinoma among non - smokers and women is noted in north america and europe .
globally , the overall lifetime risk of lung cancer is about 1 in 13 for men and 1 in 16 for women .
unfortunately , despite the therapeutic advances , the prognosis of patients with lung cancer ( 5-year overall survival rate of 15% ) has not changed dramatically in the past 30 years .
currently , diagnostic and treatment approaches to lung carcinoma , mainly adenocarcinoma , are undergoing a revolution .
the classification of lung carcinoma is going beyond small cell carcinoma and non - small cell carcinoma .
precise subcategorization of nsclc into adenocarcinoma and squamous cell carcinoma has a direct impact on patient management and prognosis .
presence of egfr mutations in adenocarcinoma is a predictor of responsiveness to egfr tyrosine kinase inhibitors .
the diagnosis of lung carcinoma is a multidisciplinary process requiring correlation with clinical , radiologic , molecular and surgical information .
the world health organization has published guidelines for the classification of lung cancer in resection specimens based on the examinations of the entire tumor ; however , 70% of the lung cancers present at advanced stages and are unresectable and hence subjected to platinum - based chemotherapy or radiation therapy .
hence , a new classification has been proposed by the iaslc / ats / ers for small biopsies and cytology samples .
this new classification emphasizes the use of histochemical ( mucin staining ) and immunohistochemical stains ( ttf-1 , p-63 ) and molecular studies apart from routine histomorphology on hematoxylin and eosin - stained slides .
morphologic diagnosis forms the basis and is further supplemented by a panel of immunohistochemical markers . tumor cells in adenocarcinoma are positive for ttf-1 , napsin and cytokeratin-7 .
ttf-1 is a protein that regulates transcription of genes specific for thyroid , lung and developing central nervous system ( diencephalon ) .
it is encoded by the nkx2 - 1 gene and is seen in chromosome 14q .
it is produced by clara cells and type ii pneumocytes of the peripheral bronchioalveolar unit .
it is a highly specific ( 97 - 100% ) but not very sensitive ( 54 - 75% ) marker for adenocarcinoma .
tumor protein p-63 is encoded by tp63 gene , a member of the p-53 family of transcription factors .
it is expressed in the normal respiratory epithelium of the central air conducting system and does not carry any prognostic implications in nsclc patients .
the sensitivity of p-63 ranges from 75% to more than 95% , whereas the specificity for squamous cell carcinoma is between 70% and 100% .
napsin a is moderately sensitive ( 79 - 85% ) and highly specific ( 100% ) for adenocarcinoma .
mucin stains are valuable markers , but the sensitivity and specificity for adenocarcinoma can be variable .
ntrk1 and ntrk2 are highly specific for squamous cell carcinoma , but are rarely used outside research laboratories . ttf-1 and
p-63 can be used as a reliable diagnostic tool in subtyping these tumors in routine practice .
difficulty arises in subtyping nsclc in small biopsies due to sampling error and tumor heterogeneity .
loo et al . reported that the combination of ttf-1 , alcian blue / pas , p-63 and ck5/6 in small biopsies is best in differentiating adenocarcinoma from squamous cell carcinoma .
nicholson et al . also used pas with diastase , p-63 , ttf-1 and ck5/6 to subtype nsclc into adenocarcinoma and squamous cell carcinoma .
we used a panel of two markers , ttf-1 and p-63 , in subtyping 84 cases of poorly differentiated carcinomas .
ttf-1 was positive in 55% of adenocarcinoma and p-63 was positive in 27% of squamous cell carcinomas . ttf-1 and p-63 were negative in 18% of the cases and favored metastatic adenocarcinoma .
terry et al . studied the expression of nine immunohistochemical markers on 588 lung carcinomas , of which 200 cases were adenocarcinoma and 225 were squamous cell carcinoma .
the sensitivity of ttf-1 was found to be 62% and the specificity was found to be 92% .
p-63 is considered as the single best marker to separate squamous cell carcinoma and adenocarcinoma , with a sensitivity of 84% and a specificity of 85% .
screening for egfr expression and mutation analysis would be of great value in designing treatment protocols in nsclc .
patients with these tumors survive significantly longer on egfr - tki therapy than with conventional chemotherapy .
mutations were frequently found in women ( 69.7% ) , in those who never smoked ( 66.6% ) and in those with adenocarcinomas ( 80.9% ) .
the presence of kras , her2 , braf , pi3k , lkb1 and shp2 mutations is associated with a lack of response to egfr - tkis in the treatment of lung cancer .
therefore , additional mutational analysis of genes other than egfr may be necessary to improve patient selection for egfr - targeted therapies , and egfr amplification and/or overexpression are also predictors of response to tki treatment
. the egfr overexpression accounts for about 43 - 83% of nsclc , being more common in squamous cell carcinoma ( 70% ) , followed by adenocarcinoma ( 50% ) and , to a lesser extent , in large cell carcinoma .
the egfr mutation status is best determined by fluorescence in situ hybridization ( fish ) or chromogenic in situ hybridization ( cish ) and protein expression determined by ihc with mutation - specific antibodies .
egfr is expressed in 50% of nsclc , and its expression is correlated with poor prognosis .
ruschoff et al . evaluated the interobserver reproducibility of the egfr ihc scoring system based on both the tumor cell membrane staining intensity ( graded 0 to 3 + ) and the percentage of cells staining at each intensity .
this allowed a highly reproducible allocation of nsclcs into clinically relevant high or low egfr expression groups .
our study revealed a much higher percentage of egfr expression ( 89% ) , indicating the importance of screening our patients with adenocarcinoma lung .
the current therapeutic strategies for lung cancers require accurate morphological differentiation between adenocarcinoma and squamous cell carcinoma . a panel of immunohistochemical markers , ttf-1 and p-63 , help in subtyping the poorly differentiated carcinomas .
p-63 immunostain were extremely useful in making a conclusive diagnosis , especially in small biopsies with a poorly differentiated morphology .
egfr mutation is a predictive biomarker for egfr - tki therapy . in our study ,
although the status of egfr has been assessed by ihc , cish and fish , mutational analysis has been shown to be the best predictor of tumor response to egfr - tki , which will be assessed in future studies .
although the status of egfr has been assessed by ihc , cish and fish , mutational analysis has been shown to be the best predictor of tumor response to egfr - tki , which will be assessed in future studies . | background : lung cancer is a highly aggressive malignancy causing high morbidity and mortality . an increasing incidence of lung cancer has been observed in india .
currently , the classification of lung carcinoma has gone beyond small cell lung carcinoma and non - small cell lung carcinoma ( nsclc ) .
precise subtyping of poorly differentiated nsclc into adenocarcinoma and squamous cell carcinoma has a direct impact on patient management and prognosis . with this background ,
many molecules are under study for developing targeted therapies .
epidermal growth factor receptor ( egfr ) is one such biomarker considered to be useful in targeted therapy for adenocarcinoma.objective:the aim of this study was to subtype poorly differentiated nsclc based on the expression of thyroid transcription factor-1 ( ttf-1 ) and p-63 and to evaluate egfr expression in adenocarcinomas.materials and methods : a retrospective analysis of 84 cases of poorly differentiated carcinomas of the lung was performed .
paraffin sections were immunostained with ttf-1 and p-63 and the tumors were subtyped .
egfr expression was assessed in adenocarcinomas by immunohistochemistry.results:fifty-five percent of the nsclc were adenocarcinoma , with a peak incidence between 61 and 70 years of age and a male predominance .
egfr was expressed in 89% of the adenocarcinomas.conclusions:poorly differentiated non - small cell carcinoma can be subtyped by immunohistochemical markers and hence has a direct impact on the current therapeutic strategies . |
optical approaches to compensate for presbyopia include reading glasses , monovision , multifocal contact lenses , multifocal intraocular lenses ( miols ) , and accommodating iols .
none of these can restore accommodation , but all are compromises that establish a fair quality of near vision at the expense of good far vision .
even a small amount of astigmatism provides some pseudoaccommodation in pseudophakic patients.1 several methods to restore accommodation with scleral expansion near the ciliary body were designed ; however , none has proven to be effective.24 corneal procedures for the compensation of presbyopia include presbyopic laser in situ keratomileusis ( presby - lasik ) , conductive keratoplasty , intracorneal implants ( or inlays ) , and the intracor and supracor ( technolas perfect vision gmbh , munchen , germany ) procedures .
we describe a case series of patients with a history of prior phakic iol implantation surgery in whom an intracorneal inlay ( kamra ) ( model aci 7000pdt , acufocus inc , irvine , ca , usa ) was implanted in the nondominant eye after creating a femtosecond laser corneal pocket .
the kamra small - aperture intracorneal inlay is designed to increase the depth of focus in the implanted eye , based on the principle of small - aperture optics.5 the inlay restores near and intermediate visual acuity without a significant impact on distance vision.68
this study conforms to the ethics codes established by the ethical board committee of japan .
all patients read and signed informed consent forms , which explained the surgical procedure , possible risks , and patients rights .
this case study includes three eyes with a history of prior phakic iol implantation surgery .
the aim of the present study was to show the efficacy of the kamra small - aperture intracorneal inlay in presbyopic patients with prior refractive surgeries , which was one of the exclusion factors in previous reports.5,810 the preoperative and postoperative assessment of patients and surgical techniques is already described in our previous study.9 patient satisfaction after surgery was estimated from the patients satisfaction questionnaires , as was done previously.9 a 55-year - old woman had presbyopia correction with inlay implantation in her left eye .
her preoperative visual acuity was evaluated using a snellen chart for distance and log - mar for near vision ( which was converted to a jaeger chart thereafter ) .
preoperative spherical equivalent ( se ) was 0.25 diopter ( d ) ; uncorrected distance visual acuity ( udva ) and corrected distance visual acuity ( cdva ) were 20/20 and 20/16 , respectively ; and uncorrected near visual acuity ( unva ) and corrected near visual acuity ( cnva ) were j4 and j1 , respectively .
the patient underwent a comprehensive preoperative examination.9 during surgery , the corneal pocket creation was done for the kamra inlay implantation with a crystal line femto ldv ( ziemer ophthalmic systems ag , port , switzerland ) .
the kamra inlay implantation was done with a 230 m pocket depth , not 200 m as is usual , due to the presence of 603 m central corneal thickness .
the inlay was carefully placed in the pocket over the estimated line of sight based on the midpoint of the first purkinje image and the pupil center with coaxial patient fixation.9 three - month results showed an improvement in unva to j2 , with even better outcomes for udva , which improved to 20/16 ( table 1 ) .
se was 1.0 d. cdva and cnva remained stable during the follow - up period .
the patient did not report needing reading glasses in any light conditions . a 54-year - old woman with a history of a lasik procedure that was done soon after her
the patient underwent a rigorous ophthalmic examination.9 udva and cdva were both 20/16 ; unva and cnva were j6 and j1 , respectively .
the corneal pocket formation was done for her kamra inlay implantation with a crystal line femto ldv ( ziemer ophthalmic systems ag ) .
the inlay was placed in the pocket the same way as has already been described.9 three - month results showed an improvement in unva to j4 with minimal change in udva , which was 20/20 .
se was 0.38 d. cdva and cnva remained stable during the follow - up period .
regarding patient satisfaction , the patient complained of a slight worsening of her near vision at night .
a 51-year - old woman was a candidate for presbyopia treatment by kamra inlay implantation surgery .
one month before inlay surgery , the patient underwent a lasik procedure in order to correct the refractive error after her first phakic iol implantation procedure .
se just before inlay implantation surgery was 0.00 d. udva and cdva were both 20/16 .
the kamra inlay was implanted the same way as with the case 2 patient at a depth of 200 m . there was an improvement in unva at the 3-month follow up , from j10 to j5 . a minimal change in udva
se was 0.5 d. cdva and cnva remained stable . at the 3-month follow - up
a 55-year - old woman had presbyopia correction with inlay implantation in her left eye .
her preoperative visual acuity was evaluated using a snellen chart for distance and log - mar for near vision ( which was converted to a jaeger chart thereafter ) .
preoperative spherical equivalent ( se ) was 0.25 diopter ( d ) ; uncorrected distance visual acuity ( udva ) and corrected distance visual acuity ( cdva ) were 20/20 and 20/16 , respectively ; and uncorrected near visual acuity ( unva ) and corrected near visual acuity ( cnva ) were j4 and j1 , respectively .
the patient underwent a comprehensive preoperative examination.9 during surgery , the corneal pocket creation was done for the kamra inlay implantation with a crystal line femto ldv ( ziemer ophthalmic systems ag , port , switzerland ) .
the kamra inlay implantation was done with a 230 m pocket depth , not 200 m as is usual , due to the presence of 603 m central corneal thickness .
the inlay was carefully placed in the pocket over the estimated line of sight based on the midpoint of the first purkinje image and the pupil center with coaxial patient fixation.9 three - month results showed an improvement in unva to j2 , with even better outcomes for udva , which improved to 20/16 ( table 1 ) .
se was 1.0 d. cdva and cnva remained stable during the follow - up period .
a 54-year - old woman with a history of a lasik procedure that was done soon after her prior phakic iol implantation surgery complained about her near vision .
the patient underwent a rigorous ophthalmic examination.9 udva and cdva were both 20/16 ; unva and cnva were j6 and j1 , respectively .
the corneal pocket formation was done for her kamra inlay implantation with a crystal line femto ldv ( ziemer ophthalmic systems ag ) .
the inlay was placed in the pocket the same way as has already been described.9 three - month results showed an improvement in unva to j4 with minimal change in udva , which was 20/20 .
se was 0.38 d. cdva and cnva remained stable during the follow - up period .
regarding patient satisfaction , the patient complained of a slight worsening of her near vision at night .
a 51-year - old woman was a candidate for presbyopia treatment by kamra inlay implantation surgery .
one month before inlay surgery , the patient underwent a lasik procedure in order to correct the refractive error after her first phakic iol implantation procedure .
se just before inlay implantation surgery was 0.00 d. udva and cdva were both 20/16 .
the kamra inlay was implanted the same way as with the case 2 patient at a depth of 200 m . there was an improvement in unva at the 3-month follow up , from j10 to j5 . a minimal change in udva
se was 0.5 d. cdva and cnva remained stable . at the 3-month follow - up
the inlay s polyvinylidene difluoride material incorporates nanoparticles of carbon to make it opaque.5 this permeable material has a light transmission rate of 5% .
the inlay has a pseudorandom microperforation pattern consisting of 8,400 holes ranging in size from 5 m to 11 m in diameter , to allow water and nutrition flow .
the inlay is 5 m thin and has a 3.8 mm total diameter and a 1.6 mm diameter central aperture .
there is no refractive power in the central aperture.9 the kamra inlay can be implanted under a flap or into an intrastromal pocket , both created by a femtosecond laser .
all three patients of our study had corneal pocket formation during the inlay implantation . in the postphakic eye of case 1 , the main point to mention is that the kamra inlay implantation was with a 230 m depth pocket due to 603 m central corneal thickness appearing .
the unva improved by two lines at 3 months postoperatively in this patient . no need for reading glasses was reported from this patient . in all presented cases ,
the corneal inlay was implanted as per the normal post - lasik procedure using a pocket .
we saw near vision improvement in all patients with postphakic eyes . only one patient ( case 2 ) reported some worsening of her near vision at night .
the reason for this may be the decreasing of the depth of focus with a larger pupil diameter .
figure 2 presents the unva change during the entire follow - up period for all three cases .
thus , based on our results , with improved near visual acuity , minimum impact on udva , and the appearance of clear corneas , with no opacities or changes in epitheliums , we may consider that kamra inlay surgery in postphakic iol patients appears to be safe .
additionally , it should be noted that there are other options for presbyopia corrections , such as intrastromal femtosecond laser correction and miol .
but we consider , based on the reported study,11 that intrastromal femtosecond laser correction has more impact on udva than does kamra inlay .
miol implantation might be another option for these patients , but as the patients of our study did not have any cataract formation , and knowing that cataract surgeries are not as safely reversible as kamra inlay surgeries , the patients of our study were not good candidates for miol surgery .
in summary , our case report study results suggest that the implantation of a kamra corneal inlay may improve monocular near visual acuity in presbyopic patients with a history of a prior phakic iol implantation surgery , based on our short - term follow - up . | we report a series of three case reports of kamra inlay implantation procedures in presbyopic patients with a history of prior phakic intraocular lens implantation surgery .
three - month results showed a two to five - line improvement for uncorrected near visual acuity .
the absolute uncorrected near visual acuity change for case 1 was from j4 to j2 , for case 2 was from j6 to j4 , and for case 3 was from j10 to j5 .
no significant change of uncorrected distance visual acuity was observed in all three cases . |
mites of the genus demodex are commonly found in the hair follicles and sebaceous glands of most mammals . in general , demodex are considered to be host - species specific and some hosts can be infested with two or more distinct species ( e.g. , d. canis , d. injai , an undescribed short form demodex sp . in dogs , d. brevis and d. folliculorum in humans , d. odocoilei in white - tailed deer ( odocoileus virginianus ) , and d. bovis in cattle ) [ 15 ] . in general , demodex infestations can vary widely in clinical presentation ( from asymptomatic animals to cases with variable extends of alopecia , varying degrees of thickening of the skin , to cutaneous nodular lesions and severe dermatitis / furunculosis ) . in small animals ,
some animals have concurrent immunosuppression ; however , it is unclear what role immunosuppression plays in generalized clinical demodicosis .
in addition , some individuals or species , especially cervids in europe , as well as cattle , develop a nodular demodicosis [ 3 , 6 ] . in a longitudinal study of cattle , these nodules waxed and waned both in number and size over time .
this infested deer exhibited hair loss and thickening of the skin on the head , neck , and shoulders .
a second incidence of demodicosis was reported in oklahoma in 1971 from a white - tailed deer with alopecia .
demodex odocoilei , currently the only known demodex sp . from white - tailed deer in the united states , was described using material from skin scrapings of deer in georgia , virginia , and oklahoma .
histology of the skin samples did indicate hair loss and distention of the hair follicle and sebaceous glands , but the presence of mites was not associated with inflammation .
mites morphologically consistent with d. odocoilei were reported from columbian black - tailed deer ( odocoileus hemionus columbianus ) and mule deer ( odocoileus hemionus hemionus ) from the western united states and canada [ 9 , 10 ] .
recently , demodex kutzeri , a species of european cervids , was reported from three species of cervids ( one mule deer and one rocky mountain elk ( cervus elaphus nelsoni ) from colorado and a white - tailed deer from south dakota ) as well as a white - tailed deer from south carolina ( since 1971 ) . throughout the southeastern united states ,
numerous other cases of demodicosis have been diagnosed in white - tailed deers ( scwds , unpublished data ) ; however , the causative species of demodex was not determined .
demodex is currently classified in the subclass acari , superorder acariformes , order prostigmata , superfamily chelyetoidea , and family demodicidae .
although pcr and sequence analysis have served as vital techniques to investigate relationships of numerous organisms , including mites , there is currently only genetic information available for four demodex species , d. folliculorum and d. brevis from humans , d. canis from a dog , and d. musculi from mice in genbank ( accession number jf834894 ) .
the increased availability of sequence data would allow researchers to investigate the diversity of demodex species infecting different or the same host species , host specificity , and investigate any geographic variability among demodex ( e.g. , d. canis from various continents ) . a sample of demodex from a white - tailed deer clinical case submitted to the southeastern cooperative wildlife disease study ( scwds )
was found to differ morphologically from d. odocoilei and other demodex species reported from cervids . in this study ,
our goal was to characterize this demodex sp . using a combination of morphological and molecular techniques .
the morphologic characters of this demodex sp . were compared to other cervid demodex including d. odocoilei , d. acutipes , and d. kutzeri [ 3 , 4 , 12 ] . for our molecular analysis , we compared the demodex sp .
from the white - tailed deer with two demodex samples from dogs , demodex ursi from a black bear , and related sequences available in genbank from demodex and related mites .
in october 2002 , a 2.5-year - old female hunter - killed deer from lee county , south carolina ( usa ) was submitted to scwds for diagnostic evaluation because of grossly visible skin lesions .
a complete necropsy was performed and samples of skin were fixed in 10% buffered formalin , embedded in paraffin , sectioned , and stained with hematoxylin and eosin .
demodex mites removed from nodules were placed in immersion oil and covered with a cover slip .
mites were examined on 400x power and measurements of all life stages ( adult male and female , nymph , larva , and ovum ) were taken using a calibrated micrometer .
measurement of the adults included total body length , gnathosomal length , podosomal length , opisthosomal length , and presence of and length of aedeagus or vulva as appropriate .
measurements of ova , larvae , and nymphs included total length and width for ova . a total of 38 ova , 38 larvae , 40 nymphs , 42 female , and 44 male mites were measured .
skin nodules from the white - tailed deer clinical case had been preserved in 100% ethanol , skin samples of d. ursi from a black bear from florida ( usa ) with generalized demodicosis were stored at 20c until analysis , and samples of d. canis and a short form demodex sp . from dogs from clarke county , georgia ( usa )
each individual sample was placed into a sarstedt o - ring tube with two copper bbs .
the sample was macerated for 2 one - minute cycles at 1/2-speed setting with a mini beadbeater 8 ( biospec products , bartlesville , ok ) .
phosphate - buffered saline ( 200 l ) was added to each tube and the samples were vortexed thoroughly .
dna was extracted using the gfx genomic blood dna purification kit ( amersham biosciences , piscataway , nj , usa ) according to the manufacturer 's directions for direct blood .
because the primers were designed for ticks , we used three tick species as positive controls .
dna was extracted from three individual ticks ( dermacentor variabilis , amblyomma americanum , and haemaphysalis leporispalustris ) as described for the mites except that they were individually frozen in liquid nitrogen before being macerated .
water controls were included in each step of the pcr analyses ( extraction , primary reaction , and secondary reaction ) to serve as negative controls .
overlapping primer pairs that amplify the 18s rrna gene of the ticks and other mites were selected to provide near full - length sequence data for demodex .
overlapping nested and heminested secondary pcr reactions with primer pairs ns1/ns2 , ns12+/ns2 , ns1/ns4 , and ns58.1/ns8 were conducted as described .
briefly , each 25 l reaction contained 11 l molecular biology grade water , 2.5 l 25 mm mgcl2 , 5 l 5x colorless buffer ( promega , madison , wi ) , 0.25 l of 20 mm dntps ( promega ) , 0.5 l each primer ( 50 m ) , and 0.25 l gotaq flexi ( promega ) .
reaction conditions consisted of 1 min at 92c followed by 10 cycles of 1 min at 92c , 1 minute at 48c , and 90 sec at 72c , then 32 cycles of 1 min at 92c , 35 sec at 54c , and 90 sec at 72c , and a final extension time of 7 min at 72c .
amplicons were purified using the qiaquick gel purification kit ( qiagen , valencia , ca , usa ) and bidirectionally sequenced at the integrated biotechnology laboratories ( university of georgia , athens , ga , usa ) .
sequences obtained from this study and from related organisms stored in genbank were aligned using the multisequence alignment clustal algorithm in mega version 3.1 .
genbank accession numbers for the demodex sp . from white - tailed deer , d. canis , the short form demodex sp . from a dog , and d. ursi are kc010483 , kc010485 , kc010484 , and kc010482 , respectively .
phylogenetic analyses were conducted using mega version 3.1 program using the minimum evolution algorithm with the kimura 2-parameter model .
the deer weighed 58 kg and was in good physical condition with adequate fat deposits around the kidneys and within the mesentery .
the only gross lesion noted during necropsy was the presence of multiple ~1 - 2 cm tan cutaneous nodules on the head , legs , and lateral aspects of the thorax and abdomen , but no alopecia was noted ( figure 1 ) .
the nodules , when examined microscopically , were shown to contain several hundreds to thousands of intrafollicular demodex mites ( figures 2 and 3 ) .
histologically , hair follicles were extremely enlarged and the cysts were lined by stratified squamous epithelium varying between three and more than 20 cells in thickness ( figure 4 ) . the majority of follicles were not surrounded by inflammatory cells ( figures 2 , 3 , and 4 ) , but some cysts were disrupted resulting in infiltration with lymphocytes , plasma cells , and eosinophils ( figure 5 ) .
the demodex sp . from the white - tailed deer in this study was most similar morphologically to d. kutzeri and d. acutipes from european red deer [ 3 , 12 ] and is easily differentiated from d. odocoilei from white - tailed deer ( figures 6 and 7 ) .
numerous morphological measurements of larval , nymphal , and adult mites can be used to differentiate the demodex sp . from d. odocoilei from white - tailed deer ( tables 1 and 2 ) .
this demodex sp . can also be differentiated from d. acutipes and d. kutzeri of red deer by some measurements of adult mites ( e.g. , width of podosoma and opisthosoma and length of aedeagus ) ( table 2 ) .
overlapping sequences obtained from the demodex sp . of wtd , demodex canis , and the demodex sp .
( short form ) from the dog were 1,035 , 1,029 and 1,020 bp , respectively .
the two canine demodex sequences only differed by a single base and differed from the demodex from the wtd by 34 bp .
the 766 bp obtained from d. ursi from the black bear was identical to the overlapping sequence of the demodex sp . of wtd .
the final alignment of the demodex spp . from the wtd and dogs with related organisms ( shown in figure 8) was 1,093 bp ; 405 bp was variable and of those 267 was parsimonious informative .
the samples from the genus demodex formed a monophyletic clade with d. brevis from humans at the base .
the demodex sp . of wtd formed a monophyletic clade with d. musculi from laboratory mice ( jf834894 ) and these were in a clade with d. folliculorum from humans ( figure 8) .
basal to the demodex clade was neochelacheles messersmithi , another mite in the superfamily chelyetoidea , which inhabits spore tubes of polypore fungi and preys on astigmatic mites .
the separation of demodex spp . from n. messersmithi was well supported ( bootstrap 100% ) .
since 1977 , 16 cases of demodicosis in white - tailed deer have been documented at scwds ( unpublished data ) , and when combined with previously documented cases , demodex - infested white - tailed deer have been detected throughout the host range ( maine , south dakota , texas , and numerous southeastern and midwestern states ) [ 4 , 5 , 7 , 8 , 1618 ] . in only a few of these cases were the mites morphologically determined , and most were identified as d. odocoilei , but recently , three deer were infested with d. kutzeri .
infection of individual mammalian species with multiple species of demodex is common ( e.g. , three species in domestic dogs , two in domestic cats ) ; thus the finding of a third demodex species infesting white - tailed deer is not unexpected .
the demodex sp . identified in this clinical case was on average 30% larger than demodex odocoilei but was morphologically similar to d. acutipes and d. kutzeri , both parasites of red deer ( cervus elaphus ) from europe , and the latter was recently reported from three species of cervids from north america .
however , the demodex sp . can be differentiated from both by several morphologic measurements .
similar to the initial description of d. odocoilei , the demodex sp . in this paper
similar to observations with d. odocoilei , little to no inflammation was associated with the majority of hair follicles and sebaceous glands containing the mites . however ,
in contrast to d. odocoilei - infected deer , the demodex sp .- infected deer had hair follicles that were severely distended and contained thousands of mites within giant cystic , follicular structures .
the gross presentation was more similar to that described for d. kutzeri and d. acutipes from red deer in europe [ 3 , 12 ] and d. kutzeri from three species of cervids from colorado , south dakota , and south carolina .
the case material used in this study was obtained from a clinical case submission of a noncaptive hunter - killed deer ; therefore , we were unable to investigate the chronicity of lesions associated with the demodex sp . in white - tailed deer .
similar to previous reports of d. kutzeri in cervids , the current clinical case presented with nodular demodicosis which tends to be rare in cases of d. odocoilei infestation .
interestingly , demodex from deer , dogs , mice , and humans ( d. folliculorum ) formed a clade separate from the other human - infesting demodex species ( d. brevis ) ( current study and ) .
the monophyletic demodex group was related to n. messersmithi , another member of the superfamily chelyetoidea supporting the classical relationships of this superfamily of mites and other recent studies on the phylogenetics of the chelyetoidea [ 15 , 19 ] .
use of the 18s rrna gene was useful in distinguishing most species in the current study and one previous study on human and canine species .
however , based on partial 18s rrna gene sequence , we were unable to distinguish the mites from white - tailed deer and bear as different demodex species ; however , demodex ursi from black bears and the demodex sp . from white - tailed deer
, demodex sequence data of more variable gene targets from additional species of demodex obtained from a diverse set of hosts will allow studies on the diversity of demodex sp . | demodex mites , although usually nonpathogenic , can cause a wide range of dermatological lesions ranging from mild skin irritation and alopecia to severe furunculosis .
recently , a case of demodicosis from a white - tailed deer ( odocoileus virginianus ) revealed a demodex species morphologically distinct from demodex odocoilei .
all life cycle stages were considerably larger than d. odocoilei and although similar in size to d. kutzeri and d. acutipes from european cervids , numerous morphometrics distinguished the four species .
adult males and females were 209.1 13.1 and 225.5 13.4 m in length , respectively .
ova , larva , and nymphs measured 65.1 4.1 , 124.9 11.6 , and 205.1 19.4 m in length , respectively . for phylogenetic analyses ,
a portion of the 18s rrna gene was amplified and sequenced from samples of the wtd demodex sp .
, two demodex samples from domestic dogs , and demodex ursi from a black bear .
phylogenetic analyses indicated that the wtd demodex was most similar to d. musculi from laboratory mice .
a partial sequence from d. ursi was identical to the wtd demodex sequence ; however , these two species can be differentiated morphologically .
this paper describes a second demodex species from white - tailed deer and indicates that 18s rrna is useful for phylogenetic analysis of most demodex species , but two morphologically distinct species had identical partial sequences .
additional gene targets should be investigated for phylogenetic and parasite - host association studies . |
the cooccurrence of hiv and unintended pregnancy has prompted a relatively recent body of work on dual protection , the simultaneous protection against hiv and unintended pregnancy .
dual protection can be achieved through single method use ( condoms ) or dual method use ( condoms + another contraceptive method ) .
most studies measure dual protection with condom use , but dual protection definitions must expand to capture condom use consistency and a wider variety of contraceptive methods . thus far , dual protection studies have focused on samples of women from the general population , have concentrated on developed or african countries , and have not considered hiv - related factors as influential in dual protection behavior .
studies that have considered the benefits of dual protection for people living with hiv show that dual protection can be an effective strategy to prevent hiv transmission to partners and to promote safe childbearing [ 25 ] , which warrants more research in this population .
worldwide , hiv is the leading cause of death for women of childbearing age , and up to 64% of all pregnancies are unintended [ 6 , 7 ] .
brazil has over a third of all people living with hiv in latin america and the caribbean .
although the epidemic has become stabilized in brazil ( at 0.5% prevalence ) , the male to female ratio of new hiv infections fell from 27 to 1 in 1985 to 1.4 to 1 in 2010 .
women represent 35% of the 608,230 people living with hiv / aids , and most female infections are in women of childbearing age ( 83% ) and via heterosexual transmission ( 96% ) .
little is known about the dual protection behavior of women living with hiv in brazil .
although a substantial proportion of women living with hiv have children after being diagnosed with hiv , women living with hiv in brazil report lower reproductive intentions than women in the general population [ 10 , 11 ] .
access to and provision of family planning to these women are essential to enable fertility control and to prevent unintended pregnancy . although no global estimates exist for unintended pregnancy among women living with hiv , an estimated 30 to 64% of all pregnancies are unintended , with women in south america enduring the greatest burden worldwide .
some studies indicate that the incidence of induced abortion in women living with hiv may be greater than that found in the general population .
other studies find that women living with hiv are often exclusively offered condoms , because they provide dual protection , to the exclusion of being offered a complete basket of contraceptive options [ 8 , 13 ] . for almost a decade
, studies report an unmet need for family planning among women living with hiv in brazil [ 12 , 14 , 15 ] .
although dual protection is not explicitly promoted in brazil , most adults in the general population and women living with hiv make clear distinctions between contraceptive methods and the dual protection offered by condoms [ 16 , 17 ] .
given these distinctions it is critical that we better understand dual method use , a much less studied form of dual protection that grants women greater control over their fertility than condom use alone . from a public health perspective
, there is resistance to promoting dual methods due to a notion that we call the dual method hypothesis , which purports that women who use dual methods for dual protection will use either method or both methods more inconsistently than if they just used one sole method .
several studies provide support for this hypothesis but test it among the general population of women [ 18 , 19 ] .
this study tests the dual method hypothesis and reveals how the dual method hypothesis plays out in a sample of women living with hiv in brazil .
we also explore how hiv - related factors like antiretroviral therapy ( art ) and partner 's hiv status affect sexual behavior , condom use , and contraception .
a study in africa found that , within a three - month period , most people living with hiv ( 89% ) had more than one sexual partner and some ( 36% ) had acquired a new sexual partner .
although the overall effect of art on sexual behavior and dual protection is relatively unexplored , we know that art lowers viral load , that art together with condoms helps to prevent hiv transmission to a sexual partner , and that art together with family planning is the most effective way to reduce the vertical transmission of hiv .
since 1996 , the brazilian national hiv program has offered universal access to art and hiv care to people living with hiv . clinical status and cd4 + count
generally , art is initiated when cd4 + count 500 cells / m or when the person is symptomatic , coinfected with hepatitis b , at high risk for ( or has ) cardiovascular disease , being treated for cancer , or pregnant .
brazil also has protocols in place to prevent vertical transmission , which has resulted in a rapid decline in infants with hiv . despite the availability of art
, a study in so paulo , brazil , finds that less than half of the women living with hiv report using condoms .
the effects of partner 's hiv status on condom use is mixed depending on the sampled population .
studies find that condom use is typically more consistent among hiv - serodiscordant than seroconcordant couples [ 21 , 25 ] , but one study reports that serodiscordant partners have greater odds of changing sexual partners than seroconcordant partners .
although statistics for brazil are unavailable , one study reports that serodiscordant partnerships are on the rise and may make up 60% of all relationships .
condom nonuse by hiv - seroconcordant couples may pose a risk for coinfection with other sexually transmitted diseases ( stds ) which is linked to decreased cd4 + count and increased viral load [ 27 , 28 ] .
this study first describes the characteristics of our sample of women living with hiv in brazil .
second , we test the dual methods hypothesis that women who use dual methods will do so less consistently than women who use only condoms .
data are from a cross - sectional sample of hiv - positive women attended in specialized sti / aids public health service clinics in 13 municipalities from all five regions of brazil between 2003 and 2004 .
women were eligible for the study if they could read and write , were over 18 years , and were hiv positive .
a total of 1,777 women completed a self - administered questionnaire and deposited it anonymously into a secure ballot box .
the self - administered ballot box technique is associated with fewer refusals , nonresponses , and false responses to sensitive questions and is associated with a greater frequency of reporting socially sanctioned behavior than face - to - face interviews and at times acasi [ 30 , 31 ] .
the ballot box is a preferred technique to survey sexual behavior in brazil because it is associated with the confidentiality of political elections , is a validated method in brazil [ 31 , 32 ] , and is recommended for low - literacy populations ( 28% of our sample has an elementary school or less education ) .
our sample includes hiv - positive women who are bi-/heterosexual , of reproductive age , who report at least one sexual partner in the past six months , and with condom use data ( n = 1 , 087 ) .
we conducted a sensitivity analysis , removing bisexual women , and results did not differ with the exception that sexual violence became a statistically nonsignificant predictor of consistent condom use .
further , only women who reported using condoms ( n = 834 ) were included in our analysis of consistent condom use .
we also conducted a missing data analysis and used multiple imputation to ascertain that omitted cases were not significantly different than those included .
the research protocol was approved by the irb of the so paulo sti / hiv reference and training center .
the consistent condom use measure asked , in the last six months , you used condoms : never , always , or sometimes ?
respondents who said they always used condoms were coded as using consistent condoms , whereas respondents who marked sometimes used condoms were coded as using inconsistent condoms .
sexual and reproductive health variables include method of contraception , desire to have children , number of live children , stable partner , history of std in last six months , and lifetime history of sexual violence .
method of contraception comprises three categories ( excluding condom use ) : no method , irreversible method , and reversible method .
reversible methods include birth control pills , intrauterine device ( iud ) , hormonal injection , and diaphragm .
we classified contraceptive method based on a hierarchy where female - controlled methods took precedence over vasectomy and irreversible methods took precedence over reversible methods . desire to have children is a binary variable in which the responses yes and do not know were collapsed into one category .
number of live children includes the number of children a woman had before and after testing positive for hiv .
women were categorized as having a stable partner if they responded positively to having a husband , fiance , or steady boyfriend . lifetime history of sexual violence measured any experience of being forced to engage in sexual relations .
we used stepwise logistic regression to select dual protection predictor variables related to sociodemographic characteristics , sexual and reproductive health , fertility , and hiv - related variables .
we employed descriptive and logistic regression techniques to describe dual protection and to identify important predictors of dual protection in our sample .
our first set of analyses uses the chi - square coefficient to assess significant differences related to consistent condom use .
our second set of analyses uses logistic regression to examine the bivariate and adjusted associations between consistent condom use and predictors .
prior to entering each variable into an adjusted model , we conducted a bivariate analysis to assess the association between each independent variable and dual protection .
results for significant predictors of dual protection are displayed in the logistic models , but all independent variables are included in adjusted models .
table 1 describes the characteristics of our sample . women averaged 32 years old and most had a junior high school education or more ( 73% ) .
most women either used no method of contraception ( 51% ) or an irreversible method ( 34% ) , did not desire to have children ( 58% ) , had 2.11 children on average , and were in a stable relationship ( 87% ) .
the majority of women had no history of stds in the past six months ( 81% ) and had no history of sexual violence ( 78% ) .
most women were in an hiv - serodiscordant relationship ( 47% ) and were taking art ( 72% ) .
dual protection was prevalent in our sample with most women using condoms consistently ( 77% ) .
women who used only condoms for dual protection reported more consistent condom use ( 82% ) than women who used dual methods ( condoms + another method ) for dual protection ( 72% ) . among dual method users , 74% of the women who use an irreversible method report using condoms consistently versus only 66% of women who use a reversible method .
women not using dual protection ( 13% ) either used no contraceptive method ( 42% ) , an irreversible method ( 37% ) , or a reversible method ( 22% ) .
women who use consistent condoms are slightly older and more educated than women who do not .
women who do not desire to have children report more consistent condom use ( 80% ) than women who desire to have children ( 74% ) . women with no lifetime history of sexual violence report more consistent condom use ( 79% ) than women with a history of sexual violence ( 71% ) . women with an hiv - negative partner report more consistent condom use ( 86% ) than women who do not know their partners ' hiv status ( 71% ) and women with hiv - positive partners ( 68% ) .
women on art report more consistent condom use ( 80% ) than women who are not on art ( 68% )
.
table 3 presents the bivariate and adjusted models of independent variables on the odds of consistent condom use .
women who use irreversible methods have 0.59 times the odds and women who use reversible methods have 0.48 times the odds , of using consistent condoms than women who use only condoms for dual protection , net of other factors . women who desire to have children
have 0.63 times the odds of using consistent condoms than women who do not desire children , net of other factors .
women with a lifetime history of sexual violence have 0.64 times the odds of using consistent condoms than women without a history , net of other factors .
women with hiv - negative partners have 3.23 times the odds of using consistent condoms than women with hiv - positive partners , net of other factors . women not on art have 0.56 times the odds of using consistent condoms than women on art , net of other factors .
consistent condom use ( 77% ) was much greater in our sample of women living with hiv than women in the general population at the time ( 34% in steady relationships and 66% in casual relationships ) .
one study in brazil suggests that women increase consistent condom use from prediagnosis ( 71% ) to postdiagnosis ( 86% ) .
high levels of condom use in women living with hiv may be the result of the brazilian government 's extensive effort to promote condom use in populations most vulnerable for hiv infection . despite high levels of condom use ,
23% of our sample used no dual protection and 9% of our sample used no dual protection and no form of contraception .
the lack of condom and contraception may reflect a desire to conceive [ 35 , 36 ] .
however , a closer look at our data reveals that only 60% of the women who do not use condoms or contraception report fertility desires , highlighting a need for intervention .
our study provides some support for the dual methods hypothesis and adds a caveat to the hypothesis .
the dual methods hypothesis argues that women who use dual methods for dual protection will use either one method or both methods inconsistently ; some studies indicate that the more effective the contraceptive method , the more inconsistent the condom use [ 17 , 37 ] .
we find that women who use only condoms use them more consistently than women who use dual methods ; this finding is also supported by whiteman et al . .
other studies that consider dual method use among women living with hiv demonstrate that dual method use is a viable option for dual protection but do not measure condom use consistency [ 24 ] .
mark et al . finds the adoption of a nonbarrier contraceptive method does not lead to a decline in condom use or an increase in pregnancy .
use data from a multinational prospective study of hiv - serodiscordant couples in africa to show modest declines in condom use with the uptake of another contraceptive method .
however , these studies highlight the promise of promoting dual methods for dual protection in this population .
our findings are contrary to the belief that the more effective the contraceptive method , the more inconsistent the condom use .
we find that , among dual method users , women who use an irreversible method use condoms more consistently than women who use a reversible method . a possible explanation for this finding may be the triple burden faced by women living with hiv where , on a daily basis , women must manage art protocols , prevent hiv transmission , and prevent unintended pregnancy
. a closer look at our data reveals a pattern in which condom use consistency is greatest among women who use a long - term contraceptive method ( sterilization , iud , and hormonal injection ) followed by women who use contraceptive methods that require daily attention ( contraceptive pill ) .
female sterilization may serve to alleviate women from the daily burden of contraception , allowing them to focus on consistent condom use .
moreover , for women on art , taking the oral contraceptive pill adds yet another pill to their daily medication regimens , could cause undesirable side - effects , and could interact with art to become a less effective method of contraception . in light of these factors ,
future research should consider how consistency of condom use , consistency of reversible contraceptive use , and art adherence interact ; an analysis is not permitted with our data .
there is a growing body of literature that links the ability of women living with hiv to use condoms and contraception to a positive relationship context ( i.e. , quality of relationship , partner dynamics , and communication ) [ 3941 ] .
our study finds that women with hiv - serodiscordant partners report the most consistent condom use , followed ( in order ) by women who do not know their partners ' hiv status , and women with hiv - seroconcordant partners .
our findings are supported by other studies that find the most consistent condom use is among hiv - serodiscordant couples [ 21 , 25 ] .
women in hiv - serodiscordant relationships are likely to use more consistent condoms because they have disclosed their hiv status to their partners and are in known discordant relationships .
these women are therefore clearer with their partners about the risks involved in having unprotected sex and more compelled to protect the health of their partners [ 4244 ] .
it is also possible that women who do not know their partners ' status are likely to be in less committed relationships and , therefore , are not compelled to disclose their hiv - positive status to their sexual partner , are less able to negotiate condom use , and are not as concerned for their sexual partners ' health to use consistent condoms [ 42 , 4446 ] .
women with hiv - seroconcordant couples had the lowest consistent condom use in our study , with over 30% not using consistent condoms .
relationship factors , like communication between hiv - seroconcordant partners , may lead to decisions to discontinue condoms due to the lack of perceived risk and the desire for sensation and intimacy [ 39 , 47 ] .
however , unprotected sex among hiv - seroconcordant partners could expose women to coinfection with other stds , which has been linked to decreases in cd4 + count and increases in hiv viral load [ 27 , 28 ] .
in addition , condom nonuse in hiv - seroconcordant couples may put them at risk of reinfection with other variants of hiv , including drug - resistant viral strains , which could hasten seroconversion to aids and potentially complicate art protocols .
although this phenomenon is not well understood , the potential repercussions are especially disconcerting for a context like brazil , where art is freely provided by the government .
in fact , there is so much growing concern of the emergence and transmission of antiretroviral drug resistance ( adr ) in brazil that the ministry of health has started to monitor adr in the most populated state capitals .
several studies have reported the prevalence of adr in brazil to vary from 1.4 to 12.7% [ 4951 ] , and one study in a national sample of msm found the prevalence to be as high as 2136% .
art is positively related to consistent condom use in our sample , a finding supported by other studies [ 21 , 53 , 54 ] .
data from the swiss hiv cohort study indicate that people living with hiv who are on art have lower odds of engaging in unsafe sex than those who are not on art .
art requires frequent visits to healthcare centers which may be linked to greater exposure to safe sex counseling , greater access to healthcare and to condoms , and health care seeking behavior .
other studies find that safer sexual behavior is related to art adherence [ 2 , 42 ] and that some people living with hiv believe condom use is essential for art to be effective .
evidence suggests that those who care for themselves by adhering to art regimens may have greater odds of also protecting their sexual partners by using condoms consistently . on the contrary
, we find that women who are not on art have lower odds of consistent condom use .
condom nonuse may occur in women who are not on art because they feel healthy and do not perceive the need to use , or can not negotiate , condoms .
lack of condom use may also result from a limited knowledge of hiv transmission and disease dynamics and insufficient exposure to condom messaging . in any case , women living with hiv who are not on art are also not likely to be connected with hiv services and may not know their hiv viral load , the most important determinant of hiv transmission risk in hiv - serodiscordant partners .
this finding highlights the need to counsel women , upon hiv - positive diagnosis , about transmission risk and to address barriers to condom use .
first , data are from a convenient sample of women living with hiv in brazil .
although this limits the generalizability of our findings , the specialized sti / aids health service clinics provide care to most hiv cases in each municipal ; there is no reason to assume our sample is different than the greater population .
second , data are from a cross - sectional survey and employ questions that require the respondent to recall sexual behavior . this caveat may have introduced a recall bias that could have led the respondent to inaccurately recall information .
however , the secret ballot box technique used for survey collection has been associated with a more accurate response rate to sensitive questions regarding sexual behavior than other modes of survey administration [ 3033 ] .
first , dual protection is a common practice among our sample of women living with hiv in brazil .
second , we test the dual methods hypothesis and find that women who use dual methods have lower odds of consistent condom use than women who use a single method ( condoms ) . of dual method users , we find women who use irreversible methods use consistent condoms more than women who use reversible methods .
third , we find that women who are on art and who have hiv - serodiscordant partners have greater odds of consistent condom use than their counterparts .
this study highlights dual method use as a promising form risk - reduction strategy for women living with hiv . | the cooccurrence of hiv and unintended pregnancy has prompted a body of work on dual protection , the simultaneous protection against hiv and unintended pregnancy .
this study examines dual protection and dual methods as a risk - reduction strategy for women living with hiv .
data are from a cross - sectional sample of hiv - positive women attended in specialized sti / aids public health service clinics in 13 municipalities from all five regions of brazil 2003 - 2004 ( n = 834 ) .
descriptive techniques and logistic regression were used to examine dual protection among women living with hiv .
we expand the definition of dual protection to include consistent condom use and reversible / irreversible contraceptive methods , we test the dual methods hypothesis that women who use dual methods will use condoms less consistently than women who use only condoms , and we identify predictors of dual protection .
dual protection is common in our sample .
women who use dual methods have lower odds of consistent condom use than women who only use condoms . among dual method users , we find that women who use an irreversible method use condoms more consistently than women who use a reversible method .
women on art and with an hiv - serodiscordant partner have greater odds of consistent condom use than their counterparts . |
a 70-year - old male was diagnosed with non - st - elevation myocardial infarction ( nstemi ) without signs of haemodynamic compromise and intermediate grace risk scores ( fig .
initial cardiac biomarkers were elevated with a creatine kinase of 1236 u / l and positive high sensitive troponin of 787 ng / l .
he was scheduled for coronary angiography within 24 h. one and a half hours after admission the pain had not resolved despite medical therapy , and it was decided to perform immediate angiography . to our surprise , occlusion of a large left anterior descending artery ( lad ) was found with collaterals from the right coronary artery .
after the procedure the patient remained free of symptoms and during further observation no complications occurred.figure 1a electrocardiogram on admission .
after pci with implantation of a 3.5 mm drug - eluting stent a electrocardiogram on admission . 25 mm / s , 10 mm / mv .
st - segment elevation only may not always reflect ongoing ischaemia and we should no longer focus on the presence or absence of st - segment elevation as a reliable criteria to proceed or to postpone urgent angiography and/or reperfusion therapy [ 1 , 2 ] .
future studies should focus on the nstemi acs algorithm and its identification of high - risk patients who may benefit from urgent coronary angiography and subsequent revascularisation [ 3 , 4 , 5 ] . in our opinion , the acute myocardial infarction classification based on st elevation alone should be reconsidered . | acute coronary syndromes are usually classified on the basis of the presence or absence of st elevation on the ecg : st - elevation myocardial infarction or non - st - elevation myocardial infarction ( nstemi)patients with acute myocardial infarction ( ami ) need immediate therapy , without unnecessary delay and primary percutaneous coronary intervention ( ppci ) should preferably be performed within 90 min after first medical contact .
however , in ami patients without st - segment elevation ( pre ) hospital triage for immediate transfer to the catheterisation laboratory may be difficult .
moreover , initial diagnosis and risk stratification take place at busy emergency departments and chest pain units with additional risk of
ppci delay. optimal timing of angiography and revascularisation remains a challenge .
we describe a patient with nstemi who was scheduled for early coronary angiography within 24 h but retrospectively should have been sent to the cath lab immediately because he had a significant amount of myocardium at risk , undetected by non - invasive parameters . |
neoadjuvant chemotherapy is integrated into the treatment of several cancers , including head and neck squamous cell carcinoma ( hnscc ) . in a phase iii study by licitra and vermorken ,
rates of mandibular resection were significantly lower in hnscc patients receiving neoadjuvant chemotherapy , thus demonstrating the ability of this treatment to preclude morbid and aggressive surgery .
on the other hand , neoadjuvant chemotherapy does not have an established role in differentiated thyroid cancer ( dtc ) .
patients with thyroid cancer are historically treated with surgical resection of the primary tumor and locoregional nodal metastasis followed by radioiodine ablation ( rai ) . while the majority of thyroid cancers present as locally resectable tumors with minimal surgical morbidity , occasionally the primary tumor has invaded critical structures , complicating the surgical treatment plan .
direct invasion of the larynx , trachea , pharynx , esophagus , recurrent laryngeal nerve , strap muscles , and/or carotid artery occurs in 7%16% of patients with thyroid cancer .
extensive invasion of the primary tumor into surrounding structures can necessitate relatively morbid procedures such as laryngectomy , tracheal resection , and esophageal - pharyngeal resection .
nevertheless , the importance of complete resection can not be overstated as approximately 80% of patients who die of thyroid cancer have locoregional recurrence .
certainly , a small number of patients also present with disease that may not be amenable to surgical resection , or in some cases the patient may refuse surgical resection due to the surgical morbidity . in these cases , a discussion regarding the role of neoadjuvant chemotherapy or radiotherapy is warranted . unfortunately , neoadjuvant external beam radiation therapy ( xrt ) can lead to fibrosis , which can make resection of the thyroid more challenging for the surgeon . in these situations
, there is a potential role for neoadjuvant chemotherapy and there are data that suggest that in selected patients , this strategy could prove to be beneficial for the treatment of extensively invasive dtc . in this review , we will describe the potential role of neoadjuvant therapy in the treatment of dtc .
chemotherapy generally has a limited role in dtc as it is associated with minimal survival benefit when used as single modality therapy . in the neoadjuvant
a favorable tumor response may offer the patient and surgeon significant improvement in the ability to provide an oncologic surgical resection . given
that data on neoadjuvant therapy in dtc is extremely limited , we will first discuss chemotherapy in its more established role in the metastatic setting in an effort to assess tumor response rates as they might pertain to neoadjuvant therapy .
we will then describe the results of studies demonstrating the efficacy of neoadjuvant chemotherapy in dtc as well as the data for newer , targeted agents in an effort to identify the best possible options for neoadjuvant therapy in dtc .
although a limited number of chemotherapies have been used effectively in the neoadjuvant setting , chemotherapy has not been shown to be particularly effective in the metastatic setting .
the only fda - approved chemotherapy for dtc is doxorubicin , which offers minimal benefit and is associated with toxicity for patients with metastatic dtc .
doxorubicin was initially introduced to clinical practice due to a study by gottlieb et al . , which described 6 case reports of patients with dtc treated with various chemotherapy agents given alone or in combination .
two patients ( 33.3% ) , both of whom were treated with doxorubicin , had a partial response ( pr ) .
these results led to a prospective clinical trial studying 15 patients with dtc treated with doxorubicin , in which a 33% pr was observed .
however , multiple , larger , and better - designed trials over the past 30 years have demonstrated that doxorubicin may not be as effective as previously believed .
for example , matuszczyk et al . conducted a retrospective study in which doxorubicin was given to patients with dtc , finding only a 5% pr .
given the limited efficacy of doxorubicin as monotherapy , other chemotherapy combinations have been evaluated in clinical trials , but also with disappointing results .
described a study with 22 patients with advanced thyroid cancer of all histological subtypes treated with doxorubicin and cisplatin .
they observed a 9.1% pr and the treatment was associated with considerable toxicity , including one treatment - related death .
another study by shimaoka et al . described patients that were randomized to doxorubicin with cisplatin or doxorubicin alone . among a group of 35 patients with dtc ,
the overall response rate ( orr ) for combination therapy ( 16% ) was inferior to that of monotherapy ( 31% ) .
while these results for both doxorubicin monotherapy and combination chemotherapy are discouraging for the treatment of metastatic dtc this data should be interpreted with caution .
chemotherapy is typically reserved for rai - refractory cases of dtc , and thus the biology involved in these cases may be different from that seen in the preoperative setting [ 31 , 32 ] .
the following studies , on the other hand , explore the efficacy of doxorubicin as well as various other agents given as neoadjuvant chemotherapy in dtc .
we identified three studies involving the treatment of dtc with neoadjuvant chemotherapy : two from slovenia and one from japan .
rates of residual tumor after resection are summarized in table 3 [ 11 , 12 , 14 ] .
one of the studies from slovenia described a retrospective , nonrandomized study of 29 patients with t3 or t4 follicular or hurthle cell thyroid cancers treated from 1979 to 2004 in which the tumor was considered inoperable .
mean tumor diameter was 9.3 cm and extrathyroid growth was seen in 15/29 patients ( 51.7% ) .
regional metastases were present in 6 patients ( 20.7% ) and distant metastases in 12 ( 41.4% ) .
chemotherapy consisted of vinblastine for 19 patients ( 65.5% ) , vinblastine with doxorubicin for 5 patients ( 17.2% ) , and other regimens for the remaining 5 patients ( 17.2% ) .
surgery was performed when the tumor was reduced after chemotherapy and/or xrt and the surgeon judged the tumor resectable .
the median interval between the beginning of chemotherapy and surgical procedure was 36 days ( range : 4173 days ) .
tumor size was decreased by > 50% in 13 patients ( 44.8% ) . for patients with distant metastases , tumor size decreased by > 50% in only 17% of patients , while in patients without distant metastases , tumor size decreased by > 50% in 65% of patients .
tumor resection after chemotherapy was performed in all patients , resulting in r0 ( defined as with no residual tumor ) , r1 ( microscopic residual tumor ) , and r2 ( macroscopic residual tumor ) resections in 15 ( 51.7% ) , 10 ( 34.5% ) , and 4 ( 13.8% ) cases , respectively .
total thyroidectomy was performed in 24 patients ( 82.6% ) and lobectomy was performed in 5 patients ( 17.2% ) .
similarly , the same group in slovenia described a retrospective , nonrandomized study of 16 patients with t3 or t4 papillary thyroid cancer treated from 1988 to 2005 in which the tumor was considered to be inoperable .
mean tumor diameter was 9.7 cm and extrathyroid growth was present in 13 patients ( 81.3% ) .
regional metastases were present in 10 patients ( 62.5% ) and distant metastases in 7 ( 43.8% ) .
chemotherapy consisted of vinblastine in 11 cases ( 68.8% ) , vinblastine with doxorubicin in 2 cases ( 12.5% ) , and other regimens in 3 cases ( 18.8% ) .
surgery was performed when the tumor was reduced after chemotherapy and/or xrt and the surgeon judged the tumor resectable .
the median interval between the beginning of chemotherapy and surgical procedure was 28 days ( range : 7161 days ) .
after chemotherapy , tumor size decreased by > 50% in 7 patients ( 44% ) .
r0 , r1 , and r2 resection was performed in 2 ( 12.5% ) , 10 ( 62.5% ) , and 4 ( 25% ) cases , respectively .
total thyroidectomy was performed in 11 patients ( 68.8% ) and lobectomy in 5 patients ( 31.3% ) .
interestingly , these two studies from slovenia describe very different rates of residual tumor after resection ( see table 3 ) .
given that response rates were similar between the first and second study , average tumor size was similar , and the surgeries were performed at the same institution over a similar period of time ; this difference is difficult to account for .
it is likely due to a higher rate of extrathyroid growth in the second study ( 81.3% ) when compared to that of the first study ( 51.7% ) . given this difference in extrathyroid growth and resections between the two groups , it is possible that papillary thyroid carcinoma can be more invasive and consequently more difficult for the surgeon to resect completely .
these results are also difficult to characterize given the variety of chemotherapy strategies utilized and the time period over which the reviews were performed .
an additional study from japan evaluated the effect of weekly paclitaxel chemotherapy in 3 patients with papillary thyroid cancer with a squamous cell carcinoma component ( a very aggressive tumor with behavior resembling anaplastic thyroid cancer ) .
weekly chemotherapy was performed as induction for 2 of the patients , and all patients underwent locally curative surgery with weekly adjuvant chemotherapy after surgery . the response to chemotherapy
the first patient was a 70-year - old woman with a solitary 5.9 cm thyroid tumor and multiple node metastases .
she additionally had an incomplete right laryngeal nerve paralysis , likely due to tumor invasion .
she received weekly induction paclitaxel at 80 mg / m for 3 cycles . the tumor size decreased by 45% , and lung metastases disappeared after induction .
the patient underwent total thyroidectomy with modified radical neck dissection , and the tumor was completely resected .
the second patient was a 68-year - old woman with a 4.4 cm thyroid tumor and a single cervical node metastasis , without distant metastases .
the patient received induction therapy with weekly paclitaxel ( again at 80 mg / m ) for 2 cycles . the tumor size decreased by 15% ; this result was evaluated as stable disease ( sd ) .
the patient underwent total thyroidectomy and modified neck dissection , and the tumor was completely resected . at the time of publication of this study ,
the patient was alive 29 months after her diagnosis and 27 months after surgery with no evidence of recurrence .
the results from these studies indicate that neoadjuvant chemotherapy may have some effectiveness in dtc and should be considered in select cases .
neoadjuvant regimens consisted mostly of vinblastine , doxorubicin , and/or paclitaxel and offered superior responses compared to doxorubicin alone ( or with cisplatin ) . of note , patients in these studies were typically older , with mean ages over 60 . thus , neoadjuvant chemotherapy can be both effective and tolerable in this population . despite these results , the data is limited to these two retrospective studies and two patients from a case series . interestingly , none of these studies reported toxicity associated with neoadjuvant chemotherapy . while it is possible that neoadjuvant chemotherapy was well tolerated by the patients in these studies ,
ideally , recommendations for treatment using neoadjuvant therapy would be based on randomized controlled trials ( rcts ) ; however it is unlikely that enough patients could be accrued to power such a study .
consequently , there is a need for more retrospective studies and case reports in order to enhance the current data for this treatment .
the studies described thus far in this review have all used cytotoxic chemotherapy , which is relatively indiscriminate in its toxicity .
in contrast , newer targeted therapies are based on specific genetic properties of tumors and are generally less toxic as a result . in particular , tyrosine kinase inhibitors ( tkis )
furthermore , lenvatinib was found to extend progression - free survival ( pfs ) in rai - resistant dtc in the phase iii select trial and was approved for the treatment of metastatic dtc in 2015 . although there are no adjuvant data for these drugs , the phase iii trial data is thought provoking for the potential role of targeted therapy in the neoadjuvant setting . the double - blind ,
randomized , multicenter , phase iii decision trial examined sorafenib efficacy and safety versus placebo in patients with progressive rai - refractory dtc .
a total of 417 patients were randomized ; 207 were to sorafenib and 210 to placebo .
orr in the sorafenib versus placebo arms was 12.2% and 0.5% ( all responses were partial responses ) .
stable disease 6 months was achieved in 42% versus 33% for sorafenib versus placebo , respectively .
the most common any - grade treatment - emergent adverse events in the sorafenib arm were hand - foot skin reaction , diarrhea , alopecia , rash / desquamation , fatigue , weight loss , and hypertension .
consequently , this study demonstrated a significant advantage of sorafenib over placebo with limited toxicity .
however , the 12.2% orr is still very low and is not ideal for neoadjuvant therapy .
see table 4 for a summary of studies describing targeted therapy in dtc [ 1526 ] . the multicenter , randomized , placebo - controlled , phase iii select trial presented at the american society of clinical oncology ( asco ) 2014 annual meeting examined the efficacy and safety of lenvatinib versus placebo in patients with rai - refractory dtc .
patients were allowed to have received 1 prior vascular endothelial growth factor receptor- ( vegfr- ) targeted therapy .
a total of 392 patients were randomized in a 2 : 1 ratio to lenvatinib or placebo .
complete response ( cr ) rates were 1.5% for lenvatinib and 0% for placebo , and pr rates were 63.2% for lenvatinib and 1.5% for placebo .
the most common lenvatinib treatment - related adverse events were hypertension ( 68% ) , diarrhea ( 59% ) , decreased appetite ( 50% ) , weight loss ( 46% ) , and nausea ( 41% ) .
lenvatinib - related grade 3 adverse events were hypertension ( 42% ) , proteinuria ( 10% ) , weight loss ( 10% ) , diarrhea ( 8% ) , and decreased appetite ( 5% ) . the dose was reduced in 78.5% of patients and discontinued due to adverse events in 14.2% of patients . consequently , response rates for lenvatinib were far superior to those of sorafenib . additionally , these results show that lenvatinib is a good candidate for use as neoadjuvant therapy in dtc , given its short time to response and a high response rate .
thirty - seven patients with metastatic , rapidly progressive , rai - refractory dtc received pazopanib until disease progression and/or drug intolerance .
sixteen patients ( 43% ) required dose reductions due to adverse events , the most common ( of any grade ) being fatigue ( 78.4% ) , skin and hair hypopigmentation ( 75.7% ) , diarrhea ( 73% ) , and nausea ( 73% ) .
these data demonstrate that lenvatinib and pazopanib are promising agents for neoadjuvant therapy in dtc .
the former has a documented short time to response , and both had good response rates with low toxicity .
furthermore , given that these drugs would be given for a short time period if administered as neoadjuvant therapy , long - term toxicity would also be minimal .
however , there exists the theoretical concern of selecting for tumor cell clonality resistant to these new therapies if they are used early in the course of treatment as neoadjuvant therapy .
if such a patient were to develop rai - resistant metastases , the treatment options for the patient would indeed be poor . however , this scenario is very unlikely , and its possibility should not preclude the consideration of targeted agents as neoadjuvant therapy in patients that could benefit from this treatment .
sorafenib , on the other hand , had a relatively low response rate of 12% in a phase iii trial , which is consistent with data from prior phase ii trials .
the different biochemical properties of these drugs may contribute to the considerable differences in response rates between sorafenib and lenvatinib / pazopanib ( see table 5 [ 2730 ] for a summary of the molecular targets of these tkis ) .
additional studies could be helpful in elucidating these differences . while ineffective on its own , there is the possibility that sorafenib could be effective in conjunction with other targeted therapies or cytotoxic chemotherapies .
further studies could elaborate on the potential of combining different targeted therapies with each other or with cytotoxic chemotherapy .
neoadjuvant chemotherapy can preclude the need for aggressive and morbid surgery , such as laryngectomy , tracheal resection , or pharyngoesophageal resection
. it can also render an inoperable tumor operable and can improve a patient 's prognosis by allowing a more complete resection of a patient 's tumor .
furthermore , it can act on metastatic disease if present and add additional value to postoperative rai , which is generally more efficacious for patients with only remaining microscopic disease rather than macroscopic .
the current literature describes r0 and r1 resections as offering improved survival when compared to r2 resection [ 3440 ] .
however , the respective effects of r0 and r1 resections on prognosis are more controversial .
the clinical benefits of complete resection are apparent in a retrospective single - center study by hartl et al .
, in which they described 46 patients with dtc invading the trachea , larynx , pharynx , or esophagus , none of whom received neoadjuvant chemotherapy .
r0 , r1 , and r2 surgeries were performed in 22 ( 49% ) , 24 ( 51% ) , and 0 ( 0% ) patients , respectively .
75% for r1 patients ; 5-year disease - specific survival was 95% for r0 patients and
. seems to describe a more favorable r0/r1/r2 than the two slovenian studies , despite the lack of neoadjuvant therapy in the former study .
however , the tumors described in the slovenian studies were considered inoperable at the start and consequently were likely larger and more extensive from the beginning than those described in the study by hartl et al . also of note , the studies from slovenia seem to demonstrate that neoadjuvant therapy is more effective in patients without metastases .
this is potentially due to the less aggressive biology of tumors that have not yet metastasized .
neoadjuvant therapy could also be useful in certain specialized cases , for instance , those involving deep tracheal invasion or pet - positivity .
for instance , some studies demonstrate that tracheal shaving is an effective procedure for the treatment of tracheal invasion when compared to sleeve resection ( a more aggressive but more definitive procedure ) [ 41 , 42 ] .
however , in cases of deep invasion , tracheal shaving has a greater risk of leaving gross disease behind .
thus , neoadjuvant therapy could potentially reduce the extent of invasion and reduce the risk of residual disease after surgical resection .
prior studies have demonstrated that pet - positive tumors are often aggressive and rai - resistant [ 43 , 44 ] . in these situations ,
neoadjuvant therapy might be a valuable tool in a setting in which treatment options are limited . while vegf is critical for tumor angiogenesis and growth , it is also imperative to wound healing . thus , a significant possible drawback of the use of anti - vegf tkis , such as sorafenib , lenvatinib , or pazopanib , as neoadjuvant therapy is the possibility of impaired wound healing after surgery .
consequently , the use of these agents as neoadjuvant therapy would potentially require a waiting period between drug administration and surgery , possibly allowing the tumor time to regrow .
that being said , the current literature contains few reports of impaired healing or wound dehiscence following therapy with anti - vegf tkis .
for instance , in the phase iii select trial of lenvatinib , 6 of 118 deaths were determined to be treatment - related , and none of these were related to surgical or wound problems . of note , the package insert for lenvatinib reports a 0.8% rate of impaired healing and 0.4% of wound dehiscence .
antiangiogenic therapy could also potentially lead to fistula formation in these patients given the potential for radiation and/or surgery around the trachea and esophagus .
tracheoesophageal fistula has been reported in lung cancer patients after treatment with the vegf inhibitor bevacizumab in addition to radiation [ 4850 ] . in another study ,
5 of 43 head and neck cancer patients treated with bevacizumab and chemoradiotherapy developed an aerodigestive fistula .
however , a 2014 literature review conducted by blevins et al . only yielded one case of antiangiogenic therapy for thyroid cancer resulting in aerodigestive fistula , though details regarding the patient 's history were not published . in the same paper , blevins
thus , these data demonstrate a rare risk of fistula formation in patients with dtc treated with antiangiogenic tkis .
given that there is no data regarding the use of antiangiogenic tkis as neoadjuvant therapy in dtc , it is difficult to speculate as to the possibility of postoperative wound dehiscence or fistula formation . furthermore , there are no current guidelines for these drugs on how long to wait before surgery . while we believe that , in specific circumstances , neoadjuvant therapy might be a valuable tool in the treatment of dtc , these risks should be deliberated in any treatment plan .
the possibility of a waiting period between drug administration and surgery should be considered while considering the risk of tumor regrowth .
given the lack of guidelines or data , these decisions should be made on the basis of the individual patient and tumor .
more data need to be collected regarding the possibility of impaired wound healing or fistula formation after treatment with antiangiogenic tkis in dtc before any recommendations on a waiting period between drug administration and surgery are made . in the opinion of the authors of this paper , an antiangiogenic tki
could be administered for approximately four months ( with response first assessed after two months ) , and surgery could take place 46 weeks after stopping the drug . again
, however , additional data is necessary before establishing any guidelines for duration of therapy and the waiting period .
however , our review shows that in selected situations neoadjuvant chemotherapy can be beneficial in the treatment of dtc .
moreover , the select trial shows that the targeted therapy lenvatinib is effective and safe in the treatment of dtc and could potentially be useful as neoadjuvant therapy for this disease given its high response rate and short time to effect .
pazopanib has also demonstrated promise in phase ii data , and sorafenib , while relatively ineffective alone , could possibly be useful in conjunction with other therapies .
there is still limited data on the respective effects of r0 , r1 , and r2 on prognosis .
there are also no clear indications for the use of neoadjuvant therapy in dtc , and this decision would likely be made by the surgeon based on the extent of disease and complexity of the operation for the surgeon .
it is unlikely that a sufficiently powered rct will be conducted for this treatment , and , consequently , treatment recommendations will have to rely on strong retrospective data and case reports and series .
future studies should potentially include combinations of targeted therapies with each other or other cytotoxic chemotherapies to further elucidate the possibilities for neoadjuvant therapy in dtc . | objectives . invasion of differentiated thyroid cancer ( dtc ) into surrounding structures can lead to morbid procedures such as laryngectomy and tracheal resection . in these patients
, there is a potential role for neoadjuvant therapy . methods .
we identified three studies involving the treatment of dtc with neoadjuvant chemotherapy : two from slovenia and one from japan .
results .
these studies demonstrate that in selected situations , neoadjuvant chemotherapy can have a good response and allow for a more complete surgical resection , the treatment of dtc .
additionally , the select trial shows that the targeted therapy lenvatinib is effective in the treatment of dtc and could be useful as neoadjuvant therapy for this disease due to its short time to response .
pazopanib has also demonstrated promise in phase ii data .
conclusions .
thus , chemotherapy in the neoadjuvant setting could possibly be useful for managing advanced dtc .
additionally , some of the new tyrosine kinase inhibitors ( tkis ) hold promise for use in the neoadjuvant setting in dtc . |
despite extensive study , timing of repair for macula - off rhegmatogenous retinal detachments remains controversial .
it has been suggested that visual recovery is correlated with the height of a bullous macula - off detachment.1 it has also been reported that shorter duration of macular detachment is associated with better visual acuity outcomes.2 recent studies have reported the use of spectral - domain optical coherence tomography ( sd - oct ) in the evaluation and follow - up of patients with retinal detachment and retinoschisis.3,4 in the current study , four patients with macula - off retinal detachment are reported that underwent surgical repair at various time points after symptom onset with preoperative and postoperative clinical and anatomic correlation using sd - oct .
a 70-year - old latin american woman presented for evaluation of decreased vision in the right eye for 3 days .
past ocular history was significant for bilateral uncomplicated cataract extraction with intraocular lens placement 4 months prior .
ophthalmic examination revealed best - corrected visual acuity of 1/200 in the right eye and 20/20 in the left .
the posterior segment examination of the right eye demonstrated a bullous rhegmatogenous retinal detachment caused by a superotemporal horseshoe tear ( figure 1a ) .
sd - oct revealed marked elevation of the retina extending through the macula to the optic nerve ( figure 1b ) .
the following day , 0.3 cm of 100% c3f8 ( octafluoropropane ) gas was placed in the right eye because of persistent subretinal fluid .
six months after successful retinal detachment surgery , the patient s best - corrected visual acuity in the right eye was 20/60 ( figure 2a ) .
sd - oct revealed mild foveal atrophy in the outer nuclear layer , with mild disruption of the inner segment ellipsoid layer ( figure 2b ) .
a 57-year - old latin american woman presented for evaluation of decreased vision in the right eye for 2 days .
past ocular history was significant for bilateral uncomplicated cataract extraction with intraocular lens placement 5 months prior and a retinal tear in the right eye , status post - laser retinopexy .
ophthalmic examination revealed best - corrected visual acuity of 20/70 in the right eye and 20/20 in the left eye .
intraocular pressure was 15 mmhg in the right eye and 18 mmhg in the left eye .
the posterior segment examination of the right eye revealed a shallow rhegmatogenous retinal detachment caused by two temporal horseshoe tears ( figure 3a ) .
sd - oct demonstrated temporal subretinal fluid extending under the fovea ( figure 3b ) .
the patient underwent pars plana vitrectomy with air - fluid exchange , endolaser , and placement of c3f8 ( octafluoropropane ) gas in the right eye 1 day after presentation .
one year after surgery , the patient s best - corrected visual acuity in the right eye was 20/20 ( figure 4a ) .
sd - oct revealed intact foveal contour with normal - appearing inner segment ellipsoid layer ( figure 4b ) . a 49-year - old caucasian woman presented with history of floaters in the right eye for 3 weeks and decreased vision for 1 day .
past ocular history was significant for bilateral lasik ( laser - assisted in situ keratomileusis ) for myopia 10 years prior .
ophthalmic examination revealed best - corrected visual acuity of 20/200 in the right eye and 20/20 in the left eye .
the posterior segment examination demonstrated a bullous rhegmatogenous retinal detachment caused by a superior horseshoe tear ( figure 5a ) .
sd - oct showed extensive subretinal fluid , with bullous elevation of the fovea ( figure 5b ) .
the patient underwent a successful scleral buckling procedure with placement of 0.5 cm of 100% sf6 ( sulfur hexafluoride ) gas .
one month after surgery the patient s best - corrected visual acuity in the right eye was 20/40 ( figure 6a ) .
sd - oct revealed intact foveal contour with normal - appearing inner segment ellipsoid layer ( figure 6b ) . a 50-year - old caucasian woman presented with history of gradual decrease in vision in the right eye over several months .
ophthalmic examination revealed best - corrected visual acuity of 20/100 in the right eye and 20/15 in the left eye .
the posterior segment examination demonstrated a rhegmatogenous retinal detachment caused by inferior retinal holes ( figure 7a ) .
sd - oct revealed shallow subretinal fluid extending under the fovea ( figure 7b ) .
the patient underwent a successful scleral buckling procedure with cryotherapy . at 3-month follow - up the patient s best - corrected visual acuity in the right eye was 20/200 ( figure 8a ) .
sd - oct revealed intact foveal contour with disruption of the inner segment ellipsoid layer ( figure 8b ) .
a 70-year - old latin american woman presented for evaluation of decreased vision in the right eye for 3 days .
past ocular history was significant for bilateral uncomplicated cataract extraction with intraocular lens placement 4 months prior .
ophthalmic examination revealed best - corrected visual acuity of 1/200 in the right eye and 20/20 in the left .
the posterior segment examination of the right eye demonstrated a bullous rhegmatogenous retinal detachment caused by a superotemporal horseshoe tear ( figure 1a ) .
sd - oct revealed marked elevation of the retina extending through the macula to the optic nerve ( figure 1b ) .
the following day , 0.3 cm of 100% c3f8 ( octafluoropropane ) gas was placed in the right eye because of persistent subretinal fluid .
six months after successful retinal detachment surgery , the patient s best - corrected visual acuity in the right eye was 20/60 ( figure 2a ) .
sd - oct revealed mild foveal atrophy in the outer nuclear layer , with mild disruption of the inner segment ellipsoid layer ( figure 2b ) .
a 57-year - old latin american woman presented for evaluation of decreased vision in the right eye for 2 days .
past ocular history was significant for bilateral uncomplicated cataract extraction with intraocular lens placement 5 months prior and a retinal tear in the right eye , status post - laser retinopexy .
ophthalmic examination revealed best - corrected visual acuity of 20/70 in the right eye and 20/20 in the left eye .
no relative afferent pupillary defect was noted . intraocular pressure was 15 mmhg in the right eye and 18 mmhg in the left eye . the posterior segment examination of the right eye revealed a shallow rhegmatogenous retinal detachment caused by two temporal horseshoe tears ( figure 3a ) .
sd - oct demonstrated temporal subretinal fluid extending under the fovea ( figure 3b ) .
the patient underwent pars plana vitrectomy with air - fluid exchange , endolaser , and placement of c3f8 ( octafluoropropane ) gas in the right eye 1 day after presentation .
one year after surgery , the patient s best - corrected visual acuity in the right eye was 20/20 ( figure 4a ) .
sd - oct revealed intact foveal contour with normal - appearing inner segment ellipsoid layer ( figure 4b ) .
a 49-year - old caucasian woman presented with history of floaters in the right eye for 3 weeks and decreased vision for 1 day .
past ocular history was significant for bilateral lasik ( laser - assisted in situ keratomileusis ) for myopia 10 years prior .
ophthalmic examination revealed best - corrected visual acuity of 20/200 in the right eye and 20/20 in the left eye .
the posterior segment examination demonstrated a bullous rhegmatogenous retinal detachment caused by a superior horseshoe tear ( figure 5a ) .
sd - oct showed extensive subretinal fluid , with bullous elevation of the fovea ( figure 5b ) .
the patient underwent a successful scleral buckling procedure with placement of 0.5 cm of 100% sf6 ( sulfur hexafluoride ) gas .
one month after surgery the patient s best - corrected visual acuity in the right eye was 20/40 ( figure 6a ) .
sd - oct revealed intact foveal contour with normal - appearing inner segment ellipsoid layer ( figure 6b ) .
a 50-year - old caucasian woman presented with history of gradual decrease in vision in the right eye over several months .
ophthalmic examination revealed best - corrected visual acuity of 20/100 in the right eye and 20/15 in the left eye .
the posterior segment examination demonstrated a rhegmatogenous retinal detachment caused by inferior retinal holes ( figure 7a ) .
sd - oct revealed shallow subretinal fluid extending under the fovea ( figure 7b ) .
the patient underwent a successful scleral buckling procedure with cryotherapy . at 3-month follow - up the patient s best - corrected visual acuity in the right eye was 20/200 ( figure 8a ) .
sd - oct revealed intact foveal contour with disruption of the inner segment ellipsoid layer ( figure 8b ) .
with an annual incidence of 12 per 100,000 individuals , rhegmatogenous retinal detachment remains an important cause of visual morbidity , especially in macula - off cases.5 risk factors for retinal detachment include prior retinal tears , myopia , and cataract surgery.6 options for surgical repair include traditional scleral buckling , pars plana vitrectomy , pneumatic retinopexy , and combination techniques , with regional variation in the choice between these procedures.7 the strongest prognostic indicator of postoperative visual outcome is preoperative visual acuity.8 a controversial issue surrounding surgical repair of macula - off rhegmatogenous retinal detachments is the timing of repair .
the first three patients described in the cases underwent repair 9 , 3 , and 1 day(s ) after symptom onset , respectively .
one case series demonstrated that macular detachment of less than 10 days was associated with better final visual acuity.2 more recently , one group proposed that surgical repair within 6 days of symptoms of macular detachment resulted in similar anatomic outcomes but better visual acuity outcomes as compared with repair beyond 6 days.9 however , in another study , there was no statistical difference in visual recovery in patients operated within 12 days , 34 days , or 57 days after macular involvement.10 it has been recognized that successful anatomic reattachment does not always predict visual improvement in macula - off cases . although the retina was successfully attached in all four patients , the final best - corrected visual acuity in the fourth patient was 20/200 as compared with 20/60 in the first patient , 20/20 in the second patient , and 20/40 in the third patient . on postoperative sd - oct ,
the first and fourth patients had outer nuclear atrophy and inner segment ellipsoid layer disruption , while the other patients did not . despite reattachment , there are microstructural changes within the photoreceptor layer , specifically disruption of the inner segment ellipsoid layer , in sd - oct.11 one study demonstrated that the status of the inner segment ellipsoid layer as well as the outer nuclear layer thickness are significantly correlated with final visual acuity outcomes after retinal detachment repair.12 it has been shown that increased preoperative foveal thickness and outer retinal corrugation are associated with worse visual prognosis , perhaps due to ellipsoid layer disruption.3,13 however , the majority of cases , including in these studies , were not bullous retinal detachments .
these four patients with macula - off retinal detachments demonstrate anatomical findings that correlate with final visual acuity outcome using sd - oct .
this technology is usually applied to conditions without bullous retinal detachment , but this case series shows that the technology can also be applied in this condition . while the exact timing of repair for macula - off detachments remains controversial , sd - oct and other newer imaging modalities may be helpful as prognostic tools both before and after retinal detachment surgery .
further study , such as a larger retrospective series with masked reading of postoperative sd - oct images , is recommended . | the timing of repair and the utility of spectral - domain optical coherence tomography ( sd - oct ) for macula - off retinal detachment remains a controversial issue .
four patients who presented with macula - off retinal detachment underwent repair at various time points after symptom onset .
postoperative sd - oct of the macula demonstrated correlation with final visual acuity . |
extensive global evidence highlights a lack of communication skills in doctors that leads to dysfunctional consultations .
it has also been seen that doctors lack skills required for patient - centered consultations .
literature confirms that communication skills can be learned and taught and indicates that patient centeredness decreases as medical students progress into the clinical years .
effective communication is a key competency required by international regulatory and accreditation bodies for both under and postgraduates .
various communication models and teaching methodologies for teaching and learning have been reported in international literature .
pakistan medical and dental council , the national regulatory authority , stresses the development of effective communication skills .
however , among the over ninety registered medical colleges , communication skills are taught in a handful only . in 2002 - 2003 , a private medical college in pakistan instituted problem - based learning with an integrated longitudinal communication skills program .
the cambridge - calgary model of communication skills is used as the foundational framework for this program . in the first 2 years
, teaching involves the development of basic to complex communication skills including disclosing bad news , communicating with angry patients , and dealing with myths and misconceptions .
the skills are reinforced in the subsequent 3 years within the clinical rotations of the medical students .
learning strategies include plenary lectures , small group experiential sessions , video discussion , and reflective assignments .
formative and summative assessment of communication skills takes place annually through specific objective structured clinical examination ( osce ) stations .
the objective of this study was to evaluate the effectiveness of the communication skills program by comparing communication skills of its medical students ( medical college 1 [ college with integrated longitudinal communication skills program ] ) with medical students of a matching medical college without formal communication skills teaching ( medical college 2 [ comparable college without communication skills program ] ) .
a private medical college ( medical college 2 ) without formal communications skills teaching was chosen as the comparison group .
it matched regarding socioeconomic background , curriculum , and academic schedule with the college teaching communication skills ( medical college 1 ) .
communication skills of the students of the two medical colleges were assessed longitudinally through an osce conducted in years three and five on the same cohort . for each osce , we invited all students available at the time according to the curricular schedules to participate in the osce .
the osce 's consisted of four communication skills stations as this number has been found to measure communication skills with a good level of reliability .
each station had a specific patient perspective built into the scenario and required an empathetic , nonjudgmental , and honest approach to elicit patients ideas , fears , and expectations [ table 1 ] .
patient scenarios in the 4-station objective structured clinical examination validity was ensured through multidisciplinary development of the stations on commonly encountered clinical presentations and was reviewed by faculty involved in teaching communication skills .
to ensure inter - rater reliability , equal numbers of examiners were drawn from both the institutions .
examiner training and standardization was conducted through specially made teaching videos demonstrating desirable communication skills on the selected scenarios .
standardized patients were trained by examiners before each osce to ensure uniformity , and the angoff method was used for setting the standard of each station .
ethical approval was granted by the ethics review committees of both participating universities ( 1182-fm / erc - aku and 0769-zu ) .
we used a rating scale based on the cambridge - calgary guide 1 for each station to assess the communication skills .
this contains six communication skills constructs in the form of subscales with individual items in each .
the constructs included initiating the session ( its ) , gathering information ( gi ) , understanding patient perspective ( upp ) , providing structure to the consultation ( psc ) , building the relationship ( btr ) , and closing the session ( cts ) . for the disclosing bad news station , breaking the news ( btn )
we analyzed the mean construct scores and the mean total scores with 95% confidence interval for each station of both osces .
results of each osce station were converted into percentage scores derived from the sum of the construct scores divided by the total score and multiplied by 100 .
smirnov and shapiro wilk tests were used to observe normality of the scores . as the scores were not distributed normally , the mann
whitney u - test was used to compare differences in the distribution of construct scores and percentage scores by osce stations , universities , and year .
cronbach 's alpha was calculated for each osce and for each station at the end of both years to test reliability .
spss statistics for windows , version 21.0 , ibm , armonk , new york , was used for data entry and statistical calculations .
we used a rating scale based on the cambridge - calgary guide 1 for each station to assess the communication skills .
this contains six communication skills constructs in the form of subscales with individual items in each .
the constructs included initiating the session ( its ) , gathering information ( gi ) , understanding patient perspective ( upp ) , providing structure to the consultation ( psc ) , building the relationship ( btr ) , and closing the session ( cts ) .
for the disclosing bad news station , breaking the news ( btn ) construct was used instead of gi .
we analyzed the mean construct scores and the mean total scores with 95% confidence interval for each station of both osces .
results of each osce station were converted into percentage scores derived from the sum of the construct scores divided by the total score and multiplied by 100 .
smirnov and shapiro wilk tests were used to observe normality of the scores . as the scores were not distributed normally , the mann
whitney u - test was used to compare differences in the distribution of construct scores and percentage scores by osce stations , universities , and year .
cronbach 's alpha was calculated for each osce and for each station at the end of both years to test reliability .
spss statistics for windows , version 21.0 , ibm , armonk , new york , was used for data entry and statistical calculations .
students of medical college 1 had higher mean percentage scores on all stations in both years .
there was an increase in the scores from third to fifth year on stations focusing on history taking and patient 's perspective in both medical colleges .
the scores on stations focusing on the exploration of the patient 's perspective decreased from third to fifth year in both medical colleges .
the difference in scores between the two groups reduced in the fifth year . at the end of the third year , 21 ( 31.34% )
( 10 male and 11 female ) from the medical college with the longitudinally integrated communication skills program ( medical college 1 ) and 31 ( 46.26% ) ( 19 female and 12 male ) students from ( medical college 2 ) consented out of the 67 available students on each site . similarly , at the end of the fifth year , out of the available 65 students , 19 students ( 29.3% ) ( 8 male and 11 female ) from medical college 1 and 22 ( 34% ) ( 11 male and 11 female ) students from medical college 2 participated after giving consent . at the end of the third year
, medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p < 0.001 ) . at the end of the fifth year
, the overall mean total station score of medical college 1 remained significantly higher , although the difference had reduced from 9.2% to 7.1% ( 70.2% ) ( sd = 13.7 ) versus 63.1% ( sd = 15.2 ) ( p = 0.004 ) [ table 2 ] .
mean percentage scores and mean p values of the constructs and total objective structured clinical examination scores of both medical colleges at the end of the third year , the mean overall percentage score of each construct was significantly higher in medical college 1 . at the end of the fifth year
, medical college 1 received higher scores in the btr construct on worried patient presenting with fever ( wppf ) and patient presenting with positive hepatitis c report ( phcr ) stations with p = 0.026 and p = 0.017 , respectively . in the upp construct , marginally significantly higher scores were obtained on two stations again ,
anxious patient presenting with headache ( apwh ) and wppf with ( p = 0.052 ) and ( p = 0.083 ) , respectively . in the breaking the news(btn ) construct on the phcr station , the difference in scores between the two medical colleges was insignificant .
reliability was checked by calculating cronbach 's alpha for each osce at the end of each osce and was found to be 0.751 and 0.815 , respectively [ table 3 ] .
longitudinal comparison of the mean percentage construct and total scores for each station between and within universities the mean station scores in apwh station of medical college 1 and medical college 2 increased in the fifth year with a significant increase in medical college 2 scores with p = 0.048 and p = 0.004 , respectively .
the mean score of wppf station increased significantly in both medical colleges with p 0.001 and p < 0.001 .
however , the mean station score of medical college 1 decreased significantly in the disclosing bad news in phcr station ( p = 0.004 ) , whereas it increased insignificantly in medical college 2 ( p = 0.775 ) .
the mean station scores of patient request for faith healing therapy for diabetes mellitus station decreased in the fifth year with p = 0.0046 and p = 0.036 , respectively , for both medical colleges 1 and 2 [ table 3 ] . as this was a pilot study , the results can not be extrapolated widely .
a pretest was not realistic in such a situation where students are entering the program for the first time . increasing the number of stations to more than four
responder bias is a possibility as students with better communication skills may have been more likely to have accepted the invitation to participate in the osce .
at the end of the third year , medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p < 0.001 ) . at the end of the fifth year
, the overall mean total station score of medical college 1 remained significantly higher , although the difference had reduced from 9.2% to 7.1% ( 70.2% ) ( sd = 13.7 ) versus 63.1% ( sd = 15.2 ) ( p = 0.004 ) [ table 2 ] .
mean percentage scores and mean p values of the constructs and total objective structured clinical examination scores of both medical colleges
at the end of the third year , the mean overall percentage score of each construct was significantly higher in medical college 1 . at the end of the fifth year
, medical college 1 received higher scores in the btr construct on worried patient presenting with fever ( wppf ) and patient presenting with positive hepatitis c report ( phcr ) stations with p = 0.026 and p = 0.017 , respectively . in the upp construct , marginally significantly higher scores were obtained on two stations again ,
anxious patient presenting with headache ( apwh ) and wppf with ( p = 0.052 ) and ( p = 0.083 ) , respectively . in the breaking the news(btn ) construct on the phcr station , the difference in scores between the two medical colleges was insignificant .
reliability was checked by calculating cronbach 's alpha for each osce at the end of each osce and was found to be 0.751 and 0.815 , respectively [ table 3 ] . longitudinal comparison of the mean percentage construct and total scores for each station between and within universities
the mean station scores in apwh station of medical college 1 and medical college 2 increased in the fifth year with a significant increase in medical college 2 scores with p = 0.048 and p = 0.004 , respectively .
the mean score of wppf station increased significantly in both medical colleges with p 0.001 and p < 0.001 .
however , the mean station score of medical college 1 decreased significantly in the disclosing bad news in phcr station ( p = 0.004 ) , whereas it increased insignificantly in medical college 2 ( p = 0.775 ) .
the mean station scores of patient request for faith healing therapy for diabetes mellitus station decreased in the fifth year with p = 0.0046 and p = 0.036 , respectively , for both medical colleges 1 and 2 [ table 3 ] .
a pretest was not realistic in such a situation where students are entering the program for the first time . increasing the number of stations to more than four
responder bias is a possibility as students with better communication skills may have been more likely to have accepted the invitation to participate in the osce .
this is the first pilot study exploring the outcome of communication skills training in the undergraduate curriculum in pakistan .
improvement in communication skills as a result of formal teaching using a variety of approaches has been demonstrated repeatedly in various studies .
based on this principle , the longitudinal integrated communication skills program of medical college 1 utilizes a range of evidence - based teaching and learning strategies in delivering the content .
the cambridge - calgary guide assessment instrument has been rated very highly on the measures of psychometric properties , practicality , and overall value in a review of assessment tools .
improvement in communication skills was sustained in the medical college with the longitudinally integrated communication skills program ( medical college 1 ) , even though the difference between the two colleges decreased over time .
this can be attributed to a longitudinal program with earlier initiation of communication skills teaching in this college .
research has shown that communication skill programs introduced earlier initially lead to a greater improvement which declines over time , although it never reaches preintervention level .
this decrease was demonstrable in both colleges and can be attributed to an overall decline in patient - centeredness , increased cynicism , increased doctor centeredness , and matches previous literature findings . in our case
, this decline may also be due to the concentrated communication skills teaching sessions in the first and second year , resulting in much better scores in the first osce , followed by a reduced rate of improvement in the clinical years commensurate with the reduced curricular time for communication skills teaching .
one of the main challenges was deficiency of faculty training in teaching communication skills in clinical years in addition to lack of time in busy clinics .
limited time for communication skills teaching has often been reported as a challenge for teaching programs .
other reasons included a deficit of desirable role modeling compounded by a lack of concurrent faculty development .
the majority of the undergraduate training is based in a private , highly specialized tertiary care setting with a focus on volumes and specific organ systems in specialty clinics causing the consultation to become disease oriented .
it is well recognized that private hospitals volume and revenue - oriented goals are frequently in conflict with an ideal environment for the reinforcement of skills that are oriented to elicit the patient 's perspective that includes concerns , fears , and expectations of the patients .
the vast majority of patients complaints stem from a real or perceived lack of patient - centered communication skills and supports this position .
the consultation skills of the comparison medical college also improved longitudinally over time , and can be attributed to maturation of the students commensurate with experience .
we postulate that training in wards and clinics through role modeling and observation on real patients improves consultation skills . to counteract the drift toward the disease - oriented model ,
contextual and repetitive teaching that is patient - oriented and is supported by appropriate role modeling by trained faculty in all clinics and wards will allow greater integration .
in addition , at all stages of teaching , special focus and attention need to be given to specific communication skills required for a patient - centered consultation that can build a therapeutic relationship and elicit the patient 's perspective .
running long - term and sustainable longitudinal programs for specific communication skills teaching / learning in appropriate contexts , with the support of trained and motivated faculty , is a well - recognized challenge .
longitudinally integrated communication skills teaching in an undergraduate curriculum positively impacted communication skills of the students .
undergraduate curriculum positively impacted communication skills . community - based undergraduate training in the clinical years , supported by a longitudinal communication skills program with on - going faculty development , should ensure appropriate role modeling to reinforce a patient - centered approach . a large - scale study evaluating the effectiveness of undergraduate communication skills training in community - based settings
is required to assess the success of the above - mentioned interventions in preventing the drift toward the disease - oriented approach .
| background : evidence highlights a lack of communication skills in doctors leading to dysfunctional consultations . to address this deficit , a private medical college instituted curricular reforms with inclusion of a longitudinal communication skills program .
a pilot study was undertaken to evaluate the effectiveness of this program by comparing the consultation skills of medical students of this college with a medical college without a communication skills program.methods:a 4-station objective structured clinical examination ( osce ) was conducted in the third and final year .
mann
whitney u - test was used to compare the difference in the distribution between osce stations total and construct scores.results:at the end of the third year , 21 ( 31.34% ) , students of the study site ( medical college 1 [ college with integrated longitudinal communication skills program ] ) and 31 ( 46.26% ) students from the comparison site ( medical college 2 [ comparable college without communication skills program ] ) consented .
medical college 1 achieved a significantly higher overall mean total station score of 68.0% ( standard deviation [ sd ] = 13.5 ) versus 57.2% ( sd = 15.4 ) ( p
< 0.001 ) .
significantly higher mean scores were achieved on three stations . at the end of the final year ,
19 students ( 29.3% ) from medical college 1 and 22 ( 34% ) students from medical college 2 consented .
the difference in overall mean total station score reduced from 9.2% to 7.1% ( 70.2 ) ( sd = 13.7 ) versus 63.1 ( sd = 15.2 ) ( p = 0.004 ) .
the mean scores of both colleges decreased in patient presenting with hepatitis c report station ( p values 0.004 and 0.775 ) and in patient request for faith healing therapy in diabetes mellitus station ( p values 0.0046 and 0.036 ) , respectively.conclusion:longitudinal communication skills in an undergraduate curriculum positively impacted consultation skills .
community - based training and faculty development are required to develop effective patient - centered consultation skills . |
the first approach of using lipid microparticles was described by eldem et al . , reporting the production by high - speed stirring of a melted lipid phase in a hot surfactant solution obtaining an emulsion .
solid microparticles are formed when this emulsion is cooled to room temperature , and the lipid recrystallizes .
the obtained products were called lipid nanopellets , and they have been developed for oral administration .
lipospheres were described by domb applying a sonication process [ 35 ] . to overcome the drawbacks associated to the traditional colloidal systems , such as emulsions , liposomes , and polymeric nanoparticles , solid lipid nanoparticles ( sln ) [ 10 , 11 ]
sln are biocompatible and biodegradable and have been used for controlled drug delivery and specific targeting .
these colloidal carriers consist of a lipid matrix that should be solid at both room and body temperatures , having a mean particle size between 50 nm and 1000 nm [ 13 , 14 ] . a clear advantage of the use of lipid particles as drug - carrier systems is the fact that the matrix is composed of physiological components , that is , excipients with generally recognized as safe ( gras ) status for oral and topical administration , which decreases the cytotoxicity .
sln have been already tested as site - specific carriers particularly for drugs that have a relatively fast metabolism and are quickly eliminated from the blood , that is , peptides and proteins .
the cytotoxicity of sln can be attributed to nonionic emulsifiers and preservative compounds which are used in the production of these systems .
sln prepared up to concentrations of 2.5% lipid do not exhibit any cytotoxic effects in vitro .
even concentrations higher than 10% of lipid have been shown a viability of 80% in culture of human granulocytes .
in addition , a high loading capacity for a broad range of drugs can be achieved , especially if they have lipophilic properties [ 12 , 19 ] . due to their physiological and biodegradable properties ,
sln have been tested for several administration routes [ 20 , 21 ] , including the oral [ 22 , 23 ] and peroral [ 24 , 25 ] routes .
sln can be obtained by exchanging the liquid lipid ( oil ) of the o / w nanoemulsions by a solid lipid . in general , a solid core offers many advantages in comparison to a liquid core .
emulsions and liposomes usually show lack of protection of encapsulated drugs , and drug release as a burst ( emulsions ) or noncontrolled ( from liposomes ) .
in addition , sln are of low cost , the excipients and production lines are relatively cheap , and the production costs are not much higher than those established for the production of parenteral emulsions . at the turn of the millennium , modifications of sln , the so - called nanostructured lipid carriers ( nlcs ) , have been introduced to the literature , and these nlc represent nowadays the second generation of lipid nanoparticles .
the main difference between sln and nlc is the fact that the concept of these latter is performed by nanostructuring the lipid matrix , in order to increase the drug loading and to prevent its leakage , giving more flexibility for modulation of drug release .
this approach is achieved by mixing solid lipids with liquid lipids in nlc instead of highly purified lipids with relatively similar molecules in sln .
the result is a less - ordered lipid matrix with many imperfections , which can accommodate a higher amount of drug .
a limiting factor for in vivo performance of poorly water - soluble drugs for oral administration is their resistance of being wetted and dissolved into the fluid in the git ( apart from potential drug degradation in the gut ) .
thus , the increase in the dissolution rate of poorly water - soluble drugs is relevant for optimizing bioavailability . over the last 10 years ,
the features of lipid nanoparticles for oral and peroral delivery are related with their adhesive properties . once adhered to the git wall , these particles are able to release the drug exactly where it should be absorbed .
in addition , the lipids are known to have absorption - promoting properties not only for lipophilic drugs , such as vitamin e , repaglinide , and puerarin .
hydrophilic drugs can also be incorporated in sln ; nevertheless , the affinity between the drug and the lipid needs to be analysed . therefore
, loading hydrophilic drugs in sln is a challenge due to the tendency of partitioning the encapsulated molecules in the water during the production process of nanoparticles .
there are even differences in the lipid absorption enhancement depending on the structure of the lipids .
for example , medium - chain triglycerides ( mct ) lipids are more effective than long - chain triglycerides ( lct ) .
basically , the body is taking up the lipid and the solubilized drug at the same time .
it can be considered as a kind of trojan horse effect [ 36 , 37 ] .
oral administration of sln is possible as aqueous dispersion or alternatively transformed into a traditional dosage forms such as tablets , pellets , capsules , or powders in sachets [ 25 , 39 ] . for this route
in addition , all compounds of gras status or accepted gras status can be employed as well as from the food industry .
since the stomach acidic environment and high ionic strength favour the particle aggregation , aqueous dispersions of lipid nanoparticles might not be suitable to be administered as dosage form .
in addition , the presence of food will also have a high impact on their performance .
the packing of sln in a sachet for redispersion in water or juice prior to administration will allow an individual dosing by volume of the reconstituted sln . for the production of tablets , the aqueous sln dispersions can be used instead of a granulation fluid in the granulation process .
alternatively , sln can be transferred to a powder ( by spray - drying or lyophilization ) and added to the tabletting powder mixture . in both cases , it is beneficial to have a higher solid content to avoid the need of having to remove too much water . for cost reasons , spray drying might be the preferred method for transforming sln dispersions into powders , with the previous addition of a protectant . for the production of pellets ,
alternatively , sln can be produced directly in liquid peg 600 and put into soft gelatine capsules .
advantages of the use of sln for oral and peroral administration are the possibility of drug protection from hydrolysis , as well as the possible increase of drug bioavailability .
prolonged plasma levels has also been postulated due to a controlled , optimized released in combination with general adhesive properties of small particles .
the advantage of colloidal drug carriers described above is that they are generally linked to their size in the submicron range .
therefore , the preservation of particle size of colloidal carrier systems after peroral administration is a crucial point .
the gastric environment ( ionic strength , low ph ) may destabilize the sln and potentially lead to aggregation .
however , it is possible to produce stable sln dispersions by optimizing the surfactant / mixture for each lipid in vitro .
the drug release from sln in the git is also dependent on the lipase / colipase activity for the git digestion of the lipid matrix . the lipase / colipase complex leads to a degradation of food lipids as a prestep of the absorption . in vitro degradation assay based on pancreas lipase / colipase complex
have been developed to obtain basic information about the degradation velocity of sln as a function of lipid and surfactant used in the production process [ 45 , 46 ] .
lipid nanoparticles show great promise to enhance oral bioavailability of some of the most poorly soluble drugs .
the physical / chemical characteristics of lipid particulate systems are highly complex due to the existence of a variety of lipid assembly morphologies , the morphology - dependent solubility of drug , the interconversion of assembly morphology as a function of time and chemical structure , and the simultaneous lipid digestion .
their main characteristic is the fact that they are prepared with physiologically well - tolerated lipids . during the last ten years
, different substances have been entrapped into lipid nanoparticles ( table 1 ) , ranging from lipophilic [ 23 , 49 ] and hydrophilic molecules , including labile compounds , such as proteins and peptides .
the term lipid is used here in a broader sense and includes triglycerides , partial glycerides , fatty acids , steroids , and waxes .
however , it is required that matrix maintains the solid state at room temperature , and for this purpose , the selection of lipids is based on the evaluation of their polymorphic , crystallinity , miscibility , and physicochemical structure .
furthermore , the use of mono- and diglycerides as lipid matrix composition might increase drug solubility compared to highly pure lipids , such as monoacid triglycerides .
naturally occurring oils and fats comprise mixtures of mono- , di- , and triglycerides , containing fatty acids of varying chain length and degree of unsaturation [ 25 , 86 ] .
the melting point of these lipids increases with the length of the fatty acid chain and decreases with the degree of unsaturation .
the chemical nature of the lipid is also important , because lipids which form highly crystalline particles with a perfect lattice ( e.g. , monoacid triglycerides ) lead to drug expulsion during storage time .
physicochemically stable lipid nanoparticles will be obtained only when the right surfactant and adjusted concentration have been employed .
the choice of the ideal surfactant for a particular lipid matrix is based on the surfactant properties such as charge , molecular weight , chemical structure , and respective hydrophile - lipophile balance ( hlb ) .
the hlb of an emulsifier is given by the balance between the size and strength of the hydrophilic and the lipophilic groups .
griffin defined the lipophilic emulsifiers as low hlb values ( below 9 ) , and hydrophilic emulsifiers as high hlb values ( above 11 ) .
the hlb system is a useful method to choose the ideal emulsifier or blend of emulsifiers for the system , that is , if its required an oil - in - water ( o / w ) , water - in - oil ( w / o ) , or a double ( w / o / w ) emulsion .
matching the hlb value of the surfactant with the lipid will provide a suitable in vitro performance .
determined the hlb value for stearic acid and stearic acid capric / caprylic triglycerides to reach the best combination of surfactants ( trioleate sorbitan and polysorbate 80 ) to obtain a stable lipid nanoparticles emulsion .
the hlb value obtained for stearic acid was 15 and for stearic acid capric / caprylic triglycerides was 13.8 .
sorbitan trioleate has an hlb value of 1.8 and polysorbate 80 of 15 , when used in the ratio 10 : 90 , respectively .
polysorbate 80 is often used in combination with sorbitan trioleate due to their appropriate compatibility attributed to the similar chemical structure ( same hydrocarbon chain length ) for the production of stable emulsions .
pharmacokinetic behaviour of drugs loaded in lipid nanoparticles need to differentiate if the drug is present as the released free form or as the associated form with lipid nanoparticles .
however , the poor aqueous solubility of some drugs turns difficult the design of pharmaceutical formulations and leads to variable bioavailability .
xie et al . reported a significant increase in the bioavailability and extended the systemic circulation of ofloxacin formulated in sln , which could be attributed to a large surface area of the particles , improving the dissolution rate and level of ofloxacin in the presence of git fluids [ 109 , 110 ] , leading to shorter tmax and higher peak plasma concentration .
in addition , lipid nanoparticles may adhere to the git wall or enter the intervillar spaces due to their small particle size , increasing their residence time .
moreover , nanoparticles could protect the drug from chemical and enzymatic degradation and gradually release drug from the lipid matrix into blood , resulting in a several - fold increase mean residence time compared with native drug .
han et al . demonstrated that 5 oral doses of tilmicosin loaded in lipid nanoparticles administered every 10 days provided an equivalent therapeutic benefit to 46 daily doses of oral free drugs . in vitro release profile demonstrated that tilmicosin loaded in lipid nanoparticles followed a sustained release profile , and in vivo results showed that nanoparticles remained effective for a longer period of time , which was attributed to sustained release of the drug and also to enhanced antibacterial activity by the sln .
developed paclitaxel loaded in sln with the aim at improving the oral bioavailability of this antineoplastic drug . in vitro studies of sln formulation exhibited an initial low burst effect within 24 h followed by a slow and sustained release .
statistical analysis of in vivo experiments concluded that the oral bioavailability of paclitaxel loaded in sln was significantly higher than the control group .
produced stearic acid - sln with a fluorescence marked for evaluation of in vivo pathway by oral administration .
about 30% of sln transport was efficient , where particles were absorbed following linear mechanism in the git .
the release profile in plasma increased with the increasing of dosage depicting two concentration peaks .
the first peak of sln in blood took place during 1 - 2 h , attributed to the fast uptake of sln from the git into systematic circulation .
drug concentration began to decrease attributed to the uptake by and the distribution of sln among particular organs .
the second peak occurred at about 68 h , and the maximum concentrations were lower than that of the first peak .
lipid nanoparticles are well tolerated in living systems , since they are made from physiological compounds leading to the metabolic pathways [ 22 , 28 ] . for this purpose ,
however , such effects often occur first at rather in high concentrations and the subtler effects that arise at lower concentrations , without necessarily causing cell death , also need to be considered .
one the most important effect is dna damage , since an increased genetic instability is associated with cancer development .
the interaction with proteins and cells are an essential focus in assessing and understanding compatibility and toxicity .
cell and nanoparticle reactions of interest include cellular uptake and processing of nanoparticle in various routes , effects on cell signalling , membrane perturbations , influence on the cellular electron transfer cascades , production of cytokines , chemokines , and reactive oxygen species ( ros ) , transcytosis and intercellular transport , gene regulation overt toxic reactivity , no observable toxicity , and cell necrosis or apoptosis . in vitro culture of cell lines or primary cells on plastic plates
are employed in a wide varieties of assays and reflect the variety of possible physiologic responses to nanoparticles in vivo and all possible cell processing routes and natural reactions .
silva et al . studied the toxicity of sln and risperidone loaded sln with caco-2 cells by ( 4,5-dimthylthiazol-2-yl)2,5-diphenyl - tetrazolium bromide ( mtt ) assay .
the results suggest that all formulations evaluated are biocompatible with caco-2 cells and well tolerated by the git .
this test evaluates the mitochondrial function as a measurement of cell viability , which allows the detection of dead cells before they lose their integrity and shape .
the amount of viable cells after sln exposure was performed by the mtt assay with caco-2 cell models , which are a well - established in vitro model that mimics the intestinal barrier and is often used to assess the permeability and transport of oral drugs .
other authors have also reported that sln show biocompatibility , which increase their attractiveness for drug - delivery applications .
since early nineties , researchers turned their attention to lipid nanoparticles because of their nontoxicity and cost / effectiveness relationship . in spite of the advantages , formulating with lipid nanoparticles has been suffering some drawbacks .
because of the git conditions , most of promising drugs do not reach clinical trials .
the stability of particles must be comprehensively tested due to ph changes and ionic strength as well as the drug release upon enzymatic degradation .
lipid nanoparticles absorption through git occurs via transcellular ( through m cells or enterocytes ) or paracellular ( diffusion between cells ) .
if the major drug uptake occurs through m cells , the portal vein to the liver is bypassed , resulting in higher drug concentrations to the lymph rather than to plasma . despite the low number of lipid nanoparticles formulations on the market for drug delivery , mucosolvan retard capsules ( boehringer - ingelheim ) is a story of success .
it was produced by high - speed stirring of a melted lipid phase in a hot surfactant solution obtaining an emulsion .
this emulsion was then cooled down to room temperature obtaining the so - called lipid nanopellets for oral administration .
successful in vivo studies also include rifampicin , isoniazid , and pyrazinamide that are used in tuberculosis treatment .
the methodology employed for production is acceptable by the regulatory agencies and has been addressed by various papers and patents .
poor water - soluble drugs , as camptothecin , vinpocetine , and fenofibrate , can have their solubilization improved if incorporated into sln [ 124 , 128 ] .
injections are often painful and must be administered daily , which result in low patient compliance .
unfortunately , oral administration of insulin , produced by solvent emulsification - evaporation method based on a w / o / w double emulsion , has limitations such as low bioavailability due to degradation in the stomach , inactivation and degradation by proteolytic enzymes , and low permeability across the intestinal epithelium because of lack of lipophilicity and high molecular weight [ 124 , 129 ] .
the main advantages of incorporate insulin into sln would be the enhancement of transmucosal transport and protection from the degradation in the git .
lipids and lipid nanoparticles are promising for oral and peroral administration route for drugs , proteins , and peptides .
theses matrices are able to promoting controlled release of drugs in git and reducing absorption variability .
in addition , these matrices can be absorption as food lipids together with drugs improving the bioavailability .
these systems present several advantages , including drug protection and excipients of gras status , which decreases the danger of acute and chronic toxicity .
in addition , the oral administration of lipids nanoparticles is possible as aqueous dispersion or alternatively transformed into a traditional dosage forms such as tablets , pellets , capsules , or powders in sachets . | lipids and lipid nanoparticles are extensively employed as oral - delivery systems for drugs and other active ingredients .
these have been exploited for many features in the field of pharmaceutical technology .
lipids usually enhance drug absorption in the gastrointestinal tract ( git ) , and when formulated as nanoparticles , these molecules improve mucosal adhesion due to small particle size and increasing their git residence time . in addition ,
lipid nanoparticles may also protect the loaded drugs from chemical and enzymatic degradation and gradually release drug molecules from the lipid matrix into blood , resulting in improved therapeutic profiles compared to free drug .
therefore , due to their physiological and biodegradable properties , lipid molecules may decrease adverse side effects and chronic toxicity of the drug - delivery systems when compared to other of polymeric nature .
this paper highlights the importance of lipid nanoparticles to modify the release profile and the pharmacokinetic parameters of drugs when administrated through oral route . |
pilonidal sinus is a disease which is very common , especially in men and usually located in sacrococcygeal area .
however , the disease can be also seen in rare localizations such as umblicus , forehead , scalp , clitoris , interdigital area and axilla . in the etiology of pilonidal sinus , acquired theory is accepted by most of surgeons instead of the congenital one .
in the present study , we purposed to report our case of hirsute turkish women aged 25 having axillary pilonidal sinus .
after application of total surgical excision with eliptical skin incision , histopathological evaluations confirmed the prediagnosis of axillary pilonidal sinus .
the patient was lost to follow - up and neither recurrens nor distance metastasis has been detected during 36 months .
in our opinion ; surgical therapy of axillary pilonidal sinus , allows to complete resection in addition to absolute histopathological diagnoses and it may be an appropriate choice of treatment especially for the disease having one or two sinuses .
pilonidal sinus is a disease which is reported by anderson et al as a hair detected in a sacrococcygeal ulcer in 1847 , defined firstly by hodges et al as a term of pilonidal sinus and mostly found in sacrococcygeal area . besides this area , it can be also seen in rare localizations such as umblicus , forehead , scalp , clitoris , interdigital area , penis , abdomen , neck and axilla[101213 ] . in the etiology of pilonidal sinus ,
congenital theory was accused by kooistra et al in 1942 , collection of hairs on midline of back was become a current issue by karydakis et al firstly in 1992 and acquired theory was reported by bascom et al in 1980 .
a hirsute turkish woman aged 25 applied to our clinic in november 2006 with the history and complaint of the intermittent small amount of leakage from her right axilla during the past year .
there was no any history of pilonidal sinus and operation of which at the other sites of her body .
on the physical examination , one small sinus sized 2 mm in diameter in her right axilla not including any hair was detected .
pilonidal sinus was defined at neither the intergluteal sulcus and nor the other regions of her body .
after application of methylene blue through the orifice of the pilonidal sinus , total excision of the tractus with the neighbouring subcutaneous tissue and the elliptical skin part containing the sinus orifice was performed . following the total surgical excision , primer suturation of the subcutaneous tissue and the skin
the tractus was limited only in subcutaneous tissue and the histopathological evaluations confirmed the preassumptive diagnosis of axillary pilonidal sinus .
any perioperative or postoperative complication and recurrence has not been detected during the 36 months follow - up . sinus tract and the section of the hair inside it ( arrows ) ( h & e , original magnification , 1020 ) .
apocrine gland structures ( thick and short arrows ) and the neighbouring section of the hair ( thin ang long arrow ) ( h & e , original magnification , 510 ) .
in the beginning , the congenital theories have been suggested for the pathogenesis of pilonidal sinus , but afterwards the acquired theory is accepted by most of surgeons[416 ] .
friction ( abduction adduction ) , suction , massage , shaving , pounding , minor infection and maceration are assorted mechanisms which play a part in acquised theory of pilonidal sinus .
mayo et al indicated that the hairs may curl back on themselves and pierces the surrounding skin with their distal ends first by growth forces . on the contrary , oryu et
al suggested that penetration of shed hairs with their proximal or distal ends first coul not be designated , so the mentioned authors could not be designate the penetration direction of hair in their 5 patients .
we come accross with axilla as a very rare body area for including pilonidal sinus[101216 ] .
malignant degeneration of pilonidal disease is a rare complication composed of approximately 0.1 % of patients with chronic untreated or recurrent pilonidal disease and is associated with a high recurrence rate and poor prognosis comparing with regular nonmelanoma skin cancer . among malignant degeneration of pilonidal sinus most cases are squamous cell carcinoma
. biological behavior of pilonidal sinus carcinoma is much more aggressive than squamous cell carcinoma at the other sites and they are deeply invasive into the subcutaneous tissue in the great majority of cases . underlying bone is involved in 8% of cases , also .
i nguinal lymph node metastasis is very poor as a prognostic sign and is associated with a median survival time of only 7 months .
according to our point of view , besides complete resection allows to appropriate choice of treatment especially for the disease having one or two sinuses , it also enables to absolute histopathological diagnoses . in conclusion ,
pilonidal disease is a very common disease , especially in men and generally located in sacrococcygeal area .
so , we aimed to present our case of hirsute young turkish women having the mentioned disease .
histopathological evaluations revealed the charecteristic structures of axillary pilonidal sinus , after application of total surgical excision via eliptical skin incision .
the case was lost to follow - up and no recurrens or complication has been detected during 36 months .
awareness of the probability of a pilonidal disease even in seldom locations like groins of axilla is important and although the malignant degeneration of the disease is very rare , it has much more poor prognosis comparing with squamous cell carcinoma and regular nonmelanoma skin cancer . | context : pilonidal sinus is a disease which is very common , especially in men and usually located in sacrococcygeal area .
however , the disease can be also seen in rare localizations such as umblicus , forehead , scalp , clitoris , interdigital area and axilla . in the etiology of pilonidal sinus , acquired theory is accepted by most of surgeons instead of the congenital one.case report : in the present study , we purposed to report our case of hirsute turkish women aged 25 having axillary pilonidal sinus .
after application of total surgical excision with eliptical skin incision , histopathological evaluations confirmed the prediagnosis of axillary pilonidal sinus .
the patient was lost to follow - up and neither recurrens nor distance metastasis has been detected during 36 months.conclusions:in our opinion ; surgical therapy of axillary pilonidal sinus , allows to complete resection in addition to absolute histopathological diagnoses and it may be an appropriate choice of treatment especially for the disease having one or two sinuses . |
primary leiomyosarcoma of the greater omentum is a rare pathologic entity , and the literature includes only 27 cases including this one ( table 1 ) . the pre - operative diagnosis of these tumors is difficult and the diagnosis is usually made post operatively .
we report a case of a 55 year old woman with clinical symptoms of abdominal distension and discomfort followed by pelvic pain .
the omental origin of the tumor could not be identified using ct and us scans .
a 55-year - old , previously fit and well woman , presented with increasing abdominal distension and discomfort for 8 months and pelvic pain that began a few days prior to presentation .
a ct scan was performed as part of the evaluation and demonstrated a huge abdominal mass , measuring more than 20 cm in cross section in the left side of the abdomen . a small amount of ascites was also noted .
the mass had some large draining vessels on the left side and there was also infiltration of the fat in the upper abdomen on the left side suggesting peritoneal disease .
tumor markers were taken as part of the evaluation and her ca-125 levels were elevated at 527 u / ml , the levels of cea , ca15 - 3 and ca19 - 9 were normal .
pelvic ultrasound scan was also performed and a 20 12 cm heterogeneous lower abdominal mass with cystic and solid components was found ( fig .
ovarian carcinoma was suspected with high probability and the patient was consented for laparotomy , total abdominal hysterectomy , bilateral salpingo - oophorectomy , omentectomy and tumor debulking . at the time of surgery ,
a 23 20 13 cm irregular mass arising from the omentum and appearing to be parasitic in nature was found , the mass had extensive recruitment of huge vessels from the omentum .
the findings were not consistent with gynecologic malignancy . the liver , diaphragmatic surface , and all peritoneal surfaces were normal .
the small bowel and colon were normal and a frozen section analysis of the mass suggested sarcoma .
omentectomy was performed and the tumor removed intact , no further omental spread was noted . following division of adhesions , total abdominal hysterectomy and bilateral salpingo - oophorectomy were also performed .
2 ) . the tumor seemed to be arising from the smooth muscle in blood vessel walls in the omentum .
histopathologic examination revealed a multinodular but smooth outer surface of the tumor and foci of fleshy and pale cream - yellow with mucoid / mixoid areas underneath .
the microscopic examination confirmed the diagnosis of sarcoma with moderately cellular interlacing fascicles of spindle cells with a high degree of mitotic figures and atypical forms along with areas of mixoid change and coagulative tumor necrosis .
immunohistochemical staining was diffusely positive for desmin and smooth muscle actin , and strongly negative for s100 and cd34 in keeping with leiomyosarcoma .
the uterus , cervix , ovaries and fallopian tubes showed no evidence of disease , a positron emission tomography ( pet ) scan demonstrated no further suspicious lesions .
the case was reviewed at the gynecologic oncology tumor board and also at the specialized sarcoma unit tumor board meetings and both advised no adjuvant chemotherapy or radiotherapy .
leiomyosarcoma of the omentum is a rare condition and can present in various ways . in this case a large pelvic tumor with omental involvement , ascites and elevated ca125 was discovered in an otherwise healthy patient .
ultrasound scan was able to detect the tumor and showed accurately the internal structure of the lesion , nevertheless , it failed to determine the site of origin .
this limitation of us may be attributed to the presence of bowel gas and attenuation of the us beam passing through a huge tumor , both factors prevent precise observation of the anatomic relationship between the lesion and the neighboring organs ( ishida et al . , 1999 ) .
ishida and ishida ( 1998 ) stated it to be the imaging of choice for greater omental tumors . a gastro - colic separation resulting from a tumor in the omentum
their ct appearance is usually multilobulated , flat , and pancake - like , with enhancing solid and multicystic densities .
however , in this case the presentation , us and ct diagnosis were suspected to represent ovarian carcinoma .
there is one other case described in the literature in which leiomyosarcoma was initially diagnosed as an ovarian malignancy ( langlieb et al . , 1992 ) .
ascites and elevated ca-125 that are sometimes associated with this tumor ( dixon et al .
, 1984 , langlieb et al . , 1992 ) can suggest the initial diagnosis of ovarian carcinoma .
angiography was described as useful in the diagnosis of greater omental tumors ( dixon et al . , 1984 , fattar et al . , 1981 , ishida et al .
can be suspected when a feeding artery to the tumor originates from the omental blood supply .
the major arterial blood supply of the greater omentum is largely from the right and left gastroepiploic arteries , which derive from the gastroduodenal and splenic arteries ( sivak , 1992 ) .
a very vascular lesion with neovascularity , is more likely to represent a malignant tumor ( ishida et al . , 1999 ) .
reported primary tumors of the omentum , include leiomyosarcoma , fibrosarcoma , hemangiopericytoma , spindle cell sarcoma , liposarcoma , leiomyoma , lipoma , desmoid tumor , fibroma , mesothelioma , and others ( fattar et al . , 1981 , ishida et al . , 1999 mar , stout et al . , 1963 ,
weinberger and ahmed , 1997 ) they derive from different elements in the greater omentum which is composed mainly of fat but contains various tissues- such as vessels and lymphatics .
altogether 27 cases ( including this one ) have been reported in the literature up to date .
only one of the previously reported cases was suspected to be ovarian carcinoma at the outset ( langlieb et al . , 1992 )
. the median age of patients with leiomyosarcoma of the omentum in the cases published in the literature was 51 years ( range : 2685 , sd : 15.3 ) .
the tumor is slightly more common among males ( 16 patients , 59.2% ) and females ( 11 patients , 40.7% ) .
ishida et al . ( 1999 ) has reviewed the cases from 1963 to 1999 and found a correlation between a symptom free mass and a better prognosis .
this correlation is not as clear when adding the cases published before 1963 , although much of the details regarding these cases are unavailable to us .
also , due to the limitations of imaging techniques in that era , early diagnosis was less likely .
although these tumors are very rare , a diagnosis of leiomyosarcoma should be considered in a patient with an abdominal mass or distention and imaging studies demonstrating a huge mass with a central cystic area in the greater omentum .
the ct scan is probably more useful than us in the diagnosis of these cases and angiography may also prove useful demonstrating omental blood supply to the tumor .
an accurate diagnosis can only be achieved with a histopathologic examination of the tumor ( ishida et al . , 1999 ) .
complete surgical excision of thee tumors if feasible is probably the best option and can lead to long periods of disease free survival .
we report a case of a leiomyosarcoma of the greater omentum , presenting as ovarian carcinoma .
these tumors are very rare and the accurate determination of their precise anatomic location is usually difficult .
the method of choice for imaging these tumors is ct scan with or without angiography .
| primary omental leiomyosarcoma is a rare tumor .
we report a case of successfully resected omental leiomyosarcoma whose presentation mimicked ovarian carcinoma .
symptoms of abdominal distension and discomfort that lasted 8 months followed by pain lead to a diagnosis of a large mass in the abdomen . physical examination revealed a large , over 20 cm tumor , suspected to be of ovarian origin .
a small amount of ascites was found on computerized tomography ( ct ) and ultrasound ( us ) scans .
total abdominal hysterectomy with bilateral salpingo - oophorectomy , omentectomy and tumor debulking procedure was planned .
laparotomy revealed normal uterus ovaries and tubes with a leiomyosarcoma of the omentum which was completely resected successfully .
only 26 cases of primary leiomyosarcoma of the omentum were previously described in the literature . a review of the literature is also presented . |
aging is associated with reductions in skeletal muscle mass and strength ( sarcopenia ) , which may lead to impaired mobility in older individuals . despite a litany of research on sarcopenia ,
comprehensive reviews of the potential mechanisms behind sarcopenia are available ( e.g. , ryall et al . 2008 ) . in brief , declines in muscle - specific stem cells ( satellite cells ) ; increases in myostatin , a negative regulator of muscle growth ; changes in circulating hormone levels ; impairment of neuromuscular function ; increased myonuclear apoptosis have all been proposed as mechanisms underlying sarcopenia .
creatine ( c4h9n3o2 ) supplementation is a popular dietary supplement with recreational and professional athletes .
it has been shown to increase exercise performance [ 2 , 3 ] and strength .
creatine is a naturally occurring molecule in skeletal muscle and can be synthesized or obtained in the diet .
. increased intramuscular levels of pcr may permit increased temporal buffering of atp during high - intensity muscle contraction , and increased creatine levels may allow increased pcr resynthesis . in so doing , creatine supplementation may indirectly increase muscle mass and strength by allowing training at higher workloads .
however , the mechanisms behind the enhancement of performance and increases in muscle strength by creatine supplementation have not been fully elucidated .
the use of a dietary supplement , such as creatine , to counteract sarcopenia is not unprecedented .
high protein diets may be beneficial for stimulating protein synthesis in the muscles of aging individuals .
the use of creatine in elderly populations is of interest because of its potential to increase energy production , fat - free mass , and muscle mass .
combined , these effects would increase the ability of the elderly to perform everyday tasks such as rising from a seated position and walking .
creatine supplementation for five days in aging population had no effect on isometric strength [ 11 , 12 ] .
similarly , five days of creatine supplementation had no effect on pedaling performance in cycling - trained elderly subjects .
conversely , 5 days of creatine supplementation increased anaerobic power and work capacity of sedentary elderly subjects .
short - term creatine supplementation has also produced increases in strength and fat - free mass of elderly men and women .
longer - term creatine supplementation combined with resistance training in the elderly has also demonstrated varied responses .
the combined treatments resulted in anything from no extra benefit after 8 weeks of strength training and supplementation , to small increases in muscle strength after 12 weeks , to moderate increases in strength and creatine levels after 14 weeks , beyond resistance training alone .
it has recently been shown that low - dose creatine supplementation combined with protein supplementation and resistance training for 10 weeks increases lean tissue mass but not leg press strength .
furthermore , there was a greater response in creatine combined with protein supplementation than creatine alone or placebo .
conversely , neither creatine supplementation , protein supplementation , or a combination of protein and creatine was shown to provide further benefit beyond 16 weeks of isotonic resistance training alone in elderly subjects . in light of these controversial results , an animal model of hypertrophy ( surgical ablation / compensatory hypertrophy ) was chosen to compare the effects of creatine supplementation on hypertrophy in young and aging rats .
surgical ablation is an animal model involving surgical removal of a muscle , thereby overloading its synergistic muscles and causing them to hypertrophy in compensation [ 2123 ] .
the authors are aware of one other study that used this model to study creatine supplementation .
. showed no further hypertrophy in muscle fibers with creatine supplementation above compensatory hypertrophy alone .
our study was designed to investigate whether creatine supplementation alone , surgical ablation alone , or a combination of creatine supplementation and surgical ablation would lead to hypertrophy in individual fiber types of the plantaris muscle .
furthermore , surgical ablation has been associated with increased expression of heat shock protein 70 ( hsp70 ) , which has been linked to myoprotection [ 25 , 26 ] and anabolism [ 27 , 28 ] .
therefore , the study examined whether these experimental treatments would alter hsp70 levels and whether hypertrophic and/or hsp70 responses would differ in young compared to older animals after these interventions .
it was hypothesized that creatine supplementation would primarily increase csa in fast fibers ( iia , iib / d ) and that this would be attenuated in aging muscle .
we further hypothesized that hsp70 levels would be elevated in treatment groups undergoing surgical ablation but that creatine alone would not significantly alter hsp70 levels , regardless of age .
young ( 5 months at muscle excision , n = 27 ) and aging ( 24 months at muscle excision , n = 35 ) adult male fisher 344 rats from national institute on aging rodent colony ( harlan labs , indianapolis , in ) were used .
animals were divided into eight groups : ( 1 ) young control ( yc , n = 6 ) ; ( 2 ) young supplemented with creatine ( ycr , n = 7 ) ; ( 3 ) young overloaded ( yo , n = 7 ) ; ( 4 ) young overloaded and supplemented with creatine ( yocr , n = 7 ) ; ( 5 ) aging control ( ac , n = 8) ; ( 6 ) aging supplemented with creatine ( acr , n = 9 ) ; ( 7 ) aging overloaded ( ao , n = 9 ) ; ( 8) aging overloaded and supplemented with creatine ( aocr , n = 9 ) .
temperature was maintained at 22c 2c , and conditions consisted of a 12-hour : 12-hour dark - light cycle .
animals were allowed 10 days of recovery from shipping and to become accustomed to their surroundings prior to experimentation . at that point , four experimental groups ( yo , yocr , ao , and aocr ) underwent surgical ablation surgery to induce overload by compensatory hypertrophy .
rats were anesthetized intraperitoneally by injection of either veterinary pentothol ( 0.07 mg / kg body weight ) or a combination of xylazine and ketamine ( 0.01 g / kg body weight xylazine ; 0.01 g / kg body weight ketamine ) .
when there was no reflex response , an incision ( 2.5 cm ) was made through the skin of the posterior compartment of the leg to expose the underlying muscle .
care was taken to ensure blood supply and innervation to remaining muscles were not compromised .
the incision was sutured with 4.0 black braided silk and the stitches were approximately 0.3 cm apart , ensuring the incision was completely closed .
after overnight recovery from the anesthetic , the animals were returned to their cages . after returning to their cages , dietary creatine supplementation began for those in the creatine groups ( ycr ; yocr ; acr ; aocr ) .
rats were given creatine monohydrate , 4.45 g / l ( sigma - aldrich , st .
louis , mo ) with a 5% dextrose solution ( sigma - aldrich , st .
the dextrose solution was chosen because creatine uptake is increased when combined with a carbohydrate solution .
four weeks after the surgical ablation and/or beginning of creatine supplementation , the animals were killed , and the plantaris muscles were prepared for analysis .
samples were mounted for cross - sectioning in a mixture of tragacanth gum and optimal cutting temperature ( oct ) cutting medium ( sakura finetek co. , torrence , ca ) and frozen in methyl butane ( fisher chemicals , fair lawn , nj ) , cooled with liquid nitrogen .
samples were cryosectioned at 20c to thicknesses of 8 m for atpase analysis and 6 m for immunohistochemistry .
sections were placed onto cover slips coated with poly - l - lysine and stored at 40c until the completion of histochemical analysis .
serial sections were immunostained using primary antibodies against dystrophin ( sigma - aldrich , st .
louis , mo ) and laminin ( santa cruz biotechnology , santa cruz , ca ) ; heat shock protein 70 ( n27f3 - 4 ; santa cruz biotechnology , santa cruz , ca ) ; or myosin heavy chains ( a4.591 and n2.261 ; developmental studies hybridoma bank , iowa city , ia ) .
the serial section stained for dystrophin and laminin were blocked with blocking serum , incubated with the primary antidystrophin antibody ( 4c ) overnight , washed with washing buffer , incubated in biotinylated secondary antibody ( vector , burlingame , ca ) , washed , and labeled with the avidin - biotin complex ( abc ) reagent conjugated with peroxidase .
the sections were then incubated with antilaminin antibody ( 22c ) for 60 minutes and the same series of reactions repeated .
the sections were then incubated in nickel - enhanced diaminobenzidine ( dab ; pierce chemical , rockford , il ) , counterstained with delafield 's hematoxylin and dehydrated in a graded alcohol series , before clearing and mounting .
all other serial sections were incubated in primary antibody for one hour at 37c , followed by the biotinylated secondary antibody , abc reagent , dehydration and mounting as described above .
, serial sections were preincubated in a solution made to ph 4.15 or ph 4.45 .
these ph preincubations allowed type i , type iia , and type iib / d fibers to be characterized .
myosin immunohistochemistry was performed on a subset of muscle samples to validate the matpase - based fiber type delineation .
cross - sectional area ( csa ) was measured for each muscle fiber on a computer using a video captured microscope image of the cells stained with dystrophin and laminin and nih image software .
cross - sectional area was calculated for at least 300 fibers ( from 3 random microscope frames 0.56 mm ) , or all the fibers in the sample .
the myonuclei were counted at 1000x magnification and were identified due to their location inside the sarcolemma and the basal lamina .
the total number per fiber type and total number per 100 fibers ( % ) were counted .
adjacent sections prepared for myofibrillar atpase were matched with these fibers to characterize the fiber type and its properties .
within a fiber type , the myonuclei / cytoplasm ( n / c ) ratio was determined to estimate myonuclear domain ( mean csa / mean myonuclei ) as previously published [ 31 , 32 ] .
digital images of samples stained for hsp70 were acquired using video - capture microscopy ( figure 1 ) .
light and diaphragm settings were held constant for all image acquisition . to assess hsp70 content
, pixel density was determined on at least 50 fibers , of each major fiber type , per sample using nih image software .
all pixel density figures were normalized to an acellular background on the coverslip of the sample .
all statistical analyses were performed using systat 11 . a two - way analysis of variance ( anova ) was used to compare the dependent variables ( creatine and overload ) across ages and muscle fiber types .
significance was set at p 0.05 . where significance was found for main effects , fisher 's least significant difference method was used for post hoc analysis .
one - way anova was used to compare creatine consumption , muscle mass , and body mass across ages .
student's - tests were used for animal and muscle mass analyses . significance was set at p 0.05 . where significance was found for main effects , fisher 's least significant difference method was used for post - hoc analysis .
in the interest of full disclosure , some data from the control groups ( both young and aging ) have been previously reported in acta physiologica .
however , no data from either age category within the treatment groups ( cr , o , or ocr ) have been previously published , apart from conference abstracts .
furthermore , this manuscript contains new data from the control groups ( e.g. , hsp70 ) .
older animals took in more creatine solution than young ( p < 0.05 ) . for the duration of the study , all animals had consistently more creatine intake than recommended for loading dose ( 0.3 g / kg body weight / day ) and maintenance dose ( 0.03 g / kg body weight / day ) as advised in the literature by kreider and colleagues .
body mass was significantly heavier in the aging rats , regardless of the treatment group ( table 2 ) .
the within - subject difference in body mass between pre- and posttreatment showed both aging groups that underwent surgical overload ( ao and aocr ) lost mass .
compared to the young overloaded animals ( yo and yocr ) , these differences were significant ( table 2 ) . in both the young and aging animals , the plantaris muscle of ocr group was heavier than control or creatine ( young 30% heavier and older 18% heavier ; p < 0.05 ; table 3 ) .
the ratio of plantaris weight / body weight was also greater in the ocr group than control or creatine in both young and aging animals . in each experimental group ,
samples from aging animals had a higher percentage type i fibers and a lower percentage type iia fibers than young animals ( table 4 ) .
the percentage of type iib / d fibers was similar between young and aging animals .
csa data for type i , iia , and iib / d fibers are presented in figure 2 . there were no differences in csa between young and older control animals for any fiber type .
csa for iib / d was greater than i or iia for all treatment groups ( p < 0.05 ) .
myonuclear domain size of type iib / d fibers was significantly larger than i or iia fibers within age groups of control animals ( p < 0.05 ; figure 3 ) .
there was no difference in myonuclear domain between type i and iia fibers , within age groups .
type i and iia fibers of young control animals had significantly higher hsp70 content than in those same fiber types of aging control animals ( figure 4 ) . in the faster fibers of the plantaris
, there was no significant difference in hsp70 content between young and aging animals of the control cohorts .
fiber csa did not significantly differ in animals receiving creatine compared to control rats , regardless of fiber type or age .
however , trends toward significance were seen between yc and ycr treatment groups for csa of type iia ( p = 0.057 ) and iib / d ( p = 0.087 ) fibers ( 2059.00 115.28 m versus 1724.26 380.61 m , 28% difference and 3955.57 264.98 m versus 3642.043 648.06 m , 17% difference , resp . ;
the effects of creatine supplementation on myonuclear domain were both age and fiber type specific . in type i fibers of young animals , the myonuclear domain size was not different between creatine supplementation and control animals . in young animals ,
the myonuclear domain sizes of type iia and iib / d fibers were significantly larger in creatine supplemented rats than in control rats ( iia ycr = 1667.02 274.34 m versus yc = 1463.63 400.77 m and iib / d ycr = 2386.83 368.47 m versus yc = 1930.17 405.09 m ; figure 3 ) .
creatine supplementation had no effect on myonuclear domain size in aging animals compared to control animals , regardless of fiber type .
myonuclear domain was significantly different between fiber types within age groups of creatine - supplemented animals ( p < 0.05 ) , except there was no difference between type i and iia in ycr ( p = 0.266 ) .
creatine supplementation alone had no significant effects on hsp70 content for any age or fiber type .
in the overload group , young animals had significantly more type i fibers than control animals of a similar age ( table 4 ) .
there was no difference between the percentages of type i fibers in young and aging animals of the overload treatment .
this was different from the other groups in which the older animals had higher percentages of type i fibers than the young animals ( table 4 ) .
regardless of age , the percentage of type iib / d fibers was lower in the overloaded group than in the control group .
surgical overload significantly increased the csa of iia fibers in the plantaris of young rats ( 35% difference , 2270.01 142.84 m versus 1724.26 380.61 m , figure 1 ) .
the csas of type i and iib / d fibers from overloaded rats were larger than those fibers in control animals , but these data failed to reach significance due to large variability in the control animals ( i 29% ; 1841.68 196.98 m versus 1635.95 600.12 m ; iib / d 12% ; 3747.01 263.40 m versus 3642.04 648.06 m ; figure 1 ) . in the older animals , overload induced fiber hypertrophy of the slow - twitch fiber ( 21% ; 1879.37 212.47 m versus 1491.10 299.40 m ) and iia ( 16% ; 2112.83 226.32 m versus 1768.15 209.31
however , csa of type iib / d fibers of aging animals were not significantly different between overloaded and control groups ( figure 2 ) .
only type iia fibers of young , overloaded rats experienced a significant increase in myonuclear domain ( 1858.96 287.91 m versus 1463.63 400.77 m ; figure 3 ) .
overload did not cause any other significant changes in myonuclear domain size for any fiber type , irrespective of age ( p > 0.05 ; figure 3 ) .
myonuclear domain size was smallest in type i fibers ; type iia and iib / d had significantly larger domain sizes than type i in both young and older animals .
myonuclear domain sizes were larger in type iia fibers than type i fibers of older animals compared to young animals ( p < 0.05 ) .
however , overload did not affect the hsp 70 content of type iid / b , regardless of age group .
a combination of overload and creatine supplementation did not have the same influence on fiber type composition as overload alone . in young rats ,
the yocr treatment group had a significantly lower percentage of type i fibers than the yo treatment .
furthermore , the percentage of type i in the yocr group was similar to young controls . in young rats ,
the overloaded plantaris had a significantly higher percentage of type i fibers than overloaded plantaris from rats that also received creatine
. however , similar to the control group , older animals in the aocr group had a significantly higher percentage of type i , and a significantly lower percentage of type iia fibers than young animals ( table 4 ) .
by contrast , overload alone did not result in significant changes to fiber type composition in aging animals .
in young animals , the ocr treatment significantly increased fiber csa in type iia and iib / d fibers , relative to young controls ( iia 28% , yocr
= 2401.51 199.17 m versus yc = 1724.26 380.61 m ; and iib / d 20% , yocr = 4352.28 519.12 m versus yc = 3642.04 648.06 m ; resp . ) . in older animals , only type i and iia fibers of the ocr group
were significantly larger than those fibers in the aging control animals ( i 23% , aocr = 1949.01 180.77 m versus ac = 1491.10 299.40 m ; iia17% , aocr = 2125.66 205.74 m versus ac = 1768.15 209.31 m , resp . ) .
type iib / d fibers of older animals in the ocr group did not change fiber sizes over controls ( figure 2 ) .
the combined effects of creatine and overload differentially affected the myonuclear domains in the plantaris muscle of young and aging rats . for type iia and iib / d fibers ,
myonuclear domain was significantly larger in fibers of the yocr group than in fibers from yc ( iia 24% , yocr = 1934.58 185.16 m versus yc = 1463.63 400.77 m ; iib / d 25% , yocr = 2559.80 510.02 m versus yc = 1930.17 405.09 m ) . in older rats , the myonuclear domain was not significantly affected by combined creatine and overload , regardless of the fiber type ( p > 0.05 ) .
myonuclear domain was significantly different between fiber types within age groups of animals in the overload plus creatine supplementation groups , except there was no difference between type i and iia in yocr ( p = 0.102 ) .
the combination of creatine and overload also resulted in significantly greater fiber csa than creatine alone in fiber types i and iia , regardless of age .
for the csa of type iib / d fibers , there was a trend toward significance between yocr and ycr groups ( p = 0.051 ) .
the csa of type iib / d fibers were significantly larger in yocr than in yo animals , and there was a similar trend in the csa of type i fibers ( p = 0.057 ) . in aging animals
, there were no significant differences between the csa of aocr and ao for any fiber types .
however , hsp70 content of iia fibers of yocr did not differ in type iia fibers of yc or ycr cohorts .
creatine plus overload did not affect hsp70 expression in the aging group , regardless of fiber type .
research has shown that creatine supplementation , in combination with resistance training , can be beneficial in increasing lean body mass and strength ( reviewed in ) .
fewer still have examined the fiber type - specific effects of creatine supplementation on csa of aging muscle fibers .
this study reports that the plantaris muscles of young and aging rats respond similarly to surgical overload .
this is true for fiber type composition , cross - sectional area , and myonuclear domain size .
further , our data suggest that creatine supplementation alone had no significant effect on fiber csa or hsp70 content , regardless of age or fiber type and the combination of overload and creatine also had minimal additional effect , relative to overload alone .
one specific aim of this research was to assess the efficacy of creatine supplementation in aging muscle .
creatine supplementation without exercise did not enhance anabolic signals or significantly alter fiber csa .
our data agree with these findings - creatine supplementation alone had no significant effect on fiber csa in young or aging animals , regardless of the fiber type . in vitro
, creatine has been shown to increase myosin heavy chain expression at both mrna and protein levels , but our data did not agree with these findings .
creatine supplementation alone had no effect on csa or myonuclear domain for any fiber type in the plantaris of aging animals .
the authors are not aware of any previous research that investigated fiber - specific changes subsequent to creatine supplementation in aging muscle , so we propose the following possible explanations for our findings .
creatine supplementation significantly increases the myoprotective effects of carnosine and anserine in young , but not older animals .
additionally , although creatine supplementation increased muscle phosphocreatine concentrations in both young and older adults , this response was significantly greater in the young adults
. nonsupplementation studies also indicate that aging muscle is less capable of synthesizing or storing creatine .
compared to young skeletal muscle , aging muscle has fewer stores of pcr and cr and a lower rate of pcr resynthesis .
a second specific aim of the current research was to examine the ability of aging animals to respond to a hypertrophic stimulus .
current results indicate that the slower fiber phenotypes of aging animals maintain their ability to hypertrophy in response to synergist ablation .
these data also indicate that the overload - induced fast - to - slow fiber transition remains intact in older animals .
these findings are in agreement with much of the literature related to compensatory hypertrophy of young muscle [ 23 , 39 ] .
the young and aging animals in this study responded similarly to the overload surgery , demonstrating that older muscle maintains the ability to hypertrophy .
these findings are in contrast to a few studies that found a blunted response to overload in elderly animals [ 4042 ] .
these differences may be explained by the age difference in the aging group versus their elderly animals .
although the muscle fibers of the 24 month older rats did not exhibit age - associated atrophy , they did present other characteristics associated with aging , such as a slower fiber phenotype .
in addition , the plantaris of the older groups had significantly higher muscle mass compared to the young groups .
the increased muscle mass in the absence of fiber hypertrophy may reflect a reduction in muscle quality .
infiltrations of fat and noncontractile material are often noted in aging muscle [ 43 , 44 ] . a third specific aim of this research was to examine the response of aging muscle to a combination of creatine and overload .
compared to the control group , the ocr treatment resulted in significant hypertrophy and significant increase in myonuclear domain for iia and iib / d fibers in the young plantaris muscle . in aging plantaris muscle , the combination of creatine and overload resulted in significant hypertrophy of types i and iia fibers , but myonuclear domain sizes were not significantly different between control animals and animals in the ocr group for any fiber types . compared to creatine alone , ocr groups had significantly larger csa of type iia fibers , regardless of age .
the additional benefit of creatine in the ocr group over the o group was minimal .
it is not immediately clear why the combination of creatine and overload did not result in significantly greater hypertrophy than overload alone .
a combination of creatine supplementation and resistance training has previously been shown to result in larger fiber csa [ 4547 ] . however , the present findings are in agreement with r. e. young , and j. c. young , who demonstrated that creatine supplementation provided no additional hypertrophy beyond synergist ablation alone in soleus and extensor digitorum longus muscles .
it is possible that the overload model is not appropriate for examining the effects of creatine on resistance exercise .
the proposed benefit of creatine supplementation is to increase pcr stores , thereby increasing the capacity to regenerate atp stores during short bursts of exercise . in this scenario ,
the main benefit of creatine supplementation is to sustain high - intensity work for a longer duration . in turn , the enhanced capacity to anaerobically train the muscle leads to increases in muscle mass and strength . in the synergist ablation model ,
the overload imposed upon the muscle is permanent , and therefore can not be considered anaerobic exercise .
creatine supplementation combined with overload surgery also seems to influence the shift in muscle phenotype to a slower phenotype .
interestingly , although both overloaded muscle and aging muscle demonstrated an increase in slow fibers , creatine supplemented animals of the aging group did not show that fast - to - slow shift in fiber types in either acr or aocr groups .
this appears to show that the fiber type shift in aging animals can potentially be altered or reduced .
there is a different response between young and older animals with regards to fiber type shift in response to overload .
the young animals have an increase in the percentage of both type i and type iia fibers after overload , which is similar to previous reports [ 23 , 39 ] .
however , in the aging overload , and the young and aging ocr groups , there is a shift from iib / d to iia , without a shift from iia to i. although the young overload group had the greatest fast - to - slow shift in fiber phenotype , this was not significantly different from the percent type i fibers in the aging animals with overload alone .
the aging animals had already undergone an age - related fast - to - slow fiber shift , and the overload did not produce an additional phenotype shift .
creatine appears to blunt the overload - induced fast - to - slow fiber transition in the yocr group .
however , since creatine alone does not cause any significant shift in fiber type , we can only speculate that it must be an interaction between overload and creatine that negated the fast - to - slow shift seen in the yo group .
in addition , the young ocr group had the greatest percent increases in csa indicating the greatest response to the interventions .
a further specific aim of this project was to examine the age - specific effects of creatine and compensatory hypertrophy on myonuclear domain size .
creatine supplementation alone resulted in a significant increase in myonuclear domain of types iia and iib / d fibers in young animals whereas it had no effect on myonuclear domain size in aging animals .
an increase in myonuclear domain implies that fiber size increased at a disproportionately higher rate than myonuclear addition .
the results of this study indicate that creatine alone resulted in trends toward hypertrophy in young , but not aging , muscle fibers .
therefore , it is not surprising that the creatine - induced increase of myonuclear domain occurred only in the young animals .
it is not clear why the myonuclear domain size of type i fibers did not significantly increase with creatine supplementation ; however , fast - twitch fibers have relatively larger myonuclear domain sizes than slow - twitch fibers .
the concept of myonuclear domain implies that there is an optimal amount of genetic material to govern the structural proteins in that domain .
it has been reported that if a fiber hypertrophies beyond 26% , myonuclei are added to maintain a constant myonuclear domain . in young animals , type iia fibers from the ycr , yo , and yocr treatment groups were all hypertrophied more than 26% .
of these , the myonuclear domain size of the iia fibers was also increased in all three treatments .
it is possible that the fibers were in the process of increasing in size , and the myonuclei had not yet had a chance to proliferate .
indeed , transgenic overexpression of hsp70 in aging rats was able to rescue damage - associated force deficits and speed recovery of the muscles .
this atrophy - attenuating quality of hsp70 is due , at least in part , to the repression of the foxo3a - induced transcription of atrogin-1 .
our data demonstrate a significant decrease in hsp70 content in type i and iia fibers of aging rats , relative to young rats .
these findings partially agree with naito and colleagues , who found an insignificant decrease in hsp70 content in the plantaris muscle of aging rats , relative to young rats . in that study , hsp70 content was measured in muscle homogenates , not individual fiber types .
given the high percentage of type iid / b fibers in the plantaris and the low hsp70 content in type iid / b fibers , it is reasonable to conclude that homogenizing the muscle , as done in , diluted any age - related differences that may have occurred in hsp70 content .
conversely , chung and ng demonstrated a significant increase in hsp70 content in aging rats .
it is not possible to directly compare the two studies due to differences in muscle ( plantaris versus gastrocnemius ) , technique ( histochemistry versus western blot ) , and ages used ( 5 m24 m versus 16 m29 m ) .
further , our data demonstrate a creatine - induced attenuation to the overload - associated increase in hsp70 content , as evidenced by the lack of significance between c and ocr groups . to our knowledge , these data are the first to examine the effects of creatine supplementation on hsp70 content .
exercise resulted in increased hsp70 expression of glycogen - depleted skeletal muscle but not in glycogen - containing control muscle .
it has also been demonstrated that adp acts as a cofactor in hsp70-peptide binding interactions , and binding of hsp70 is directly correlated to the adp / atp ratio .
therefore , the attenuation of hsp70 content in ocr groups , relative to overload alone , may be explained by the ability of creatine to maintain atp levels . in the current study
, the aging muscle shows an inability to respond to creatine and ( in comparison with the young animals ) a blunted response to overload .
if creatine acts directly on satellite cells , as has been shown in cell culture , then the age - associated loss of responsiveness to creatine may be a result of dysfunctional satellite cells .
in young individuals , creatine increased the quantity of both satellite cells and myonuclei in young males , contributing to enhanced growth .
many age - related studies that have used a rat model have documented an increase in body mass throughout the animal 's lifespan .
fisher 344 rats were selected for this study , because the body mass of this strain plateaus .
24 months of age was selected for the aging group , because the nih survival curves indicate a 50% survival for fisher 344 rats at this age .
furthermore , 24-month - old fisher 344 rats are analogous to 80-year - old humans .
age - associated changes in fiber type and csa often accompany animals in this age range [ 42 , 61 ] . while the skeletal muscle of the aging animals in this study did not exhibit some of the characteristics commonly associated with sarcopenia , the aim of this study was to determine whether the c / n characteristics and responsiveness to overload and creatine change during the aging process .
the data demonstrate that these age - associated changes did occur , despite the absence of age - associated declines of fast fiber types and fiber csa .
therefore , it is not clear whether age - specific results were due to the inability of aging muscle to take in creatine or to respond to the creatine .
secondly , the compensatory hypertrophy model is an involuntary model of extreme overload . though it is a valid , common technique for inducing hypertrophy in murine models , humans
furthermore , creatine is believed to confer its effect on muscle by replenishing anaerobic energy stores , thereby allowing users to perform an additional repetition of resistance exercise .
as such , creatine supplementation may result in greater gains if the training intensity can be increased throughout the training period .
for example , significant increases in fat - free mass and strength were seen when creatine supplementation was coupled with resistance training , modified to maintain maximal effort .
nevertheless , compensatory hypertrophy is a form of permanent overload that can not be modified incrementally and is a limitation of the study .
creatine supplementation has been linked to protein synthesis ; therefore , measuring these indices may have enhanced the study . given the prevalence of age - associated sarcopenia , the search for a preventative supplement is of vital importance .
the data presented here indicate that creatine supplementation alone does not affect fiber hypertrophy in aging muscle .
furthermore , this study found that a combination of creatine and overload was no more effective than overload alone on hypertrophy .
in fact , the creatine supplementation appeared to result in a small , but significant , attenuation of overload - induced hsp70 expression . | attenuation of age - related sarcopenia by creatine supplementation has been equivocal . in this study ,
plantaris muscles of young ( y ; 5 m ) and aging ( a ; 24 m ) fisher 344 rats underwent four weeks of either control ( c ) , creatine supplementation ( cr ) , surgical overload ( o ) , or overload plus creatine ( ocr ) .
creatine alone had no effect on muscle fiber cross - sectional area ( csa ) or heat shock protein ( hsp70 ) and increased myonuclear domain ( mnd ) only in young rats .
overload increased csa and hsp70 content in i and iia fibers , regardless of age , and mnd in iia fibers of yo rats .
csa and mnd increased in all fast fibers of yocr , and csa increased in i and iia fibers of aocr .
ocr did not alter hsp70 , regardless of age .
mnd did not change in aging rats , regardless of treatment .
these data indicate creatine alone had no significant effect .
creatine with overload produced no additional hypertrophy relative to overload alone and attenuated overload - induced hsp70 expression . |
despite current advances in cancer screening , diagnosis , and therapeutics , pancreatic cancer remains a highly malignant disease with a dismal 5-year survival rate of < 5% . in 2012
, approximately 44,000 people in the united states will be diagnosed with pancreatic cancer with more than 37,000 expected to die from this disease , making pancreatic cancer the fourth leading cause of cancer - related deaths in the country . pancreatic cancer is often diagnosed at an advanced stage owing to nonspecific symptoms and a lack of reliable biomarkers . because of the high number of locally advanced and metastatic cases at the time of diagnosis , only 1520% of patients can undergo potentially curative surgical resection .
in addition , response to chemotherapy and radiotherapy is often very poor , making management of the disease extremely challenging for clinicians .
resistance to the drug is widespread and may be due to a number of factors including poor drug delivery and inherent chemoresistance of pancreatic tumor cells .
combination chemotherapy with leucovorin , fluorouracil , irinotecan , and oxaliplatin is an emerging treatment that offers a better survival outcome compared to gemcitabine , albeit with increased toxicity . in a study of 342 patients ,
folfirinox treatment resulted in a median 11.1-month overall survival compared to 6.8 months for gemcitabine treatment .
it is clear that novel chemotherapeutic approaches are necessary to improve the clinical outcomes of patients with this devastating disease . for these strategies to be viable for clinical use ,
this article summarizes recent literature ( 1995 to 2012 ) and clinical trials highlighting the current progress on new therapeutic agents and drug delivery methods , and the potential targets for the treatment of pancreatic cancer .
a number of therapeutic agents and drugs may serve as alternative molecular approaches to conventional chemotherapies ( table 1 ) . among these agents are small - molecule inhibitors , oncolytic viruses , monoclonal antibodies , suicide genes , and small interfering rna ( sirna ) that target abnormal gene expression , cell - surface receptors , and cell - signaling pathways .
studies of these alternative agents may generate improved therapeutic strategies and facilitate progress towards improving the clinical outcome of pancreatic cancer patients . through gene therapy , cancer cell growth
can be inhibited by replacing or silencing key genes in pancreatic cancer cell growth pathways involving p53 , retinoblastoma protein ( rb ) , p21 , p16 , kras , and bcl-2 .
modulation of kras expression has been shown to inhibit growth of pancreatic cancer cells in vitro and growth of intraperitoneal tumors in xenograft nude mice in vivo .
research has also demonstrated that re - expression of the tumor suppressor gene p53 in a subcutaneous nude mouse model inhibits pancreatic tumor growth .
sirna and short hairpin rna ( shrna ) can be utilized to post - transcriptionally silence gene expression in a sequence - specific manner .
these molecules are being investigated in cancer biology and other fields as powerful tools for regulating expression of genes essential for cellular processes such as survival , proliferation , and drug resistance .
shrna silencing of zinc transporter zip4 has been shown to inhibit tumor growth and extend the survival of nude mice with pancreatic cancer xenografts .
in addition to re - expressing tumor suppressor genes , gene therapy strategies can also deliver suicide genes to tumor cells , which encode for drug - activating enzymes .
pancreatic cancer cells transfected with cytosine deaminase , which converts 5-fluorocytosine ( 5-fc ) to its active form fluorouracil ( 5-fu ) , showed decreased tumor growth when subsequently treated with 5-fu . however , despite strong potential , the success of gene therapy agents , especially in clinical studies , has been limited , perhaps because of the complex nature of pancreatic tumors .
simultaneous targeting of multiple gene mutations may be necessary to overcome the effects of extensive crosstalk .
the epidermal growth factor receptor ( egfr ) inhibitor erlotinib is fda approved in combination with gemcitabine to treat metastatic pancreatic cancer based on clinical trial results that showed significantly increased overall survival and progression - free survival compared to gemcitabine monotherapy in this patient population [ 10 , 13 ] .
egfr , involved in cancer growth and metastasis , promotes epithelial - to - mesenchymal transition for decreased cell adhesion and increased cell migration .
overexpression of egfr has been shown to be common in pancreatic cancer and may indicate the potential aggressiveness of the cancer .
also , several studies have utilized carbon nanotubes for imaging as a significantly better contrast agent .
other investigational therapeutic agents include monoclonal antibodies against targets such as egfr , vascular endothelial growth factor receptor ( vegfr ) , mucin 1 ( muc1 ) , and mesothelin .
antibody therapies have the potential to inhibit tumor growth and angiogenesis and provide a method of treating otherwise resistant cancers .
oncolytic viral therapies , often using adenovirus and herpes simplex virus , are engineered to replicate selectively in tumor cells , targeting the cells for lysis , cell - to - cell fusion , or immune response .
commonly used delivery vehicles for cancer therapies include liposomes and nanoparticles . however , there are few clinical trials using these newer delivery methods in pancreatic cancer .
most current pancreatic cancer chemotherapies work systemically and thus are subject to a number of physiologic and chemical limitations that decrease efficacy . unfavorable pharmacokinetics and poor targeting of cancer cells among other issues significantly affect the ability of these drugs to provide greater clinical benefit , and toxicity and side - effects limit acceptable dosages . the newer delivery vehicles not only target specific organs but also offer increased drug solubility , protection against degradation and elimination , decreased toxicity , and reduced resistance .
liposomes are vesicles composed of single or multiple phospholipid bilayers that can be loaded with a variety of content including genetic materials and chemotherapeutic drugs .
liposomes can improve drug solubility and stability , are biodegradable , and exhibit low toxicity .
they have already proven to be viable clinically , with several fda - approved liposome formulations in existence for cancer treatment .
liposomes have been used for in vitro gene transfection to protect antisense oligonucleotides , sirna , and shrna from degradation and improve transfection efficiency and targeting .
for example , liposomal delivery of both pancreatic and duodenal homeobox 1 ( pdx-1 ) and zip4-targeted shrna was shown to inhibit tumor growth in immunodeficient mice [ 16 , unpublished data ] .
the addition of surface ligands can help target liposomes to cells of interest , thereby helping to reduce the toxicity of therapeutic agents and enrich concentrations in target tissues .
liposomes can also be pegylated to increase stability and prolong the circulation time of a drug . in a study by cosco and colleagues ,
pegylated liposomes loaded with gemcitabine increased survival and reduced tumor growth and toxicity in severely compromised immunodeficient ( scid ) xenograft mouse models of pancreatic cancer compared to controls treated with standard gemcitabine .
improved liposomal delivery efficiency may further allow lower concentrations of drugs to be used for the same effect , prolong circulation times , improve drug internalization , and decrease unwanted toxic side - effects .
the benefits of nanoparticles are a large surface - to - mass ratio and the ability to bind to a number of substances , provide sustained release of drugs , and improve drug circulation and concentration .
stearoyl gemcitabine nanoparticles have been found to enhance the effect of gemcitabine on pancreatic cancer cells in vitro , and suppress tumor growth more effectively than normal gemcitabine in a mouse model .
nanoparticles can also be targeted to specific locations through the addition of monoclonal antibodies or use of magnetic guidance .
recently , carbon nanotubes have emerged as an exciting potential modality for drug delivery and imaging .
they are able to absorb a large amount of drug , which can be slowly released , and their stable structure can be modified for improved targeting of therapies .
carbon nanotube - delivered cisplatin modified to target egfr - expressing cells was found to selectively kill head and neck squamous carcinoma cells ( hnsccs ) .
to improve the targeting of new therapeutic agents to pancreatic tumor cells , the unique expression profile of pancreatic cancer cells can be exploited to differentiate normal , healthy cells from cancer cells .
liposomes , nanoparticles , and carbon nanotubes can be modified with monoclonal antibodies or ligands to allow the therapeutic agents to be directed towards specific cells , thus increasing the accumulation of drugs in the target organ and decreasing toxicity to normal cells .
potential targets that can be used include egfr , urokinase plasminogen activator receptor ( upar ) , transferrin receptor , her-2 , ca125 , mesothelin , and muc1 .
these potential delivery targets have been shown to be overexpressed in pancreatic cancer cells . however , because of the heterogeneity of pancreatic cancer and the variable expression profiles among pancreatic cancer cells , further research is warranted to identify more effective targets for drug delivery .
increasing evidence suggests that the tumor microenvironment may play a critical role in pancreatic cancer cell resistance and explain the disparity between preclinical and clinical study results .
dense desmoplastic stroma and poor perfusion of the tumor prevent drugs from properly penetrating pancreatic tumors . inhibition of hedgehog signaling has been found to decrease stromal tissue and increase perfusion of the tumor , thus improving survival in a mouse model . for novel therapeutics to be successful ,
it is likely that there will be a need for a greater understanding of the microenvironment and how it can be overcome for better drug delivery .
recent advancements in the development of therapeutic agents and delivery modalities for improved targeting , efficacy , and clinical outcomes in pancreatic cancer are extremely promising . however , because the data are mostly preclinical and the clinical trials are few , more research is necessary to further refine these methods , confirm their safety , and improve delivery specificity . even if targeted delivery is accomplished and the drug enters the cell , it is possible that there will be a response failure .
therefore , a greater understanding of pancreatic cell biology is also crucial since the presence of multiple mutations may make single - target treatments insufficient .
emerging targeted therapeutic approaches may be exciting , viable opportunities that will tangibly improve the management of this devastating disease . | pancreatic cancer is an incredibly challenging disease due to its high rates of resistance to traditional chemotherapy and radiotherapy .
there has been little improvement in the prognosis of pancreatic cancer cases in the past decades , highlighting the crucial need for more effective therapeutic approaches .
erlotinib , an egfr inhibitor , and gemcitabine , a nucleoside analog , are currently used in combination for chemotherapy treatment , but new developments in drug delivery systems using liposomes and nanoparticles may be promising new modalities for management of the disease .
in addition to standard chemotherapeutic drugs , these delivery systems can be utilized to deliver therapeutic agents such as sirna , oncolytic viruses , small molecule inhibitors , antibodies , and suicide genes .
further work is required to elucidate how ligands and antibodies could be used to enhance the targeted delivery of drugs , thus increasing specificity , improving stability , and reducing the effect of the drugs on healthy tissue . despite significant preclinical data ,
there are currently very few clinical trials involving pancreatic cancer targeted drug delivery .
this article summarizes current developments in targeted pancreatic cancer drug delivery , focusing on delivery systems , targets , and therapeutic agents . |
a previously healthy 22-year - old woman initially presented with right hemiparesis and episodes of right - sided sensory abnormalities , both of which had begun one year earlier .
she also had a one - year history of intermittent loss of consciousness unrelated to these symptoms .
routine electroencephalography ( eeg ) demonstrated diffuse cerebral dysfunction over the left hemisphere , but no epileptic or epileptiform discharge was noted .
mr studies of the brain using a 1.5-t system revealed a well demarcated broad - based mass , 2 cm in size , on the falx cerebri and prominent peritumoral edema .
realative to gray matter , the mass was hypointense on t1-weighted mr images ( fig .
the tumor 's cystic structure was isointense relative to cerebrospinal fluid , and it was thus thought to be a peritumoral cyst ( figs .
surgery revealed that the tumor was located in the brain parenchyma , adhered to the falx cerebri , and had an extra - axial component .
there was no necrosis or calcification within the mass , and microscopic examination showed that it was composed of relatively mature ganglion cells and abundant collagen - rich stroma in which spindle stromal cells and lymphocytes were observed ( fig .
both the cytoplasm of the ganglion cells and stromal cytoplasmic processes stained positively with antibody for synaptophysin , though glial fibrillary acidic protein ( gfap ) staining failed to reveal a glial component . finally , gangliocytoma was diagnosed .
a previously healthy 59-year - old woman presented with stubborn headache and dizziness which had begun six months earlier .
she also complained of intermittent facial palsy and had a seven - year history of hypertension .
the results of neurological examination were perfectly normal , and routine eeg showed no epileptic or epileptiform discharge .
mr images of the brain demonstrated a small dural - based mass , and adjacent to it , in the right parieto - occipital region , was a small amount of subdural hygroma .
the tumor appeared to have both intra- and extra - axial components ; tumor - cell infiltration of brain parenchyma was thought to arise from the intra - axial portion ( fig .
gadolinium - enhanced t1-weighted sagittal images showed that the major portion of the tumor was located intra - axially ( fig .
the preferred preoperative radiologic diagnosis was an extra - axial mass such as atypical meningioma .
surgery revealed that the tumor was located in the cortical area of the brain parenchyma , showed partly exophytic growth , and adhered to the adjacent leptomeninges .
its cut surface was soft and whitish , and it contained yellowish friable granular material .
microscopic examination showed that it was composed of ganglion cells , and there was marked desmoplastic reaction and lymphocytic infiltration .
the ganglion cells varied considerably in size and shape , but a glial component was not clearly demonstrated .
numerous eosinophilic granular bodies were also observed . microscopic examination ( low - power field ) showed that the mass was attached to the dura mater ( fig .
immunohistochemistry demonstrated positive staining of the cytoplasm and process of some tumor cells with synaptophysin , but negative staining with gfap .
a previously healthy 22-year - old woman initially presented with right hemiparesis and episodes of right - sided sensory abnormalities , both of which had begun one year earlier .
she also had a one - year history of intermittent loss of consciousness unrelated to these symptoms .
routine electroencephalography ( eeg ) demonstrated diffuse cerebral dysfunction over the left hemisphere , but no epileptic or epileptiform discharge was noted .
mr studies of the brain using a 1.5-t system revealed a well demarcated broad - based mass , 2 cm in size , on the falx cerebri and prominent peritumoral edema .
realative to gray matter , the mass was hypointense on t1-weighted mr images ( fig .
the tumor 's cystic structure was isointense relative to cerebrospinal fluid , and it was thus thought to be a peritumoral cyst ( figs .
surgery revealed that the tumor was located in the brain parenchyma , adhered to the falx cerebri , and had an extra - axial component .
there was no necrosis or calcification within the mass , and microscopic examination showed that it was composed of relatively mature ganglion cells and abundant collagen - rich stroma in which spindle stromal cells and lymphocytes were observed ( fig .
both the cytoplasm of the ganglion cells and stromal cytoplasmic processes stained positively with antibody for synaptophysin , though glial fibrillary acidic protein ( gfap ) staining failed to reveal a glial component . finally , gangliocytoma was diagnosed .
a previously healthy 59-year - old woman presented with stubborn headache and dizziness which had begun six months earlier .
she also complained of intermittent facial palsy and had a seven - year history of hypertension .
the results of neurological examination were perfectly normal , and routine eeg showed no epileptic or epileptiform discharge .
mr images of the brain demonstrated a small dural - based mass , and adjacent to it , in the right parieto - occipital region , was a small amount of subdural hygroma .
the tumor appeared to have both intra- and extra - axial components ; tumor - cell infiltration of brain parenchyma was thought to arise from the intra - axial portion ( fig .
gadolinium - enhanced t1-weighted sagittal images showed that the major portion of the tumor was located intra - axially ( fig .
the preferred preoperative radiologic diagnosis was an extra - axial mass such as atypical meningioma .
surgery revealed that the tumor was located in the cortical area of the brain parenchyma , showed partly exophytic growth , and adhered to the adjacent leptomeninges .
its cut surface was soft and whitish , and it contained yellowish friable granular material .
microscopic examination showed that it was composed of ganglion cells , and there was marked desmoplastic reaction and lymphocytic infiltration .
the ganglion cells varied considerably in size and shape , but a glial component was not clearly demonstrated .
numerous eosinophilic granular bodies were also observed . microscopic examination ( low - power field ) showed that the mass was attached to the dura mater ( fig .
immunohistochemistry demonstrated positive staining of the cytoplasm and process of some tumor cells with synaptophysin , but negative staining with gfap .
ganglion cell tumors include gangliogliomas , gangliocytomas , dysplastic cerebellar gangliocytomas ( lhermitte - duclos disease ) and desmoplastic infantile gangliogliomas ( 2 ) . among these ,
a tumor is designated ' ganglioglioma ' if it has a neoplastic glial component , while one in which only abnormal neurons are present is a gangliocytoma .
considering the group of differentiated ganglion cell tumors of the cns as a whole , it is apparent that in the glial component there are varying degrees of neoplastic evolution , and this variability makes clear - cut separation of gangliocytomas and gangliogliomas difficult . in the two cases described in this paper , the tumors were composed mainly of ganglion cells , while immunohistochemical staining for gfap failed to unequivocally identify a glial component . in both cases , ' gangliocytoma ' is thus an appropriate designation .
gangliocytomas are extremely rare , with a reported frequency of only 0.1% ( 5 ) .
they are almost always benign , so preoperative study of their radiological features is important ( 2 ) .
supratentorial gangliocytomas have on occasion been cited as the cause of epileptic foci ( 3 , 6 ) , though discussion of their specific clinicoradiological features has been limited to individual case reports ( 3 , 4 , 7 ) .
previous reports have failed to document clearly the imaging findings of supratentorial gangliocytoma . according to reports by sherazi and peretti - viton et al .
( 4 , 7 ) , mr imaging of gangliocytomas revealed masses of low signal intensity on t1-weighted images and high signal intensity on t2-weighted images , while gadolinium - enhanced scanning frequently demonstrated enhancement . in terms of mr signal intensity and degree of contrast enhancement ,
our two cases were similar to those of sherazi and peretti - viton et al .
altman ( 3 ) , however , reported that mr imaging of supratentorial gangliocytomas demonstrated intra - axial tumors of mixed signal intensity on t1-weighted and proton - density images , low signal intensity on t2-weighted images , and little mass effect in three cases involving children with intractable seizure .
altman suggested that the low signal intensity seen on t2-weighted images might be the result of large nuclei and prominent nucleoli with long - chain nucleic acids . the high signal intensity seen on t2-weighted images , as in our two cases and two previous reports ( 4 , 7 ) ,
reported that ct scanning of these tumors often revealed calcification and cysts . in our second case report , calcification was observed . in both our cases ,
, a broad - based mass was attached to the falx cerebri and had a dural tail , mimicking falx meningioma . in the second case ,
the tumor showed partly exophytic growth , mimicking an extra - axial tumor , and adjacent to it there was a small amount of subdural hygroma . according to altman and peretti - viton et al .
( 3 , 7 ) , supratentorial gangliocytomas occurred in cortical and subcortical locations . in the case described by itoh et al .
( 8) , the gangliocytoma was also cortically located . in previous reports ( 3 , 7 , 8) and in our cases ,
this predominantly cortical location might be because the tumor originated from ganglion cells in the cortex .
in previous reports , however , mr findings suggesting an extra - axial or exophytic component such as a dural tail or subdural hygroma , as in our cases , were not mentioned , and the tumors seen on ct or mr images were evidently considered to be intra - axial . the dural tail of the tumor in our first case might have resulted from fibrous adhesion to the falx . in both our cases , microscopic examination revealed that tumor cells were diffusely positive for masson 's trichrone staining and there was a marked desmoplastic reaction , characteristics which might result in fibrous adhesion to the falx or meninges .
the subdural hygroma adjacent to the tumor , as seen in our second case , might be secondary to the exophytic component of the tumor and the fibrous adhesion to the leptomeninges .
we believe that the presence of these extra - axial components and fibrous adhesion to the falx or leptomeninges could be important imaging findings of supratentorial gangliocytoma .
the differential diagnoses of supratentorial gangliocytoma include ganglioglioma or meningioma , but a rigid separation between gangliocytoma and ganglioglioma on either histological or clinical grounds is difficult .
as already described ( 4 , 7 ) , the mr and ct findings of gangliocytomas showed significant overlap with those of gangliogliomas , and differentiation between them on the basis of imaging findings is thus difficult or impossible .
since meningiomas usually show iso or low signal intensity on t2-weighted images , high signal intensity in these circumstances can provide a means of differentiation . in summary , supratentorial gangliocytoma , a very rare benign neuronal tumor , can manifest as a cortical or subcortical lesion which shows high signal intensity on t2-weighted mr images and homogeneous enhancement on contrast - enhanced images , and calcification may be observed .
it can , however , as we report here , mimick an extra - axial tumor such as meningioma . | we report two cases of supratentorial gangliocytomas mimicking an extra - axial tumor .
mr imaging indicated that the tumors were extra - axial , and meningiomas were thus initially diagnosed .
relative to gray matter , the tumors were hypointense on t1-weighted images and hyperintense on t2-weighted images .
on contrast - enhanced t1-weighted images , homogeneous enhancement was observed , while ct scanning revealed calcification in one of the two cases . |
several factors contribute to the heightened recent interest in understanding the role of home health care in the continuum of services for medicare beneficiaries .
first , medicare home health agencies ( hhas ) command growing importance in the medicare budget , almost doubling expenditures from $ 1.6 billion in fiscal year ( fy ) 1983 to $ 3.2 billion in fy 1989 ( approximately 6 percent of the total expenditures ) and almost doubling again in the next 2 years to $ 5.7 billion in fy 1991 ( letsch et al . , 1992 ) .
second , the increasing numbers of the very old among the medicare beneficiaries are expected to contribute to more demand beyond the 2.2 million hha clients in 1991 ( prospective payment assessment commission , 1993 ) .
third , the number of medicare - certified providers also has been growing steadily over the past several years , increasing 43 percent from 1983 to 1989 to 6,100 hhas ( u. s. general accounting office , 1989 ) and leveling off at 6,000 in 1991 . this growth in the number of providers
is expected to continue as hospitals continue to enter this market and as recent legal and administrative changes increase the medicare services available to medicare beneficiaries .
last , the incentives of medicare 's hospital prospective payment system ( pps ) have also contributed to increased use ; shortened stays and more care needs at discharge have contributed to greater home care demand post - discharge ( noether , 1988 ) .
all these factors have increased concerns about the growing expense of home care and , hence , interest in promoting its efficiency .
pps and capitation systems are viewed as complimentary ways to redirect incentives from the current cost - based system , with its traditional inflationary incentives , to more cost - conscious behavior among the providers .
an initial step towards promoting efficiency , either by pps , a capitated managed care system , or any other policy redirection , is to document systematically the characteristics of current hha clients , the actual services they receive , the charges for these services , and more importantly any significant associations between client characteristics and types , amount , and charges for services received .
a prior analysis of the present data examined the concordance between planned and approved visits during the first 60 days of home care ( branch , goldberg , and cheh , 1991 ) .
the data for this analysis were obtained in 1987 from a sample of the medicare - certified hhas that the health care financing administration ( hcfa ) had recruited in 1985 to participate in an hha prospective payment demonstration that was not implemented at that time .
these agencies had been recruited within 10 states ( california , connecticut , florida , illinois , massachusetts , ohio , pennsylvania , tennessee , texas , and wisconsin ) from the universe of known hhas in those states that met three inclusion criteria ( urban location , non - government operated , and established before january 1 , 1983 ) .
a total of 235 agencies met the criteria and were approached ; 127 expressed initial interest in participating in this data collection effort ; and of these , 86 agencies actually submitted data .
each agency submitted copies of hcfa forms-485 and 486 ( figures 1 and 2 ) for a sample of clients who began an episode of care in 1986 and submitted copies of the agency 's medicare cost reports for 1986 . from each of the participating agencies , a systematic sample of clients
was selected , designed to yield approximately 80 to 200 medicare hha clients per agency who were admitted for a new episode of care during calendar year 1986 . of those clients for whom
data were received , only those who had verified medicare claim numbers ending in 0 , 4 , 5 , or 8 ( and , therefore , whose complete hha billing information would be available on the hcfa bureau of data management and strategy [ bdms ] hha 40-percent bill skeleton file ) and for whom data on allowed visits were actually received were considered conditionally eligible for this analysis ( unweighted n = 3,614 clients ) .
the sampling fraction for each hha varied ; consequently , the individual responses were weighted by the inverse of the sampling fraction .
the sample sizes indicated in the tables are the number of respondents ; the percents are based on the weighted data . for each client admitted to hha care , hcfa forms-485 and 486 are completed to establish medical necessity for home health care and to document treatment plans and other aspects of the case .
hcfa forms-485 and 486 also include a certification from and to date . however , because of inconsistencies in agency practice , the from date on hcfa form-485 was found not necessarily to be the starting date of a new episode for every client .
therefore , for this analysis , the conceptual definition of a new episode was that the client had not been receiving hha services for the preceding 60 days . the operational definition of the start of a new episode relied on the hha 40-percent bill skeleton file in the following way .
date on hcfa form-485 , each patient 's billing record was examined retrospectively to identify a 60-day interval with no home health services . the day after this 60-day billing gap
was designated as the episode start date for this analysis . for nearly three - fourths of the conditionally eligible clients ( 72 percent ; unweighted n = 2,615 clients ) , the episode start date thus defined was identical to the from date on hcfa form-485 ; and for another 7 percent ( unweighted n = 258 ) , the episode start date was within 5 days of the from date on hcfa form-485 .
these 2,873 clients comprise the analytic group for this analysis , and their data were appropriately weighted .
the end of an episode was defined as the last day of home health care following the start date that preceded another 60-day gap in the hha 40-percent bill skeleton file .
treatment plans for every 60-day period following the initial end date on hcfa form-485 are recorded on a series of hcfa forms-486 for each client .
the hha 40-percent bill skeleton file included all claims processed through september 1987 , which is 9 months after the start date of the last possible new episode during the study window of calendar year 1986 .
however , previous experience has shown that a 6-month lag in the processing of home health claims can exist , which could cause some distortions in exceedingly long episodes and some of the episodes that began in late 1986 .
table 1 presents the weighted distribution of episode lengths of stay according to this definition .
more than 40 percent of the medicare hha clients completed their home care within 30 days ( 41.2 percent ) , and another one - third completed their episodes within 30 - 60 days ( 30.9 percent )
. only about 5 percent of the clients had episodes lasting 6 months or longer .
although there were a few exceptions , the information provided on hcfa forms-485 and 486 is without missing data in nearly all fields . as figure 1 indicates , the variable living arrangement
it was not possible to categorize household composition on 46 percent of the cases , either because their episode was short and they did not have a hcfa form-486 or because nothing
the variable admission source had missing data in 1 percent of the records . for three variables ( morbidity restrictions , medications , and factual limitations ) , the category
none was inferred from the absence of a response ; because 3.6 percent , 5.2 percent and 1.3 percent , respectively , of these variables was judged consistent with expected prevalence rates , any problem resulting from missing data is assumed to be negligible , with the exception of living arrangement .
hcfa form-485 provided sex and age ( categorized as under 65 years of age , 65 - 74 years , 75 - 84 years , and 85 years of age or over in this analysis information ) . for the 54 percent with information on usual living arrangement , the categories were
admission source had four categories : from a hospital ( medical ) , from a hospital ( surgical ) , from a nursing home , or
mobility restrictions were categorized based on responses to the hcfa form-485 item indicating activities permitted ; the four categories developed for this analysis were complete bed - rest , wheelchair - bound , required cane or crutch or walker , or has none of the previous three restrictions .
information from hcfa form-485 on number of medications was categorized in this analysis as none , to 1 to 3 current medications ,
nutritional status was obtained from the hcfa form-485 notation of physician 's orders for special diets , and each individual was categorized as needing a
low sodium diet , another special diet , or no special diet .
functional limitations in each of the following 11 areas were available from hcfa form-485 : endurance , ambulation , respiration , vision , hearing , mental , bowel or bladder , speech , paralysis , contracture , or amputation .
the principal international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) ( public health service and health care financing administration , 1980 ) diagnosis was reported on hcfa form-485 , and the 16 most common diagnoses are included in this analysis .
the most common diagnosis was malignant neoplasms ( 11.5 percent of the clients ) , and the 16th most common diagnosis was chronic skin ulcers ( 2.5 percent of the clients ) .
the dependent variables were allowed visits and allowed charges , both in total and by type of service for the whole episode of care .
type of services included skilled nursing , home health aides , physical therapy , occupational therapy , speech therapy , medical social work , and other .
rates of service use were calculated both for the total sample and for the subset who received the specific type of service .
the data for this analysis were obtained in 1987 from a sample of the medicare - certified hhas that the health care financing administration ( hcfa ) had recruited in 1985 to participate in an hha prospective payment demonstration that was not implemented at that time .
these agencies had been recruited within 10 states ( california , connecticut , florida , illinois , massachusetts , ohio , pennsylvania , tennessee , texas , and wisconsin ) from the universe of known hhas in those states that met three inclusion criteria ( urban location , non - government operated , and established before january 1 , 1983 ) .
a total of 235 agencies met the criteria and were approached ; 127 expressed initial interest in participating in this data collection effort ; and of these , 86 agencies actually submitted data .
each agency submitted copies of hcfa forms-485 and 486 ( figures 1 and 2 ) for a sample of clients who began an episode of care in 1986 and submitted copies of the agency 's medicare cost reports for 1986 .
from each of the participating agencies , a systematic sample of clients was selected , designed to yield approximately 80 to 200 medicare hha clients per agency who were admitted for a new episode of care during calendar year 1986 . of those clients for whom data were received , only those who had verified medicare claim numbers ending in 0 , 4 , 5 , or 8 ( and , therefore , whose complete hha billing information would be available on the hcfa bureau of data management and strategy [ bdms ] hha 40-percent bill skeleton file ) and for whom data on allowed visits were actually received were considered conditionally eligible for this analysis ( unweighted n = 3,614 clients ) .
the sampling fraction for each hha varied ; consequently , the individual responses were weighted by the inverse of the sampling fraction .
the sample sizes indicated in the tables are the number of respondents ; the percents are based on the weighted data .
for each client admitted to hha care , hcfa forms-485 and 486 are completed to establish medical necessity for home health care and to document treatment plans and other aspects of the case .
hcfa forms-485 and 486 also include a certification from and to date . however , because of inconsistencies in agency practice , the from date on hcfa form-485 was found not necessarily to be the starting date of a new episode for every client .
therefore , for this analysis , the conceptual definition of a new episode was that the client had not been receiving hha services for the preceding 60 days . the operational definition of the start of a new episode relied on the hha 40-percent bill skeleton file in the following way .
date on hcfa form-485 , each patient 's billing record was examined retrospectively to identify a 60-day interval with no home health services . the day after this 60-day billing gap
was designated as the episode start date for this analysis . for nearly three - fourths of the conditionally eligible clients ( 72 percent ; unweighted n = 2,615 clients ) , the episode start date thus defined was identical to the from
date on hcfa form-485 ; and for another 7 percent ( unweighted n = 258 ) , the episode start date was within 5 days of the from date on hcfa form-485 .
these 2,873 clients comprise the analytic group for this analysis , and their data were appropriately weighted .
the end of an episode was defined as the last day of home health care following the start date that preceded another 60-day gap in the hha 40-percent bill skeleton file .
treatment plans for every 60-day period following the initial end date on hcfa form-485 are recorded on a series of hcfa forms-486 for each client .
the hha 40-percent bill skeleton file included all claims processed through september 1987 , which is 9 months after the start date of the last possible new episode during the study window of calendar year 1986 .
however , previous experience has shown that a 6-month lag in the processing of home health claims can exist , which could cause some distortions in exceedingly long episodes and some of the episodes that began in late 1986 .
table 1 presents the weighted distribution of episode lengths of stay according to this definition .
more than 40 percent of the medicare hha clients completed their home care within 30 days ( 41.2 percent ) , and another one - third completed their episodes within 30 - 60 days ( 30.9 percent ) .
although there were a few exceptions , the information provided on hcfa forms-485 and 486 is without missing data in nearly all fields . as figure 1 indicates , the variable living arrangement
it was not possible to categorize household composition on 46 percent of the cases , either because their episode was short and they did not have a hcfa form-486 or because nothing
the variable admission source had missing data in 1 percent of the records . for three variables ( morbidity restrictions , medications , and factual limitations ) , the category
none was inferred from the absence of a response ; because 3.6 percent , 5.2 percent and 1.3 percent , respectively , of these variables was judged consistent with expected prevalence rates , any problem resulting from missing data is assumed to be negligible , with the exception of living arrangement .
hcfa form-485 provided sex and age ( categorized as under 65 years of age , 65 - 74 years , 75 - 84 years , and 85 years of age or over in this analysis information ) . for the 54 percent with information on usual living arrangement , the categories were
admission source had four categories : from a hospital ( medical ) , from a hospital ( surgical ) , from a nursing home , or unknown .
mobility restrictions were categorized based on responses to the hcfa form-485 item indicating activities permitted ; the four categories developed for this analysis were complete bed - rest , wheelchair - bound , required cane or crutch or walker , or has none of the previous three restrictions .
information from hcfa form-485 on number of medications was categorized in this analysis as none , to 1 to 3 current medications ,
nutritional status was obtained from the hcfa form-485 notation of physician 's orders for special diets , and each individual was categorized as needing a low sodium diet , another special diet , or no special diet .
functional limitations in each of the following 11 areas were available from hcfa form-485 : endurance , ambulation , respiration , vision , hearing , mental , bowel or bladder , speech , paralysis , contracture , or amputation .
the principal international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) ( public health service and health care financing administration , 1980 ) diagnosis was reported on hcfa form-485 , and the 16 most common diagnoses are included in this analysis .
the most common diagnosis was malignant neoplasms ( 11.5 percent of the clients ) , and the 16th most common diagnosis was chronic skin ulcers ( 2.5 percent of the clients ) .
the dependent variables were allowed visits and allowed charges , both in total and by type of service for the whole episode of care .
type of services included skilled nursing , home health aides , physical therapy , occupational therapy , speech therapy , medical social work , and other .
rates of service use were calculated both for the total sample and for the subset who received the specific type of service .
approximately two of every three hha clients were female ( 62.6 percent ) , and 5.7 percent were under 65 years of age , whereas 21.9 percent were 85 years of age or over .
living arrangement was unknown for nearly one - half of the clients ( 45.3 percent ) ; but of the remainder , approximately one of every three lived alone ( 34 percent ) , nearly one of every two lived with a spouse ( 42 percent ) , and 24 percent lived in other arrangements . concerning the source of admission to the medicare hha
, about one of every five came directly from the community ( 19.6 percent ) ; three of every four came from a hospital , with approximately one - third having been surgical patients ( 32.0 percent ) and slightly more having been medical patients ( 44.0 percent ) ; whereas 3.4 percent had been discharged from a nursing home .
concerning their mobility restrictions , very few had none ( 3.6 percent ) , and the vast majority ( 79.1 percent ) relied on a cane or crutch or walker .
approximately 1 of every 10 ( 11.7 percent ) relied on a wheelchair , and 1 in every 20 ( 5.6 percent ) required total bed - rest .
the vast majority of clients were receiving some prescription medications ( only 5.2 percent were not ) .
nearly one - half ( 46.4 percent ) had no indication of a special diet , approximately one in every four ( 28.1 percent ) required a low sodium diet , and another one in every four ( 25.5 percent ) had another type of special diet required .
eleven categories of functional limitations were identified for each of the hha clients . the vast majority of the clients were reported to have endurance limitations ( 85.0 percent ) and ambulation limitations ( 80.6 percent ) .
the next most common limitations were reported for approximately one in every four clients ( vision limitations for 27.4 percent and respiration limitations for 26.7 percent ) .
slightly less than one of every five clients were reported to have hearing limitations ( 19.8 percent ) , mental limitations ( 18.3 percent ) , and bowel or bladder limitations ( 16.5 percent ) .
the remaining four limitations were reported for less than 10 percent of the clients : 7.5 percent had speech limitation , 5.9 percent had paralysis limitations , 3.3 percent had contracture limitations , and 2.8 percent had amputation limitations . simply summing across the number of limitations that any individual hha client
was reported to have , it is interesting to observe that the clients in general had multiple limitations . only 1 in every 10 ( 9.9 percent ) had only one limitation reported , and only 1.3 percent had no limitations ( these probably reflect incomplete hcfa form-485 completion ) .
nearly one of every three ( 31.3 percent ) had two limitations , and approximately one - third ( 28.7 percent ) had three limitations .
the last 30 percent had four limitations or more , including 4.9 percent who had six limitations or more .
five to 10 percent had heart disease - other ( 8.3 percent ) , cerebrovascular diseases ( 7.8 percent ) , digestive system disease ( 6.2 percent ) , arthropathies ( 5.3 percent ) , ischemic heart disease ( 5.2 percent ) , or diabetes mellitus ( 5.0 percent ) .
table 2 presents the distribution of allowed visits and charges for a total episode during 1986 for these medicare hha clients .
the vast majority of hha clients received skilled nursing visits ( 90.1 percent ) , whereas approximately one of every three received care form a home health aide ( 35.2 percent ) or a physical therapist ( 32.8 percent ) . the remaining types of skilled care were provided to much smaller percents of clients , ranging from 14 percent receiving medical social work services to 2 percent receiving speech therapy .
the second column of table 2 presents the mean number of visits during the whole episode by type of visit for the total sample of clients .
the mean number of visits for all clients was 22.71 , with more than one - half provided by skilled nurses ( 12.62 ) and one - fourth provided by home health aides ( 5.79 ) .
three of the services ( occupational therapy , speech therapy , and medical social work ) provided less than one visit on average to these clients .
the next column presents the mean number of visits for the subgroup that used the specific type of visit . among users , home health aides provided the highest number of visits per regimen at 16.48 on average , followed by skilled nurses at 14.00 and speech therapists at 11.45 per regimen .
both physical therapists and occupational therapists provided about 8 to 10 visits , on average , to the subset who received them .
medical social work was used both by a small percent ( 13.9 percent ) and in small amounts ( 2.04 visits per regimen ) .
the next column presents the mean allowed charges for all these clients , with an average total episode charge of $ 1,237.82 per client in 1986 .
the mean charges by type of service for all clients in large measure reflect the mean number of visits because the charges per visit were approximately equal ( averaging $ 57 to $ 59 per visit ) in four of the major types ( skilled nursing , physical therapy , occupational therapy , and speech therapy ) .
the two outliers were home health aide visits , which averaged less at $ 42 per visit , and medical social worker visits , which averaged more at $ 77 per visit .
notwithstanding that medical social workers had the highest charge per visit , on average , the last column of table 2 indicates that the mean charges for a regimen of medical social work therapy was by far the least expensive service at $ 156.70 compared with the next lowest average charge for the users of a specified therapy ( $ 498.52 for those receiving occupational therapy ) .
skilled nursing services had the highest mean charges at $ 819.83 for the users . table 3 presents the distribution among these medicare hha clients of allowed visits and allowed charges during a whole episode by the type of visit . inspecting the total visit column , the range varies considerably according to the type of visit the client received .
the mean number of total visits among all clients was nearly 23 visits . however , those receiving speech therapy received almost 54 visits on average , with 11 to 12 visits on average from the speech therapist contributing to their increased amount
. regimens of nearly 14 and 11 visits for skilled nursing and physical therapy also contributed to their 54-visit average .
an occupational therapy regimen of approximately 8 to 9 visits was also the norm of those receiving either speech therapy or occupational therapy , contributing to the total mean visits of 54 for the former and 47 visits on the average for the latter group .
for the 90 percent receiving skilled nursing services , their total mean visits was nearly 24 , with more than one - half their visits ( 14.00 ) provided by skilled nurses and over one - fourth ( 6.17 ) provided by home health aides .
the same patterns emerge when inspecting table 3 from the perspective of allowed charges , suggesting that variation in charges were minimal , as implied by the homogeneity of charges per visit for skilled nursing and physical , occupational , and speech therapy as mentioned previously .
those receiving skilled nursing services had mean allowed charges per episode of $ 1,286.56 , whereas those receiving occupational therapy and medical social work services had approximately twice the mean charges ( $ 2,665.42 and $ 2,259.49 , respectively ) , and those receiving speech therapy had even higher mean allowed charges per episode at $ 2,883.54 .
table 4 presents the distribution of allowed visits during an episode according to the characteristics of the client .
the patterns of allowed visits and charges as a function of the characteristics of the hha client are very similar because of the homogeneity of charges among four of the professional therapies .
furthermore , the large sample size lends itself to observing frequent statistical significance between client characteristic and number of specific services , as indicated by a chi - square analysis .
consequently , statistical significance is not indicated in this descriptive report , and the authors ' judgments concerning practical significance are .
inspecting table 4 for differences in total visits or types of visits associated with characteristics of the client suggests some interesting patterns .
the sex of the client was associated with both the number of allowed visits and the charges for an episode , with females receiving slightly more skilled nursing visits in particular and more visits in general .
an exception was observed with speech therapy , for which male clients received approximately two to three times more visits than female clients .
however , the prevalence of speech therapy is low even for males ( 0.39 versus 0.14 mean visits for males and females , respectively ) .
the age of the client also had a moderate association with the number of allowed visits and charges .
those under 65 years of age received fewer visits in general and fewer skilled nursing and home health aide visits in particular .
there were few differences in type or amount of services among the age subgroups of those 65 years of age or over .
those living alone received fewer total services ( 20.50 ) than either those living with spouse or in other specified households ( 24.15 and 22.89 , respectively ) . those living alone received fewer home health aide , physical therapy , occupational therapy , and speech therapy visits than the other household compositions , whereas those living with spouse received slightly more speech therapy services .
there were few differences in total services or type of individual services among those admitted from the community , from hospitals as medical patients , or from hospitals as surgical patients .
those whose admission source was unknown ( 1.0 percent of the clients ) had substantially lower total services ( 14.61 total mean visits versus 22.71 for the total sample ) .
those admitted from nursing homes ( again , a small subgroup at only 3.4 percent ) had 50 percent more total services , on average , than the other clients ( 34.29 versus 22.71 total mean visits ) , including substantial increases in home health aide and physical therapy visits compared with the other clients .
the relationship between the client 's mobility restrictions on volume and cost of allowed services was extremely variable .
those with wheelchair restrictions received substantially more services ( 37.72 mean total allowed visits compared with 22.71 for the total group ; total allowed charges of $ 2,070 compared with $ 1,238 for the total group ) .
as would be expected , those confined to bedrest had substantially less physical therapy visits and charges than the other clients .
occupational therapy visits were also substantially associated with mobility restrictions ; those with no mobility restrictions or those confined to bed received very few occupational therapy visits , whereas those requiring the ambulatory aids of canes , crutches , or walkers or those in wheelchairs received substantially more . those with no mobility restriction received substantially more skilled nursing services as well , contributing to a higher than average total mean visits ( 31.30 versus 22.71 ) .
the association between medications and the number of visits and charges was uniform and positive .
those receiving no medications received 13.15 visits , on average , and had $ 789 in total approved charges . those receiving 1 to 3 medications had an average of 20.68 visits and $ 1,133 total charges . those taking 4 to 6 medications had 23.69 total mean allowed visits and $ 1,302 in total charges . those receiving 7 medications or more had 25.61 total mean visits and $ 1,356 in total charges .
the nutritional status of the client also was associated with the allowed visits and charges in a uniform fashion .
total visits ranged from nearly 21 , to 24 , to nearly 25 for those with no special diet , low sodium diets , and other special diets , respectively .
total costs reflected the same trend , at $ 1,137 , $ 1,307 , and $ 1,346 for the three groups , respectively .
the functional limitations of the clients also were associated with the allowed visits and charges for these hha clients .
home health aide services were elevated for those with bowel or bladder limitations or paralysis limitations .
physical therapy services were elevated for those with paralysis limitations or amputation limitations and reduced for those with respiration limitations . occupational therapy visits and charges
were elevated for those with paralysis limitations or speech limitations , whereas speech therapy was elevated principally for those with speech limitations and secondarily for those with paralysis limitations .
overall , there seem to be three clusters of total allowed costs among the types of reported functional limitations .
six of the functional limitations were associated with total charges between $ 1,158 and $ 1,317 for the total episode ( endurance , ambulation , vision , respiration , hearing , and mental limitations ) .
it is also interesting to observe that these are the six most prevalent limitations reported as well .
four of the remaining limitations have total allowed episode charges that cluster between $ 1,404 and $ 1,603 ( bowel or bladder limitations , speech limitations , amputation limitations , and contracture limitations ) .
the last group , those with paralysis limitations , had the most costly average episode at $ 2,154 , with elevated costs for home health aides , physical therapy , occupational therapy , and speech therapy .
the number of these limitations reported per client had a slight linear association with increases in total services , with the exception that the small percent ( 1.3 percent ) who had no limitations recorded had total services at approximately 1 times the mean level for the whole group ( 34.11 visits versus 22.71 respectively ) .
this finding supports the contention that those cases with missing information on functional limitations were more likely to be incomplete records than clients without limitations . excluding those without recorded limitations , the greatest increment in approved visits and charges was between those with one limitation versus those with two limitations , with total allowed visits ranging from 15.03 to 22.80 and total allowed charges ranging from $ 816 to $ 1,236 as a function of the second limitation .
the association of principal diagnosis with total allowed services and allowed costs is interesting . those with a diagnosis of cerebrovascular disease had the highest total charge per episode at $ 1,791 .
those with either diabetes mellitus , circulatory system disease , or malignant neoplasms as the primary diagnosis formed a cluster with the fourth most costly episode of care ranging from $ 1,353 to $ 1,404 . those with a diagnosis of heart disease - other followed at $ 1,267 per episode , followed by those with central nervous system disease at $ 1,176 per episode .
a cluster of five diagnoses followed , with total episode charges between $ 1,023 and $ 1,096 ( bone fractures , hypertension , genitourinary system disease , injuries or poisoning , or chronic skin ulcers ) .
the least expensive clustering had episode charges from $ 892 to $ 955 and includes those with diagnoses of ischemic heart disease , chronic obstructive pulmonary disease , digestive system disease , or arthropathies .
for those with a principal diagnosis of cerebrovascular disease , the amounts of physical therapy , occupational therapy , and speech therapy were considerably elevated comparable with those with other diagnoses .
those with hip fractures , arthropathies , or bone fractures also had elevated physical therapy charges compared with other diagnoses .
the purpose of this analysis was to quantify systematically the characteristics of medicare home health care clients , the parameters of a total episode of care , and the bivariate associations that might exist between client characteristics and components of care .
the data available for this purpose have some weaknesses that limit some of the generalizability and utility of the findings .
the available data were designed to facilitate service delivery and payment , not for the exploratory research effort to which they were put .
furthermore , only a third of the eligible universe ( 37 percent , 87 out of 235 hhas ) provided data for this analysis . hence , the generalizability may be limited because the data came only from hhas willing to participate in a demonstration project .
checking all that apply for functional limitations requires that the analysts draw an inference from a non - response . hence
, both an intended non - response and an erroneous omission can not be distinguished .
the impact of this technical deficiency is presumably minimal , however , because of the low level of erroneous omissions ( i.e. , missing data ) in other items .
the other example of a technical deficiency was in the wording of unusual home or social environment previously mentioned , which precluded the straightforward elicitation of living arrangement and produced missing data in 46 percent of the records .
consequently , the associations reported with living arrangement must be interpreted with considerable caution . notwithstanding
those technical difficulties , hcfa forms-485 and 486 are particularly helpful to this kind of policy research because these forms have continuing use in the medicare home health care program .
this continuing use of hcfa forms-485 and 486 facilitate subsequent policy analyses at different times , at different sites , or with different contexts , but with the same data elements . in summary ,
the descriptive profile of the model client was : female , 75 - 84 years of age , living with a spouse , admitted from a hospital , used ambulation aids , took 5 prescription medications , had no special diet , had 2 functional limitations ( commonly endurance and ambulation limitations ) , and had cancer and/or heart disease .
the total episode was approximately 23 visits , including 13 skilled nursing visits , 6 from home health aides , and 3 from physical therapists .
the mean allowed charges for the total episode were $ 1,238 ( 1986 dollars ) .
the age and sex of the client had a slight association with allowed visits and charges , with those under 65 years of age and those who were male receiving fewer allowed services per episode than those 65 years of age or over or females .
those living with a spouse received more speech therapy , possibly suggesting an increased awareness of communication needs for these clients . those admitted from a nursing home had greatly increased level of services , particularly home health aide and physical therapy services .
medication use exhibited a systematic linear association with total services and allowed charges for these medicare clients .
nutritional status inferred from dietary requirements also was associated with hha total service use , with those on no special diet likely to receive fewer services per episode . both the specific type and the total number of limitations of the clients were associated with the total services provided per episode . with the exception of the few cases with no limitations listed , the association between total number of limitations and total services per episode was linear .
although those with paralysis limitations had by far the most costly care per episode , those with bowel or bladder limitations , speech limitations , amputation limitations , or contracture limitations had less but nevertheless substantially elevated total charge per episode compared with those with endurance , ambulation , vision , respiration , hearing , or mental limitations .
lastly , some of the principal diagnosis correlated with total service use , as follows . those with cerebrovascular disease as the primary diagnosis had the highest total charge per episode , followed by those with hip fractures and those with either diabetes mellitus , circulatory system disease , or malignant neoplasm .
those with heart disease - other had the next most costly episodes , followed by those with diseases of the central nervous system .
the second least costly episodes were found among those with primary diagnoses of either bone fracture , hypertension , genitourinary system disease , injuries or poisoning , or chronic skin disease .
the lowest total charges per episode were found among those with diagnoses of ischemic heart disease , chronic obstructive pulmonary disease , digestive system disease , or arthropathies .
the variability of charges and the definable service arrays among identifiable subgroup of hha clients generate cautious optimism that a multivariate case - mix model may be developed that can render subgroups of these clients homogenous with respect to charges ( branch and goldberg , 1993 ) .
the next step following case modeling or identifying homogenous subgroups with respect to charges or some other utilization - based measure is to consider prospective payment models or capitation models based on characteristics of the client as he or she enters the medicare home health care program .
based on the findings from these analyses in general and an inspection of the total authorized visits per episode reported in table 4 in particular , a prospective payment model to medicare hhas might incorporate the following steps : first , determine the mean total episode approved charges for an index year .
second , offer a prospective payment to certified medicare hhas based on an inflation - adjusted mean charge , modified by the following client characteristics present at intake : a 33-percent increment for those admitted from a nursing home . a 33-percent decrement for those under age 65 . a 50-percent increment for those confined to a wheelchair .
a 25-percent increase for those with a principal diagnosis of hip fracture . a 25-percent decrease for those with a principal diagnosis of digestive system disease . a 25-percent decrease for those with a principal diagnosis of arthropathies .
third , allow the hha to exclude post facto from the modified prospective payment a predetermined percentage of outliers ( perhaps up to 3 percent ) for adjudicated fee - for - service payments .
a capitation model could similarly incorporate modifiers to the basic compensation rate based on these demonstrated variations in the total episode of care , but additional analysis would be necessary to apply the modifier to the total eligible population and not just to the subset of hha users as suggested in the prospective payment model . | the purpose of this study was to present descriptive information on the characteristics of 2,873 medicare home health clients , to quantify systematically their patterns of service utilization and allowed charges during a total episode of care , and to clarify the bivariate associations between client characteristics and utilization.the model client was female , 75 - 84 years of age , living with a spouse , and frail based on a variety of indicators .
the mean total episode was approximately 23 visits , with allowed charges of $ 1,238 ( 1986 dollars ) .
specific subgroups of clients , defined by their morbidities and frailties , used identifiable clusters of services .
implications for case - mix models and implications for capitation payments under health care reform are discussed . |
a 21-year - old woman visited our department complaining of a large painless swelling in the left submandibular region .
on palpation , the swelling revealed a soft , painless , and fluid - containing mass .
the patient had no traumatic or surgical history , and the swelling did not cause difficulty in swallowing or speaking .
contrast - enhanced computed tomography ( ct ) scan demonstrated a large rim - enhanced fluid attenuation mass occupying both sublingual spaces with an anterior connection ( fig .
the lesion extended into the left parapharyngeal space superiorly and compressed the left submandibular gland inferiorly ( fig .
anteriorly , it extended to the right sublingual space in a horseshoe shape ( fig .
at the lower level of the lesion , several linear septa were noted ( fig .
1c ) . a fluid - fluid level , which is the interaction between two fluids with different viscosities , was also noted ( fig .
although the septation and fluid - fluid level within the lesion made the differential diagnosis from a cystic hygroma difficult , considering the location of the lesion in the sublingual space , it was diagnosed as a plunging ranula . under general anesthesia , an incision was made in the left lingual vestibule , and excision of the lesion along with extirpation of the left sublingual gland was performed . at surgery
, the cystic lesion was found to be filled with a viscous and yellowish mucous fluid .
after removal of the left sublingual gland , a cut - down tube was inserted into the middle portion of the left wharton 's duct .
the histopathologic examination of the specimen from the sublingual gland revealed ruptured acinar cells ( fig .
the cut - down tube inserted into the left wharton 's duct was removed after 2 weeks .
the descriptive term ranula normally refers to a bluish , transparent , thin - walled swelling in the floor of the mouth .
while simple ranulas are all confined to the sublingual space , plunging ranulas are centered on the submandibular space and tend to spill into one or more adjacent spaces.7 the nature of ranulas was unknown until 1956 , when bhaskar et al8 investigated the pathogenesis histopathologically and experimentally , and they concluded that ranula was produced by the extravasation of saliva from a damaged salivary sublingual gland and was not lined by epithelium . the occurrence of ranula is rare , and the reported male - to - female ratio is 1 : 1.3 , without significant side preference.9 the plunging ranula most frequently occurs in the second and third decades of life , with an age range from 3 to 61 years , and most commonly involves the right side.10,11 most giant plunging ranulas are located from the ipsilateral sublingual space to the parapharyngeal space . however , the present case of plunging ranula passed the midline and extended to the contralateral sublingual space . based on our review of the english literature ,
only two cases of huge plunging ranula which passed the midline have been reported.11,12 the previous cases which passed the midline showed an extension into the cervical space inferiorly , but the present case did not extend beyond the submandibular gland . a rare case of plunging ranula with subtle septa
these septa might have been secondary to surgical procedures or a traumatic history . in those cases ,
wall thickening and minimal internal septa formation were noted.13,14 a trauma or a surgery might lead to the development of scar tissue , and it might be detected as a subtle septation within the plunging ranula . in the present case ,
although no previous trauma or surgery was found by history taking , it might be expected that the patient was unaware of the previous trauma considering the fluid - fluid level within the lesion .
a fluid - fluid level has , in rare cases , been reported to occur in the plunging ranula and refers to the interaction between two fluids with different viscosities , commonly by hemorrhage.13 the blood vessels near the submandibular gland area are weak ; therefore , the fluid - fluid level might easily result from trauma to the area .
cystic hygroma rarely presents in the sublingual space , but if present , it reveals very similar images on ct scan to those of a plunging ranula . for cystic hygromas , the mainstay of treatment is surgical excision . the surgical team should attempt to completely remove the lymphangioma or to remove as much as possible , sparing all vital neurovascular structures.15 treatment of a plunging ranula is also surgical excision , but the difference is the complete excision of the sublingual gland .
there are several treatment options for ranula , which include excision of only the ranula and marsupialization . however , excision of the entire sublingual gland and ranula reported the lowest recurrence rate of 1.55% .
as plunging ranula and cystic hygroma are managed with different surgical approaches , the differential diagnosis is critical.14 cystic hygroma arises from the abnormal development of fetal lymphatic tissue .
it typically presents early in life , with 50% present at birth and 90% evident by 2 years of age .
cystic hygroma is characteristically infiltrative in nature and typically involves contiguous anatomic regions in the neck .
it appears as defined but poorly circumscribed , lobulated , septated , homogeneous , and with fluid attenuation on ct images .
a previously infected lesion may show heterogeneous attenuation due to previous hemorrhage or high protein content . in case of hemorrhage
therefore , the differential diagnosis between plunging ranula and cystic hygroma is often difficult on imaging .
the characteristics of cystic hygroma for lobulation , strong septation , and rarity in the sublingual space should be helpful for diagnosis . in our case , the histopathologic examination demonstrated ruptured acinar cells of the sublingual gland , which confirmed the diagnosis of the plunging ranula .
the recurrence rates of ranula were not related to the swelling patterns and surgical approaches , but intimately related to the methods of surgical procedure.16 effective treatment is removal of the involved sublingual gland or inducing sufficient fibrosis to seal the leak through which mucus extravasates.17 in the present case , the plunging ranula and ruptured sublingual gland were removed and the patient showed no recurrence 6 months postoperatively . in conclusion , as plunging ranula and cystic hygroma are managed with different surgical approaches , it would be important to differentiate them radiologically .
although a submandibular cystic lesion with septa and a fluid - fluid level would be unusual for a ranula , a plunging ranula should be considered in the differential diagnosis along with a cystic hygroma . | a ranula is a bluish , transparent , and thin - walled swelling in the floor of the mouth .
they originate from the extravasation and subsequent accumulation of saliva from the sublingual gland .
ranulas are usually limited to the sublingual space but they sometimes extend to the submandibular space and parapharyngeal space , which is defined as a plunging ranula .
a 21-year - old woman presented with a complaint of a large swelling in the left submandibular region .
on contrast - enhanced ct images , it dissected across the midline , and extended to the parapharyngeal space posteriorly and to the submandibular space inferiorly .
several septa and a fluid - fluid level within the lesion were also demonstrated .
we diagnosed this lesion as a ranula rather than cystic hygroma due to the location of its center and its sublingual tail sign .
as plunging ranula and cystic hygroma are managed with different surgical approaches , it is important to differentiate them radiologically . |
childhood disintegrative disorder ( cdd ) , also known as heller 's syndrome and disintegrative psychosis , is a rare condition characterized by late onset ( > 3 years of age ) of developmental delays in language , social function , and motor skills .
it is a complex disorder that affects many different areas of the child 's development .
it is grouped with the pervasive developmental disorders ( pdds ) and is related to the better known and more common disorder of autism .
initially cdd was considered strictly a medical disorder and was believed to have identifiable medical causes . after researchers reviewed the reported cases of cdd ; however , no specific medical or neurological cause was found to account for all occurrences of the disorder .
for that reason , cdd was included in the fourth edition of the diagnostic and statistical manual of mental disorders , or dsm - iv , in 1994 .
research findings suggest , however , that it may arise in the neurobiology of the brain .
eegs measure the electrical activity in the brain generated by nerve transmission ( brain waves ) .
cdd is also sometimes associated with seizures another indication that the neurobiology of the brain may be involved .
children with cdd have at least 2 years of normal development in all areas language understanding , speech , skill in the use of large and small muscles , and social development . after this period of normal growth
this loss usually takes place between ages 3 and 4 , but it can happen any time up to age ten .
the prevalence of cdd is 1 in 100,000 boys and ratio of boys to girls is estimated to be 8 boys to 1 girl .
a female child aged 10 years came with complaint of irritable behavior , and communication problem .
the child is a product of consanguineous marriage wherein her father is her mother 's maternal uncle .
the child is born at full term normal vaginal delivery , no intranatal maternal infections , no complications after birth , the child attained age appropriate motor and language milestones till 5 years of age .
she also used to take bath all by herself with soap and water under supervision . at the age of 4 years the patient developed a severe attack of upper respiratory tract infection for which she suffered with fever and cough for 6 months and had pleural effusion for which the fluid is drained .
the parents were instructed to use medications for 1 month with regular follow ups but they could not do so due to financial restraint .
the patient from then on started to get severe attacks of fever and was only treated by an unqualified health worker .
she stopped going to school and started to lose all the communicative milestones like talking sentences , calling her family members by name .
she used to sit alone all day self - absorbed in play and showed increased anger and irritability , wherein she would hit or bite anyone who disturbed her .
she also stopped taking personal hygiene and used to pick up bits of sticks and stones from floor and put in her mouth .
she also did not sleep all night and used to cry for no apparent reason .
she even lost the toilet training she acquired previously and started to pass stools and urine in clothes . with the above complaints the patient was brought to mamata general hospital to the psychiatric op .
the patient was admitted in psychiatry ward all the investigations done like complete blood picture to rule out and blood dyscrasias , liver function tests for any metabolic abnormalities , blood urea and creatinine for renal abnormalities , urine test for sugar and proteinuria .
computerized tomography of brain showed marked reduction of brain volume with less sulci and gyri and enlarged ventricles .
the patient was started on resperidon 1 mg once a day and over the next 3 week she improved , the symptoms of anger and irritability have reduced and she also started to sleep normally .
her motor coordination improved , and she was able to feed herself which she previously was not able to .
on subsequent follow - ups for the next 6 months child has shown improvement in communication in the form of naming objects and also her social interaction also improved wherein she started to play with other children at her home .
she has been referred to a higher center wherein she is being given special training to improve her cognitive abilities .
the child is scheduled for clinical interview for every 2 months with the above medication and training to assess the improvement . till
now 3 follow - up visits have take place and substantial improvement was found in above - mentioned areas .
cdd is most commonly diagnosed when the parents of the affected child consult the pediatrician about the child 's loss of previously acquired skills
. the doctor will first give the child a medical examination to rule out any organic cause to explain the condition .
following the medical examinations and tests , the child will be referred to a psychiatrist who will then make the differential diagnosis of cdd . to be diagnosed with cdd , a child must show loss or regression in at least two of the areas listed below with an apparently normal development for at least first 2 years after birth .
these are
receptive language skills ( language understanding)expressive language skills ( spoken language)social skills or adaptive behaviorsplay with peersmotor skillsbowel or bladder control , if previously established .
receptive language skills ( language understanding ) expressive language skills ( spoken language ) social skills or adaptive behaviors bowel or bladder control , if previously established .
the child should have abnormal functioning in at least two of following :
impaired nonverbalbehaviors , failure to develop peer relations with no social and emotional reciprocityin ability to start and maintain conversations with other people etc.)restricted , repetitive and stereotyped behavior , such as bobbing the head up and down , or other repeated movements .
these changes must not be caused by a general medical condition or another diagnosed mental disorder .
impaired nonverbalbehaviors , failure to develop peer relations with no social and emotional reciprocity in ability to start and maintain conversations with other people etc . ) restricted , repetitive and stereotyped behavior , such as bobbing the head up and down , or other repeated movements .
these changes must not be caused by a general medical condition or another diagnosed mental disorder .
speech and language therapy , occupational therapy , social skills development , and sensory integration therapy may all be used according to the needs of the individual child .
currently , there are no pharmacological interventions that specifically target the core symptoms of pdd .
however , studies have demonstrated that atypical antipsychotics and selective serotonin reuptake inhibitors may be beneficial for behavioral problems associated with pdd .
two reports have been published about the use of atypical antipsychotics in children with pdd . in both studies ,
treatment with atypical anti psychotics led to significant improvements in aggressive , self - injurious , and disruptive behavior .
many children with pdd have motor clumsiness in addition to social deficits . in this case study ,
dopaminergic dysfunction has been reported to be associated with decreased motor coordination and given that a typical antipsychotics can be thought of as a dopamine system stabilizer , this may represent the mechanism of therapeutic action .
atypical antipsychotics have also been reported to improve depression and anxiety and this therapeutic effect of is mediated by a potent partial agonistic effect at the 5ht1a receptor .
similar to their findings , the aggressive behavior was greatly reduced in this case after treatment .
it should still be observed that this effect was lost and then reestablished with the discontinuation and reintroduction of this treatment respectively . | we are presenting a case of a 10-year - old female child who presented with normal development till 5 years of age followed by deterioration in previously acquired language and social skills with stereotypic hand movements suggestive of childhood disintegrative disorder .
this case is reported as this condition is very rare . |
anal fissure is one of the most common and painful proctologic diseases that is effectively treated and prevented with conservative measures in its acute form , whereas chronic fissures may require medical or surgical therapy .
because of the disability associated with surgery for anal fissure and the risk of incontinence , medical alternatives for surgery have been sought .
most recently , pharmacologic approaches that relax the anal smooth muscle , to accomplish reversibly what occurs in surgery , have been used to obtain fissure healing [ 13 ] .
glyceryl trinitrate ointment ( 0.2% ) has an efficacy of up to 68% in healing chronic anal fissure , but it is associated with headache as the major and most common side effect .
diltiazem , a calcium channel antagonist used in the treatment of angina , hypertension , and arrhythmias , achieved healing of chronic anal fissure comparable to 0.2% glyceryl trinitrate ointment but caused fewer side effects .
concerning new therapeutic application of diltiazem , validated and stability - indicating methods should be available to determine this drug in topical preparations .
the united states and british pharmacopeias present two reversed - phase hplc methods using d-10-comphorsulfonic acid and n , n - dimethyloctylamine , respectively , as amine modifiers suitable for the quantitative analysis of diltiazem in raw material and tablet formulation .
however , the use of such ionic additives has fallen somewhat out of favor , as these can have some undesirable effects , including the difficulty of their removal from the stationary phase after use , leading to permanent alteration of its properties and even chemical reaction with some solute types . moreover ,
no method was compiled in these pharmacopoeias for the analysis of this medicine in topical preparations .
in addition , literature survey reveals little information about the quantitative determination of diltiazem in topical formulations and there is only one report dealing with the analysis of diltiazem in a transdermal gel which has not been validated according to international conference on harmonization ( ich ) stability - indicating guidelines .
furthermore , in all of the previously reported hplc methods for determination of diltiazem in pharmaceutical or biological matrixes with uv detection , acetonitrile or methanol has been used as a part of mobile phase or extraction procedure [ 713 ] .
it is worth mentioning that these solvents are ranked by us environmental protection agency ( epa ) as hazardous solvents and because of their inherent toxicity , safe detoxification of the waste solvents is essential , which may lead to high to very high disposal costs . in recent years
, green analytical chemistry has recognized momentum not only in the academic world but also in industrial and pharmaceutical laboratories .
green analytical techniques aim to minimize or eliminate the hazardous waste associated with analytical methods [ 1620 ] .
moreover , from an economic point of view , it is reasonable to replace acetonitrile by other solvents ( preferably ecofriendly ones ) because of its worldwide shortage [ 2124 ] .
testing laboratorie , who wish to comply with the requirements of iso / iec 17025:2005 need to estimate uncertainty of measurement for their quantitative methods .
estimation of uncertainty leads to better measurement reliability , renders data from interlaboratory studies comparable , and helps to assess the statistical significance of the difference between the measurement and a relevant reference value [ 25 , 26 ] .
although few methods for the determination of diltiazem in pharmaceutical matrixes have been reported , information on such metrological parameters is scarce [ 79 , 11 ] .
taking ich guidelines into consideration , the present study describes a simple , validated , and stability - indicating analytical method for the determination of diltiazem in pharmaceutical preparations , which meets the green aspects in analytical chemistry .
also , the calculation of the measurement uncertainty which is based on the validation of the analytical procedures in a laboratory is presented .
moreover , the performance of the method was evaluated and its potential for the determination of diltiazem in pharmaceutical preparations was investigated .
qualified diltiazem hydrochloride standard ( 99.50% ) and pharmaceutical grade diltiazem hydrochloride were kindly provided by arya pharmaceuticals ( tehran , iran ) .
all solvents and reagents were of gradient and analytical grade , respectively , and were purchased from merck ( darmstadt , germany ) .
identification test of desacetyl diltiazem was performed with ir and nmr spectrometry ( data not shown ) .
hplc - grade water was obtained through a milli - q system ( millipore , milford , ma , usa ) and was used to prepare all solutions . the pharmaceutical formulations ( gel 2% ) and the corresponding placebos ( mixture of all the excipients )
stock standard solution of diltiazem was prepared in methanol at a concentration of 1 mg / ml .
this solution was found to be stable for at least 1 month , when it was stored at 28c .
freshly prepared working standards at concentration levels of 0.5 , 1 , 5 , 10 , 20 , and 50 g / ml were obtained from stock solution by the appropriate dilution in hplc - grade water .
a 1.01.5 g portion of gel ( equivalent to 2030 mg of diltiazem ) was transferred into a 25 ml volumetric flask .
then , 15 ml of hplc - grade water was added to this portion and the solution was sonicated for 10 min .
thereafter the volume was adjusted to the mark with the same medium to provide a theoretical concentration of 8001200 g / ml of diltiazem .
this solution was again diluted with hplc - grade water to make final concentration of 812 g / ml .
the same procedure was applied to placebo to be sure about the selectivity of the method .
the hplc method was carried out on a shimadzu hplc system ( shimadzu , kyoto , japan ) , set to recycle the mobile phase and was equipped with an scl-10avp system controller , lc-10 advp pump , dgu-14a degasser , and a spd - m10avp pda detector .
the peak areas were integrated automatically by computer using a shimadzu class vp software program .
a 20 l volume of sample was introduced into a rheodyne model 7725i injector .
the elution was carried out on a c18 column ( 250 mm 4.6 mm , 5 m particle size ) from hector ( daejeon , south korea ) .
all analyses were performed at the column temperature of 50 1c under isocratic conditions with a mobile phase of ethanol : phosphoric acid solution ( ph = 2.5 ) ( 35 : 65 , v / v ) and a flow rate of 2.0 ml / min , using pda detection at 240 nm .
the stability - indicating capability of the method was determined by subjecting diltiazem solutions ( standard and pharmaceutical preparations ) at the concentration level of 100 g / ml to accelerated degradation by acidic , basic , heat , oxidative , and photolytic conditions to evaluate the interferences in the quantitation of diltiazem .
sample solutions prepared in 1 m hydrochloric acid and 1 m sodium hydroxide were used for the acidic and basic hydrolysis , respectively .
both solutions were heated at 70c for 12 h and then neutralized with basic or acidic solutions , as necessary . for evaluating the heat condition ,
the sample solutions were heated at 70c for 12 h. for oxidative degradation , sample solutions were exposed to a solution of hydrogen peroxide ( 3% ) and kept at ambient temperature for 4 h , protected from light .
photodegradation was induced by exposing the sample solution to uv - lamp at a wavelength of 254 nm placed in a wooden cabinet for 4 hours .
the solutions were diluted with hplc - grade water to final concentration of 10 g / ml and were injected into chromatograph .
linearity was evaluated by determining six working standard solutions at a concentration range of 0.550 g / ml .
slope , intercept , and coefficient of determination ( r ) of the calibration curves were calculated to ascertain linearity of the method .
the limit of quantification ( loq ) was defined as the lowest concentrations with the rsds lower than 5% and accuracies within 5% , considering at least ten times the response compared to that of the blank . in order to check the robustness ,
the conditions studied were flow rate ( altered by 0.2 ml / min ) , column temperature ( altered by 2c ) , and ph of phosphoric acid solution ( altered by 0.2 ) .
these chromatographic variations were evaluated for resolution between diltiazem and desacetyl diltiazem , % assay of the drug , theoretical plates , and tailing factors of the peaks . for method repeatability ,
assay of working standard solutions ( 0.5 , 5 , 10 , and 50 g / ml ) was repeatedly performed five times on the same day ( intraday ) . for reproducibility ,
freshly prepared solutions at aforementioned concentration levels were analyzed at different days ( interday ) and results were statistically evaluated in terms of % rsd . for recovery studies ,
0.125 g portions of preassayed diltiazem gel 2% were spiked with extra 0.5 , 1 , and 2 ml of stock standard solution .
these samples were handled as explained in section 2.1.2 and the final target levels of 12 , 14 , and 18 g / ml were prepared .
to assess accuracy , freshly prepared placebo of the diltiazem pharmaceutical formulations was spiked with various amounts of diltiazem to obtain the concentration levels of 0.5 , 5 , 10 , and 50 g / ml .
selectivity of this method was indicated by the absence of any excipient interference at retention times of the peaks of diltiazem .
the absence of interfering peak was evaluated by injecting a blank sample consisting of diluent and placebo .
the double check of the lack of interferences of the resulting by - products with the elution of the peaks of diltiazem was done by calculating the f factor , meaning the ratio of the uv molar absorption coefficients of diltiazem at the 240 ( peak ) and 260 ( valley ) nm , respectively , using
( 1)f = a(240)a(260 ) ,
where a(240 ) and a(260 ) are the diltiazem peaks areas obtained at 240 and 260 nm , respectively .
the resulting f factors were compared with that of the standard . moreover , the uv spectrum of each diltiazem peak was acquired during the appearance of the peak in the chromatogram .
an expanded uncertainty budget was constructed for diltiazem in pharmaceutical preparations by the rp - hplc - pda method according to previously reported procedures [ 26 , 3032 ] .
the uncertainty of the measurement standard is calculated by the quadratic addition of two terms : the uncertainty certified by manufacturer ( ustock ) and the uncertainty corresponding to its preparation by dilution or weighting ( upreparation ) :
( 2)ustandard(%)=ustock2(%)+upreparation2(% ) .
the stock uncertainty ( ustock ) is calculated from a value given by the manufacturer using
( 3)ustock(%)=(100p%)3 ,
where the purity is expressed as p% . when there are independent standard preparations at each concentration level , the upreparation term could be eliminated . in this case , the contribution of this term is included in the upreparation term [ 26 , 30 ] .
this value is calculated using
( 4)ucalibration = sx0x0 ,
where x0 is the concentration calculated from the calibration curve and sx0 is the standard deviation of the concentration , obtained from the calibration curve using
( 5)sx0=s(r)m1n+1n+(0)2m2i=1n(xix)2,s(r)=(yimxib)2n2 ,
where m is the slope of the line , b is the y - intercept of the line , n is the number of replicate unknowns , n is the number of the standards , -0 is the mean of n repeat measurements of y for the sample , - is the mean of the y values for the calibration standards , xi are the concentrations of the standards , and x- is the average concentration of the standards .
this value is calculated using
( 6)uprecision = sx0n ,
where s is the standard deviation of the experimental data for precision and n is the number of assays .
this parameter is calculated using ( 7 ) :
( 7)uaccuracy = s()n ,
where s( ) is the relative standard deviation of the recovery and n is the number of assays .
the value of the expanded uncertainty was calculated according to iso gum guidelines using ( 8) :
( 8)u = kcustandard2+ucalibration2+uprecision2+uaccuracy2 ,
where u is expanded uncertainty , k the coverage factor ( for confidence interval 95% , k = 2 ) , and c is the concentration of the drug .
the hplc procedure was optimized with a view to develop a green stability - indicating assay method while keeping the system suitability necessities according to the united states pharmacopeia , which needs the resolution between diltiazem and desacetyl diltiazem and the number of theoretical plates for the diltiazem peak to be greater than 3 and 1200 , respectively . in order to follow the first principle of green chemistry ,
acetonitrile was replaced by ethanol in the previously reported mobile phase containing acetonitrile : sodium phosphate monobasic monohydrate buffer ( ph 2.5 , 0.02 m ) ( 33 : 67 v / v ) , which was used for the analysis of diltiazem in a transdermal gel . although ethanol has some shared or similar characteristics to methanol and acetonitrile , including complete miscibility with h2o , availability in the high purity required for hplc and low chemical reactivity with most sample species as well as with hplc , instrument and column surfaces , it has some characteristics that are less favorable .
one of them is that the viscosities of ethanol : h2o solutions are higher than those of methanol : h2o and acetonitrile : h2o solutions of the same elution strength at room temperature . to cope with this problem
this change resulted in lower chromatographic pressure , better peak shapes , good separation of diltiazem and desacetyl diltiazem , and suitable theoretical plates .
however , the retention times were not satisfactory when the flow rate was 1 ml / min . therefore , this parameter was increased to 2 ml / min to resolve the aforementioned problem . according to these preliminary results , the detection wavelength of 240 nm , flow rate of 2 ml / min and the mobile phase of phosphoric acid solution ( ph = 2.5 ) : ethanol ( 65 : 35 , v / v ) , and the column temperature of 50c were selected . under the chromatographic conditions of this method , desacetyl
diltiazem and diltiazem were separated completely from each other and their peaks appeared at 4.2 and 5.8 min , respectively ( figure 2 ) , the theoretical plates for diltiazem peak was 11511.26 0.33% , the resolution between diltiazem and its impurity was more than 9 , and the tailing factor for diltiazem peak was 1.1 1.30% .
the values of theoretical plates obtained in this study are much higher than those reported previously ( 1863 4.65 ) which indicates better column efficacies at the chromatographic conditions used in this study .
before fully implemented in the quantitative determination of drug substance and pharmaceutical preparation , this method was thoroughly validated according to ich guidelines .
stability - indicating method is defined as an analytical method that accurately quantifies the active ingredients without interference from degradation products , process impurities , excipients , or other potential impurities .
diltiazem showed drastic degradation in acidic , basic , and photolytic conditions ; in that only 16.67 1.95 , 10.47 2.10 , and 48.86 1.48% of the drug remained , respectively , and at the same time a remarkable increase in the concentrations of desacetyl diltiazem was observed ( figures 3(c ) , 3(d ) , and 3(f ) ) .
the first - order hydrolysis of diltiazem in the presence of acids and bases and formation of desacetyl diltiazen in these conditions has been reported previously , which confirms the results of this study .
showed that the main photoproduct of diltiazen after irradiation of uv - a and uv - b is diltiazem - s - oxide .
however , according to the findings of the present study the main photoproduct after exposure of an aqueous solution of diltiazem to uv - c is desacetyl diltiazem , indicating a different photodegradation process . under the oxidative and heat conditions ,
the diltiazem contents decreased to 89.01 1.74 and 84.57 0.86% , respectively , with a minor increase in the amounts of desacetyl diltiazem ( figures 3(b ) and 3(e ) ) .
the degradation products of the parent compound were found to be similar for both the pharmaceutical and standard solutions .
specificity is the ability of the method to unequivocally assess the analyte response in the presence of its potential impurities that was illustrated by the complete separation of diltiazem from degradation products as shown in figure 3 .
furthermore , the decreases observed in diltiazem contents in stability studies , when degradation products appeared , proved the specificity of the method ( table 1 ) . also , the f factors of the diltiazem in stress solutions ( 3.523.55 ) show the same values of that of the standard ( 3.55 ) .
the uv spectrum obtained during the appearance of the peaks also confirms their purity ( figure 4 ) .
consequently , the forced degradation studies documented the stability - indicating power and specificity of the proposed method .
the application of the whole procedure to placebo samples in order to verify the method selectivity demonstrates that no interferences were detected ( figure 5(b ) ) .
linearity was determined by constructing five independent calibration curves , each one with six calibration points of diltiazem , including the loq , in the range of 0.550 g / ml . the peak areas of diltiazem against the respective concentrations were used for plotting the graph , and the linearity was evaluated by the least square regression analysis .
the linearity curve was defined by the following equation : y = 44446.17x + 5597.38 ( r = 0.9996 ) , which indicated the linearity of the calibration curve for the method . moreover ,
the relative standard error of slope can be used as a parameter with respect to the precision of the regression , as a general acceptance criterion for the linearity performance of the analytical procedure .
this parameter should be comparable to the calculated rsd in the evaluation of the precision . in this study , the result obtained for the rsd of the slopes was 1.22% which is comparable to mean value 1.36% of the rsd of the precision .
the method was proved to be precise , as the intra- and interday precision calculated for the concentration levels of 5 , 10 and 50 g / ml ranged from 0.78% to 1.46% and from 1.18% to 1.53% , respectively .
these values fulfill the validation criteria of an analytical method designed for quality control of pharmaceutical preparations for which rsd values
the loq is the lowest concentration that can be quantified with acceptable precision and accuracy .
the loq of diltiazem was determined to be 0.5 g / ml , considering the mean accuracy value of 96.67% and rsd value of 0.91% ( table 2 ) .
these values indicate that the proposed method is much more sensitive than what have been reported previously for analysis of diltiazem in a transdermal gel .
diltiazem is a basic compound and consequently prone to peak tailing and poor peak efficiency ; therefore , sensitivity of the method is of paramount importance to resolve the mentioned problem because one of the factors which can end up to peak asymmetry is sample overload .
the accuracy was evaluated applying the proposed method to the analysis of the in - house mixture of the gel excipients with known amounts of the drug , to obtain solutions at concentration levels of 0.5 , 5 , and 10 and 50 g / ml .
the accuracy was assessed from three replicate determinations and calculated as the percentage of the drug recovered from the formulation matrix .
the mean and rsd values calculated for the analysis of three diltiazem concentration levels of 5 , 10 , and 50
g / ml are shown in table 2 ; the mean values were found to be 100.1% , 99.06% , and 98.78% with rsds 1.32% , 0.67% , and 0.59% , respectively , demonstrating that the method is accurate within the desired range . also , the results obtained from the analysis of preassayed pharmaceutical preparations , spiked with different amounts of diltiazem stock solution , revealed acceptable recoveries with the mean value of 100.49 and % rsds < 1.61 , respectively .
chromatographic parameters including % of assay , resolution between diltiazem and its impurity , theoretical plates , and tailing factor of diltiazem peaks were not significantly affected by the slight changes in the chromatographic conditions like alteration in flow rates , ph of the aqueous solution of mobile phase , and column temperature .
analysis was carried out in triplicate and only one parameter was changed in the experiments at a time .
the results of the experimental variables evaluated were within the acceptable deviation ( rsd < 2% ) , the resolution of the aforementioned peaks was more than 9 , and the theoretical plates and tailing factor parameters were calculated to be more than 11500 and less than 1.2 , respectively , indicating that the proposed method is robust under the conditions tested .
the expanded uncertainty of the method for quantification of diltiazem in pharmaceutical preparations was calculated .
the results are shown in table 3 . among the four sources of uncertainty , which were taken into consideration ,
the uncertainty associated with calibration appears to be the most important source in the overall uncertainty .
the optimized and validated method was applied to the determination of diltiazem in marketed gels .
typical chromatogram obtained following the assay of the pharmaceutical dosage form is shown in ( figure 5(a ) ) .
the results of the assay undertaken and the calculated uncertainties are shown in table 3 .
the value of 99.46% of label claim indicates that the method is selective for the analysis of diltiazem without interference from the excipients used to formulate and produce these gels .
the aim of this study was to develop a green and specific method for the determination of diltiazem in pharmaceutical preparations .
the method was designed to be specific , selective , sensitive , robust , reproducible , accurate , inexpensive , and easy to perform .
the principal advantage of the method is the use of available environmentally friendly solvents and reagents for lc analyzing and extractions to follow the first principle of green chemistry which emphasizes waste prevention instead of remediation . to the best of our knowledge ,
this is the first method which is thoroughly green and reports the metrological parameters in quantification of diltiazem in pharmaceutical dosage forms .
in addition , recycling significantly reduced the mobile phase consumption and made the method economic . moreover ,
finally , the newly developed method was successfully performed for the analysis of diltiazem in pharmaceutical preparations and it can thus be used for routine analysis , quality control , and studies of the stability of pharmaceutical formulations containing diltiazem . | a green , simple , and stability - indicating rp - hplc method was developed for the determination of diltiazem in topical preparations .
the separation was based on a c18 analytical column using a mobile phase consisted of ethanol : phosphoric acid solution ( ph = 2.5 ) ( 35 : 65 , v / v ) .
column temperature was set at 50c and quantitation was achieved with uv detection at 240 nm . in forced degradation studies ,
the drug was subjected to oxidation , hydrolysis , photolysis , and heat .
the method was validated for specificity , selectivity , linearity , precision , accuracy , and robustness .
the applied procedure was found to be linear in diltiazem concentration range of 0.550 g / ml ( r2 = 0.9996 ) .
precision was evaluated by replicate analysis in which % relative standard deviation ( rsd ) values for areas were found below 2.0 .
the recoveries obtained ( 99.25%101.66% ) ensured the accuracy of the developed method .
the degradation products as well as the pharmaceutical excipients were well resolved from the pure drug . the expanded uncertainty ( 5.63% ) of the method
was also estimated from method validation data .
accordingly , the proposed validated and sustainable procedure was proved to be suitable for routine analyzing and stability studies of diltiazem in pharmaceutical preparations . |
natural menopause is defined as the cessation of menstrual cycles for 12 consecutive months , resulting from a lack of ovarian follicular activity .
it is a biological process occurring between approximately 40 and 60 years of age , with a mean value at approximately 51 years . in recent decades
firstly , in most western countries , a trend towards increased maternal age has been recognized . in this
context , prediction of an early menopause ( that is , early onset of subfertility or infertility ) is of clinical significance .
secondly , early onset of menopause is associated with a higher risk of cardiovascular diseases and osteoporosis , whereas a later menopausal age has been associated with an increased risk of breast cancer .
in addition , human population studies have reported that older age at menopause is positively correlated with longevity .
therefore , it is hypothesized that those factors influencing age at natural menopause ( anm ) may also play a role in the etiology and/or course of those common diseases , in addition to an association with infertility and aging .
although some environmental and lifestyle factors influencing anm have been identified , a great heritability for anm has been estimated , ranging from 31% to 53% , and 70% to 85% in singleton sisters .
thus , it is assumed that an important genetic component also accounts for the variability observed in the timing of menopause . in this minireview
the search for genetic variants influencing anm has mainly focused on candidate molecular pathways for which previous clinical and/or biological evidence suggested an involvement in ovary senescence .
for instance , given the pivotal role of estrogens in female reproduction , some genes participating in the synthesis , mechanism of action and degradation of those hormones have been analyzed and proposed as predictor factors for anm , such as esr1 ( estrogen receptor alpha ) gene , cyp17a1 ( cytochrome p450 , family 17 , subfamily a , polypeptide 1 ) gene or cyp1b1 ( cytochrome p450 , family 1 , subfamily b , polypeptide 1 ) gene .
other studies have analyzed genes with a role in thrombophilia and vascular homeostasis , since ovarian senescence is partly due to aging of the vascular supply to the ovary . in this context ,
coagulation factors v and vii have been associated with reduced and increased anm , respectively . however
, other studies could not replicate some of these findings , and the real effect of these variants on anm remains to be clarified .
other researchers have developed hypothesisfree strategies that do not focus on candidate molecular pathways . instead ,
these strategies interrogate thousands of genetic markers , spread along the genome , to identify genomic loci that could be involved in the trait under study , without prior assumptions about the underlying mechanism .
two of those strategies have been applied to the study of anm : linkage studies and genomewide association studies ( gwass ) .
van asselt et al . reported a genomewide linkage analysis in extremely concordant and discordant sibling pairs .
one year later , murabito et al . carried out a genomewide linkage analysis of anm in a populationbased sample .
the linkage signals detected in both studies were different , and thus could not be replicated .
the different study designs in the two reports are likely , at least partially , to account for these discrepancies . nonetheless , both studies proposed attractive candidate loci for age at menopause , such as the bcl2 ( bcell cll / lymphoma 2 ) gene on 9q21.3 due to its role in apoptosis , or the gnrh1 ( gonadotropinreleasing hormone 1 ( luteinizing - releasing hormone ) ) gene close to 8p22 , which is a key molecule in the hypothalamicpituitarygonadal axis that controls human reproduction .
the first stage combined 2,368 samples from the rotterdam study with 611 samples from the twinsuk study .
the second stage comprised 2,560 samples of four additional cohorts of postmenopausal females of european ancestry .
the authors identified six common single nucleotide polymorphisms ( snps ) that were significant genomewide in the combined stages 1 and 2 of their study .
four snps were located in the same haplotype block within the chromosomal region 19q13.4 ; rs236114 was located on 20p12.3 , and rs7333181 on 13q34 .
stolk and colleagues reported a joint analysis of two gwass from the nurses ' health study ( n = 2,287 ) and the women 's genome health study ( n = 15,151).they found 13 markers showing strong association in the joint analysis .
these markers were clustered in four different chromosomal regions ( 5q35.2 , 6p24.2 , 19q13.42 and 20p12.3 ) .
interestingly , two of those regions , 19q13.4 and 20p12.3 , were identified in both independent studies ( table 1 ) .
( 2009 ) maf , minor allele frequency ; snp , single nucleotide polymorphism . the effect sizes for four markers at 19q13.4 are virtually the same in both studies .
two markers ( rs12611091 and rs7246479 ) at this region were detected as significant signals only in the study by he et al . .
it is possible that two markers were not genotyped or they could have failed quality criteria in the study by stolk et al . .
interestingly , these two markers seem to be associated with a later age at menopause ( beta > 0 ) , the opposite effect to that observed for the other significant markers at the same region .
it is possible that minor alleles of rs12611091 and rs7246479 are in ' trans ' with the minor alleles of the other four markers at the same haplotypic block .
the two significant markers at region 20q12.3 show different effect sizes ( table 1 ) . according to hapmap data ( phase iii , ceu analysis panel comprising 30 trios of us residents with ancestry from northern and western europe , collected by the centre d'etude du polymorphisme humain ( ceph ) )
, both markers are in complete linkage disequilibrium ( d ' = 1 ) . therefore , they could be proxies for the same causative genetic variant .
as rs16991615 shows a larger effect size , it is tempting to speculate that rs16991615 is closer to a causal variant than rs236114 , which would account for the observed differences in the effect sizes between both markers .
these two recent gwass have highlighted the potential implications of new loci in the timing of natural menopause .
hk3 ( hexokinase 3 ( white cell ) ) is involved in carbohydrate metabolism , and uimc1 ( ubiquitin interaction motif containing 1 ) can act as a transcriptional repressor and is involved in dna damage resistance , g2/m checkpoint control , and dna repair through binding with brca1 ( breast cancer 1 early onset ) gene complexes .
sycp2l ( synaptonemal complex protein 2like ) knockout mice are sterile in male and subfertile with sharply reduced litter size in female mice .
the brsk1 ( br serine / threonine kinase 1 ) gene is required for neuronal polarization , while the mcm8 ( minichromosome maintenance complex component 8) gene is involved in dna replication .
therefore , these genes and others involved in the same molecular pathways have become very attractive candidates for further investigations in anm .
in addition , it should be borne in mind that the significant snps reported in both studies may not be the causal variants , but they could be in linkage disequilibrium with the causal variants . in this sense ,
other loci close to or within the haplotype blocks reported in both studies , such as the genes trmt6 ( trna methyltransferase 6 homolog ( s. cerevisiae ) ) , suv420h2 ( suppressor of variegation 420 homolog 2 ( drosophila ) ) , znf346 ( zinc finger protein 346 ) , hspbp1 ( hspa ( heat shock 70 kda ) binding protein , cytoplasmic cochaperone 1 ) , gcm2 ( glial cells missing homolog 2 ( drosophila ) ) or unc5a ( unc5 homolog a ( c. elegans ) ) , could be related to anm and they should also be analyzed in further studies . beyond the successful identification of two strong , consistent and novel loci for anm ,
further reanalyses of datasets produced during both projects may have a deep impact on the validation or refutation of previously proposed candidate genes for anm [ 5 - 9 ] .
moreover , the application of novel analytical techniques , such as epistasis or pathwaybased approaches to available datasets , may add new layers of knowledge and will produce novel discoveries by recycling generated genotypes .
thus , we anticipate that the results obtained in both gwass might represent only the tip of an iceberg of ongoing research .
the latest insights into anm have no immediate medical applications in young women . to date
, there is no complete model of causation able to predict , with some accuracy , the timing of menopause in women .
in fact , the markers proposed by stolk et al . and he et al . account for only 1.1% and 2.69% , respectively , of the variability observed in the samples analyzed , which shows that there must be additional environmental and genetic factors accounting for the observed variability .
this model will allow the early recognition of women at risk of early menopause , and hence a shorter reproductive life span , as well as the application of preventive measures , from the initial stages of life , to women at risk . finally , the identification of genetic variants influencing anm has potential implications for the treatment of infertile women .
those genes associated with anm could be used as targets to develop new drugs aimed at delaying the end of the reproductive capacity in women by , for instance , preventing premature depletion of the ovarian pool of oocytes .
anm : age at natural menopause ; ceph : centre d'etude du polymorphisme humain ; gwas : genome - wide association study ; snp : single nucleotide polymorphism .
fjm and jjg made substantial contributions to conception and design , acquisition of data and analysis and interpretation of data .
ar and jjg were involved in drafting the manuscript or revising it critically for important intellectual content .
fjm , ar and jjg have given final approval of the version to be published .
this work has been partially supported by the ministerio de educacin y ciencia of spain ( cit-090100 - 2007 - 57 ) and the corporacin tecnolgica de andaluca ( 07/126 ) . | the age at natural menopause shows great variability . it has been proposed that early age at menopause is a risk factor for osteoporosis and cardiovascular disease , whereas later age at menopause is a risk factor for breast cancer . in addition
, it is thought that the genetic factors accounting for the genetic variability in age at menopause could also play a role in those diseases , as well as infertility in women . in this minireview
we comment on the latest genetics and genomics insights into age at natural menopause . |
. many outbreaks of methanol poisoning have been reported from india.[13 ] being cheap and potent , it is the first adulterant of illicit liquors .
the outbreak of methanol poisoning described in this paper occurred in ahmedabad , gujarat , india in july 2009 .
most patients were received in three municipal corporation run tertiary care hospitals , government hospital and few private / corporate setups .
we , at the shardaben hospital ( municipal corporation run hospital ) , received 178 patients in four days .
initial diagnosis was made based on a clinical history with sufficient evidence of intake of toxic alcohol , as well as the presence of high anion metabolic acidosis . the accidental exposure and amount of illicit liquors
was confirmed by interviewing the patient , relatives and peripheral hospital workers . a brief initial screening examination , including vital signs , mental status and that of the eyes , was performed to identify immediate measures required to stabilize the patient .
the changes noted were dilated pupils with or without sluggish reaction to light , hyperemia of the discs , retinal congestion and edema , blurring of the disc margins and , later , optic atrophy and varying degrees of loss of vision .
all patients were treated with cofactor therapy folinic acid ( leucovorin ) 50 mg iv every 6 h to accelerate formate metabolism .
we administered supplemental thiamine ( 100 mg iv ) and pyridoxine ( 50 mg iv ) and methylcobalamin to all patients .
all patients with a ph below 7.3 were treated with 1 - 2 meq / kg of sodium bicarbonate via iv bolus and volume expansion with isotonic saline to correct acidosis and promote diuresis .
a maintenance infusion was then prepared by mixing approximately 133 meq of sodium bicarbonate in 1 l of d5 w. the infusion rate was 150 - 250 ml / h .
the appropriate rate was adjusted in individual patients according to the initial ph , fluid status and serum sodium level .
the goal of treatment was maintenance of an arterial or venous ph above 7.35 , at which point the infusion was discontinued .
as fomepizole was unavailable , patients were treated with ethanol ( 4 - 8 ml / kg of a 10% ethanol solution , followed by 0.5 - 1 ml / kg of 10% ethanol solution infused per h and 50 ml of 95% through ryle 's tube ) in any patient with documented recent ingestion of methanol or strong clinical suspicion of ingestion of methanol with an arterial ph < 7.3 , bicarbonate
< 20 meg / l , a standard protocol of therapy was initiated in all the patients to start with and was modified later according to individual patient 's needs .
our intention is to share the experience of clinical features , laboratory investigations and their relation during this tragedy .
study of treatment given , interventions and outcome is out of the purview of this study .
data was analyzed using epi2000 and t - test was used to compare the two groups ( those who survived v / s those who died after hospitalization ) . whenever required anova or mann - whitney ,
variables are expressed as mean sd , and p < 0.05 was considered to be statistically significant .
data was analyzed using epi2000 and t - test was used to compare the two groups ( those who survived v / s those who died after hospitalization ) . whenever required anova or mann - whitney ,
variables are expressed as mean sd , and p < 0.05 was considered to be statistically significant .
the liquor was brewed in the house of locale , who also died after consuming the liquor .
eight people , including three women , who were responsible for supplying the spurious brew were subsequently arrested .
initially , we were confused about the cause but as many patients started pouring in , we realized the cause can be methanol poisoning in the light of clinical presentation and history .
we got 2 patients 12 h after history of hooch ingestion and 1 patient after 18 h of ingestion ; maximum patients ( 39.8% , n = 45 ) patients in 48 h ; 22.1% ( n = 21 ) in 72 h ; 3.5% ( n = 4 ) after 72 h , 2 pt after 96 h and 1 patient after 100 h after ingestion .
mean age of presentation was 41.9 10.2 years , median was 40 years and highest number of victims were in 30 - 40 years of age - group [ graph 1 ] .
most were poor labourer ( 32% ) ; lorry driver ( 30% ) ; sweepers ( 28% ) staying in the
challis ( close congested localities ) . most were driven in by relatives or friends ; however few were carried in by non - government organizations ( ngos ) and social workers .
hundred and eight ambulance services ( 108 services which was launched in gujarat on 29 august 2007 was proven prompt enough to bring the patients to the hospital and also pioneered inter hospital transport ) .
age - group distribution of victims from those patients were history was possible to elicit , it was found that the mean time of presentation was 30 h and few were carried in as lately as 100 h after history of ingestion . however , those who were brought in late ( n = 7 ) were relatively stable ( normal vitals , no visual or neurological symptoms ) and dropped in because of panic . as per history
( whenever possible to obtain , n = 77 ) , average amount consumed by them was 150 ml .
the forensic science laboratory report revealed that the country - made liquor , consumed by these victims , had a large dose of methyl alcohol - four times the permissible dose - making it lethal .
eight three percent patients had gi symptoms , 46% had neurological symptoms , 73% had visual symptoms and 32% had dyspnoea [ graph 2 ] . among gi symptoms nausea 80% ,
n = 80 ) , vomiting ( 70% , n = 64 ) and abdominal pain ( 8% , n = 12 ) were common .
few even had taken local treatment for that without getting diagnosed outside . among the neurological symptoms ,
giddiness ( 33% , n = 24 ) , sedation ( 42% , n = 32 ) , altered sensorium ( 28% , n = 20 ) , seizure ( 15% , n = 12 ) and coma ( 15% , n = 12 ) were common .
clinical presentation of victims among those who survived and could be followed - up , we could diagnose four patients with bilateral putaminal necrosis [ figure 1 ] , delayed onset neuropathy and axonopathy .
t1w mri of a victims with bilateral necrosis among the visual symptoms most had blurring of vision ( 62% ) , 10.5% had blindness .
patients with visual symptoms had high mean level of methanol against those with normal ethanol levels ( 120.12 23.12 vs. 55.43 29.24 , p = 0.014 ) .
, 52.8% ( n = 62 ) had bilateral hyperaemia of discs , 8.4% ( n = 12 ) had bilateral disc pallor and 4.5% had papilledema ( n = 5 ) [ graph 3 ] . among the patients with hyperaemia of discs , discs pallor or papilledema , mean methanol level was higher than in patients with normal fundus examination ( 121.1 32.2 mg% v / s 70.1 23.2 mg% , p = 0.032 ) .
fundus examination findings mean packed cell volume ( pcv ) was 45.6 2.1 femtoliters / cell , hemoglobin was 10.2 1.1 mg% ; mean potassium level was 3.2 0.6 meq / l and random blood sugar level 162.2 32.8 mg / dl , mean base deficit was 18.4
mmol / l ( range 2 - 29 ) , anion gap of 25 mmol / l . however , these parameters were not significantly related with serum methanol level or mortality ( anova test , logistic regression analysis respectively ) . among the important deviation of the laboratory reports were abg derived ph , bicarbonate level and serum methanol level .
it correlated statistically significant with the methanol level [ graph 3 ] ( p < 0.05 ) and with the mortality ( p < 0.01 ) .
those who had expired had lower mean ph then those who survived ( 6.94 0.33 v / s 7.23 0.15 , p < 0.01 ) .
it correlated statistically significant with the serum methanol level [ graph 4 ] ( p < 0.05 ) .
those who expired had much lower bicarbonate level then those who survived had ( 7.5 4.1 mmol / l v / s 13.3 6.7 mmol / l , p < 0.01 ) .
ph , bicarbonate levels v / s serum methanol level mean serum methanol level was 87.1 mg / dl , the maximum was 376 mg / dl , the minimum was 12 mg / dl . those who expired had significantly higher serum methanol level ( 53.1 41 mg / dl v / s 121 92 mg / dl , p value < 0.05 ) . mean serum ethanol level was 93 m / dl in survival group v / s 154.2 in those who expired ( p value 0.39 ) . there was no statistically significant difference of serum ethanol level v / s delay of presentation ( mann - whitney two sample test , p value 0.16 ) , level of acidosis ( mann - whitney two sample test , p value 0.47 ) or bicarbonate level ( mann - whitney two sample test , p value 0.55 ) .
. many outbreaks of methanol poisoning have been reported from india.[13 ] being cheap and potent , it is the first adulterant of illicit liquors .
it goes by the names of khopadi , ladda , dalda , bewada , french polish , etc .
the mean time interval between ingestion and presentation was 30 h. this is because , upon ingestion , methanol is quickly absorbed in the gastrointestinal tract and metabolized in the liver . in the first step of degradation , methanol is transformed into formaldehyde via the enzyme alcohol dehydrogenase ( adh ) .
this reaction is slower than the next step , the transformation of formaldehyde into formic acid via the enzyme aldehyde dehydrogenase .
mean age of presentation was 41.9 10.2 years , which signifies alcohol consumption is very common in this particular age group where most of them are earning members of the families .
male are commonly affected , consistent with the indian culture , where female are less frequently consuming alcohol .
however , three female in our study says , few female even in lower socio - economic strata are also consuming such alcohol .
notably , one of the female herself was bootlegger . in our study the time interval between poisoning and ed presentation in deceased do not correlate statistically significant with mortality .
however in some studies , it is shown as even the prognostic factor of mortality .
it may be because of various confounding factors like initial outside treatment , amount of methanol consumed , co - morbid conditions , facilities at the receiving hospital and authorities and administrative commitments .
gi symptoms predominates in such tragedy , few are even brought - in because of such symptoms .
ocular changes consisting of disc oedema , blurring of the disc margins , hyperaemia of the discs and optic atrophy as a late squeal , are quite characteristic of methanol poisoning .
optic nerve demyelination has been reported to be due to formic acid destruction of myelin .
the major damage occurs at the retrolaminar optic nerve , with intra - axonal swelling and organelle destruction .
a peak serum alcohol concentration is reached within 1 - 2 h. an arterial blood gas serves as an essential triage tool when caring simultaneously for multiple patients with poisoning .
arterial blood gas derived ph value and bicarbonate levels correlate significantly with the serum methanol level and are also good prognostic factors in methanol poisoning . in our study
the serum ethanol level did not show significant correlation with mortality , ph or bicarbonate levels .
it is important to emphasize that the onset of methanol toxicity is delayed when ethanol is co - ingested .
hence , the possibility of concomitant ethanol and toxic alcohol ingestion must always be considered , particularly in alcoholics , who may ingest alcohol in any form .
ethanol was given for treatment purpose through ryle 's tube and intra - venous route to all victims and the timing of sample collection was also variable .
hence the correlation of blood ethanol level and morbidity and mortality needs to study further .
increased pcv level , elevated rbs and hypokalemia were found in this study , but did not correlate significantly with the serum methanol level or mortality .
, these variables should be taken into consideration while treating these patients . according to swartz et al .
an increase in the rbc size is probably responsible for this finding and not the hemoconcentration .
gi symptoms , neurological symptoms and breathlessness are important clue to ed physician for diagnose methanol poisoning .
blood sugar , hypokalemia are important associations to be taken into consideration while treating these patients .
methanol level takes time to be available or not possible in some settings and mortality .
hence , ph and bicarbonate level helps to manage such patients and predict the mortality risk as well .
the limitation of our retrospective study is that we have limited history about the amount of hooch consumption , exact time of consumption , co - ingestions , treatment taken from outside set - ups initially .
gi symptoms , neurological symptoms and breathlessness are important clue to ed physician for diagnose methanol poisoning .
blood sugar , hypokalemia are important associations to be taken into consideration while treating these patients .
the limitation of our retrospective study is that we have limited history about the amount of hooch consumption , exact time of consumption , co - ingestions , treatment taken from outside set - ups initially .
gi symptoms , neurological symptoms and breathlessness are important clue to ed physician for diagnose methanol poisoning . visual symptoms and fundus finding correlate well with the methanol level . increased pcv ,
blood sugar , hypokalemia are important associations to be taken into consideration while treating these patients . | introduction / purpose : the outbreak of methanol poisoning described in this paper occurred in ahmedabad , gujarat , india in july 2009 . our intention is to share the experience of clinical features , laboratory investigations and their relation during this tragedy.materials and methods : single center , retrospective study of clinical features and laboratory parameters of 178 cases of methanol toxicity treated at tertiary care hospital in ahmedabad , gujarat.results:maximum patients ( 39.8% , n = 45 ) were received in 48 h ; mean age of presentation was 41.9 10.2 years .
most of them were men ( 175 out of 178 ) . on presentation , 83% patients had gastro - intestinal symptoms , 46% had neurological symptoms , 73% had visual symptoms and 32% had dyspnoea .
62% had blurred vision , 10.5% had blindness .
patients with visual symptoms had high mean level of methanol ( 120.12 23.12 vs. 55.43
29.24 , p = 0.014 ) .
on fundus examination 52.8% ( n = 62 ) had bilateral hyperaemia of discs , 8.4% ( n = 12 ) had bilateral disc pallor and 4.5% had papilledema ( n = 5 ) .
patients with hyperaemia of discs , discs pallor or papilledema , had higher mean methanol level ( 121.1 32.2 mg% v / s 70.1 23.2 mg% , p = 0.032 ) .
mean of ph values was 7.17 0.22 and bicarbonate was 12.3 7.3 mmol / l .
both ph and bicarbonate levels correlated well with mortality and serum methanol level .
mean serum methanol level was 87.1 mg / dl , and correlated significantly with the mortality ( 53.1 41 mg / dl v / s 121 92 mg / dl , p value < 0.05).conclusion : gi symptoms , neurological symptoms and breathlessness are important clue to ed physician for diagnose methanol poisoning . visual symptoms and fundus findings correlate well with the methanol level .
arterial blood gas derived ph and bicarbonate levels correlate significantly with the methanol level and mortality . |
semiconducting metal oxide sensors are one of the most studied groups of the chemical sensors which have been designed to react with gases .
different materials such as sno2 , wo3 , zno , moo3 , tio2 , inox , and mixed oxides have been studied and showed promising applications for detecting gases such as nox , o3 , nh3 , co , co2 , h2s , and sox [ 2 , 3 ] .
therefore , improvement in the sensitivity , selectivity , rate of gas response , and reliability of oxide semiconductor gas sensors is important [ 46 ] . among various metal oxide semiconductors ,
tungsten oxide exhibits various special properties , which makes it very promising for applications in catalysis [ 7 , 8 ] and detection of the toxic gases .
wo3 based mixed oxides such as wo3-ti , wo3-pd , pt , or au , wo3-in2o3 , and wo3-bi2o3 have also been investigated for their sensing characteristics .
these mixed oxides have been especially used in fabricating selective and sensitive nox gas sensors .
this is because the w ions have different oxidation state ( w , w ) enhancing the adsorption activity of nox molecule on the surface of wo3 structures [ 14 , 15 ] .
recently , novel sensors based on tungsten oxide have been used for ozone monitoring [ 16 , 17 ] .
tungsten oxide has attracted a lot of interests as an n - type oxide semiconductor . wo3
band gap has been measured by optical absorption in the range of 2.5 to 3.2 ev and is smaller than other semiconductors [ 19 , 20 ] . for wo3-based gas sensors ,
wo3 plays a role as a sensitive layer for detecting small quantities of nox and such nanoscale assemblies can achieve high sensitivity and fast response times . despite the considerable amount of works done on wo3 crystal structure so far ,
there are still important aspects of the electronic and structural properties of the wo3 nanoclusters and nox adsorption which are unclear , especially for the small clusters of wo3 .
furthermore , the theoretical works which have been done so far are based on the standard dft methods which seriously underestimate the semiconductor band gap .
this problem can be improved by using dft in combination with the hybrid functionals which provide a satisfactory option for the description of both the electronic and the structural properties of wo3 [ 22 , 23 ] . in this work
a theoretical procedure was applied to study the nox ( x = 1,2 ) adsorption on the ( wo3)n ( n = 26 ) nanoclusters to evaluate the reactivity of wo3 nanoclusters from the quantum chemistry point of view .
knowledge of the quantum reactivity indices and their role on the sensing properties of metal oxides is important to have an insight into the adsorption process and factors involved .
in order to study the nox ( x = 1,2 ) adsorption on the cyclic ( wo3)n ( n = 26 ) nanoclusters , theoretically , dft method has been applied .
lanl2dz and 6 - 311++g(d , p ) basis sets have been applied for w and other atoms , respectively [ 2527 ] .
( wo3)n ( n = 26 ) structures were generated in the vacuum and fully optimized using two kinds of hybrid functional of b3lyp and x3lyp .
the adsorption of no and no2 molecules on the clusters was investigated to evaluate some aspects of nanoclusters and nox interactions .
for geometry optimization , nox molecules were taken relaxed but the optimized structures of ( wo3)n ( n = 26 ) were kept frozen .
the adsorption energies of nox molecule and five different substrates have been computed according to
( 1)e = enox @ wo3n ewo3nenox ,
where e[nox @ ( wo3)n ] is the total energy of the tungsten oxide cluster - nox complex and e(wo3)n and e(nox ) are the total energy of the isolated ( wo3)n and nox , respectively .
[ 28 , 29 ] was carried out to explore the distribution of the electrons into atomic and molecular orbitals .
based on these data , by homo - lumo analysis , the stabilization energies were calculated .
chemical hardness , , and charge transfer , n , have been computed by koopmans theorem , using the following , respectively :
( 2)=lh ,
( 3)n=bab+a ,
( 4)e=l+h2 ,
where
h and
l correspond to the kohn and sham one - electron eigenvalues associated with the frontier molecular orbitals of homo and lumo , respectively .
e is the electronic chemical potential . a and b subscripts stand for nox and ( wo3)n , respectively
. densities of states ( dos ) were also calculated in order to analyze the band structures and fermi level changes of wo3 clusters , during the nox adsorption .
figure 1 demonstrates all optimized cyclic structures of wo3 . in order to evaluate their abilities for using as the gas sensors
since there are different adsorption sites on wo3 clusters for no adsorption , interfacial interactions between the clusters and no molecule will be different from the energy point of view .
the most important parameters which affect the adsorption energy are the adsorption site and no conformation on the clusters .
this means that for better analysis of the systems , it is necessary to investigate different orientations of no molecule on the different sites of the clusters .
theoretical calculations confirmed that no adsorption through the n - head ( onwo3 ) is the most stable orientations through which the most adsorption energy is obtained ( table 1 ) .
figure 3 shows the potential energy diagram for no adsorption on w2o6 clusters as an example . according to figure 3 , relaxed no molecule is close to wo3 clusters from a distance of 6 .
considering table 1 , it can be concluded that x3lyp functional predicts stronger physical adsorption than b3lyp .
correlation between the size of the cluster and adsorption energy is according to n : 2 > 3 4 > 5 > 6 at the x3lyp functional .
the energy of adsorption for the most stable structures of on @ w2o6 and no @ w2o6 complexes is 0.685 and 0.340 ev , respectively .
since on @ w2o6 complex is a better candidate than others from the energy viewpoint , in the next stage of our study , we concentrated on this type of interaction and it has been fully investigated through the quantum chemistry approach .
table 2 shows the quantum chemistry reactivity indices for the stable structures . according to table 2 ,
charge transfer and chemical hardness have been increased from no to the surface with increase in the ( wo3)n clusters sizes .
higher values of the charge transfer and chemical hardness mean that increase in the size of the system makes its electronic charges unstable and lowers its flexibility .
natural population analysis confirms that there are considerable orbital overlap between w and n atoms of the clusters and no molecule , respectively .
natural charges for n and w atoms of no @ w2o6 complex are + 0.08 , + 1.5e before adsorption and + 0.12 , + 1.38e after adsorption , respectively .
this type of charge fluctuation indicates the charge flow from the adsorbate to the surface .
the influence of no adsorption on the electronic properties of the tungsten oxide clusters was also investigated .
as an example , figure 4 presents dos spectra of w2o6 cluster before and after adsorption . considering all systems
, it was found that band gap ( e
g ) changes are between 4.37 and 9.91% after the adsorption process .
these results show that the adsorption of no molecule can not significantly affect the e
g and conductivity of the nanostructures while the fermi level energy ( e
fl ) is shifted by 0.385 ev towards the higher energies . due to this effect
, metal oxide work function ( ) is decreased . finally , the dispersion corrected functional , x3lyp , shows that the theoretical results which have been computed by this method differ from the b3lyp in all cases .
this means that the inclusion of an empirical dispersion correction to the density functional amplifies the calculated energies of physisorption . since ,
in the previous section , stronger adsorptions were obtained by x3lyp functional , this method is chosen for analysis of no2 @ ( wo3)n systems .
figure 5 shows these adsorption models ( a)(d ) for no2 @ w2o6 complex as an example .
adsorption energies for no2 @ ( wo3)2 systems have been calculated and reported in table 3 . according to table 3 , model d is the best configuration for no2 adsorption from the energy point of view , 2.0 ev .
this extent of adsorption energy corresponds to no2 chemisorption on the w2o6 cluster . considering all possible models ,
it is confirmed that the reactivity order is according to the following : d > a > c > b. obtained correlation between the size of the cluster and adsorption energy is according to the following : n = 2 > 3 > 4 > 5 > 6 . different behavior of no2 adsorption on ( wo3)n ( n = 26 ) has been seen in the fermi level shifting character . as an example
, w2o6 band gap is decreased from 10.10 to 9.97 ev after adsorption . due to no2 adsorption
, fermi level is shifted by 0.26 ev towards high - energy region ( from 4.95 to 4.69 ev ) .
charge transfer and chemical hardness changes during the no2 adsorption have been calculated and reported in table 4 . according to table 4
, there is a similarity between no2 and no adsorption behavior from the chemical hardness viewpoint . considering the electronic charge transfer ,
this means that increase in the size of the cluster promotes the chemical hardness while reducing the charge transfer between no2 and clusters .
figures 6 and 7 show the obtained linear correlation of the adsorption energy - chemical hardness and adsorption energy - charge transfer , respectively .
good correlation between the adsorption energy and chemical hardness is obtained while this correlation is weak in the case of the charge transfer .
therefore , it can be concluded that the extent of chemical hardness fluctuation is one of the important parameters in sensing properties of the metal oxide clusters .
natural population analysis confirms that w atom of w2o6 in the vicinity of no2 molecule has a positive character and carries a charge of + 1.6e .
one o atom of no2 molecule which is closer to w atom of the nanocluster has obtained more negative charge than the other . due to this charge transfer from w ,
oxygen atomic charge is changed from 0.24 to 0.27e . comparing between the negative character of o atom and positive character of w atom
, it can be concluded that w and o atoms do not exist as w and o as in the bulk of wo3 .
therefore , we can conclude that there are considerable orbital overlaps between w and o atoms .
back - donation of lone pair electrons of o atoms to the vacant d orbitals of w atoms is the source of this covalent character for w
the adsorption of nox ( x = 1,2 ) molecules on the tungsten oxide clusters was investigated using density functional theory calculations by b3lyp and x3lyp functionals . during the adsorption of nox on the ( wo3)n ( n = 26 ) nanoclusters
, energy is released with a significant charge transfer from the nox to the nanoclusters .
the results showed that the adsorption energy depends on the size of the cluster . x3lyp
w2o6 cluster is the best candidate for nox adsorption from the energy point of view . in order to have a meaningful understanding of molecular changes during the adsorption process
, quantum chemistry reactivity indices such as chemical hardness and charge transfer parameters were evaluated .
obtained results from the natural bond orbital and electronic density of states analysis confirmed the electronic charge transfer from the adsorbates to the wo3 clusters and fermi level shifting after adsorption , respectively . | today 's wo3-based gas sensors have received a lot of attention , because of important role as a sensitive layer for detection of the small quantities of nox . in this research
, a theoretical study has been done on the sensing properties of different cyclic nanoclusters of ( wo3)n ( n = 26 ) for nox ( x = 1,2 ) gases .
based on the calculated adsorption energies by b3lyp and x3lyp functionals , from the different orientations of nox molecule on the tungsten oxide clusters , o
nw was preferred .
different sizes of the mentioned clusters have been analyzed and w2o6 cluster was chosen as the best candidate for nox detection from the energy viewpoint . using the concepts of the chemical hardness and electronic charge transfer , some correlations between the energy of adsorption and interaction energy have been established .
these analyses confirmed that the adsorption energy will be boosted with charge transfer enhancement .
however , the chemical hardness relationship is reversed . finally , obtained results from the natural bond orbital and electronic density of states analysis confirmed the electronic charge transfer from the adsorbates to wo3 clusters and fermi level shifting after adsorption , respectively .
the last parameter confirms that the cyclic clusters of tungsten oxide can be used as nox gas sensors . |
this infection is the leading cause of morbidity and mortality- in critically ill patients in developing countries .
respiratory tract infections ( rtis ) , which involve the upper or lower respiratory tract , frequently occurs after birth .
rtis , such as sore thoat , earache , laryngitis , common cold , otitis media , sinusitis , and mastoiditis , are the most frequently - occurred infections of all human diseases and have been frequently documented .
since these infections often seem trivial , they are more commonly discounted as being mere temporary inconveniences that cause transient discomfort .
respiratory infections in particular those occur in upper respiratory tract are seen with great frequency in both children and adults and have remarkable economic impact , related not only to lost output in the workplace but also to the frequent prescription by physicians of antibiotics , even when the causative agents of infection almost certainly are not bacteria . on the other side ,
eradication of the causative agents of respiratory tract infections is recognized as a requirement , however during the last few years , the increase in the rates of antibiotic resistance amongst the major microbial causes of the respiratory infections in the community has compromised the selection of empirical treatment for some respiratory tract infections .
it has been reported that pneumonia due to secondary bacterial infections is the most important complication of the lower respiratory tract and that surveillance of acute rtis in defined populations to monitor prevailing pathogens and to determine population groups at special risk are important for taking preventive measures .
the main bacterial pathogens recovered from complicated influenza virus infections include haemophilus influenza , staphylococcus aureus , streptococcus pneumonia , klebsiella pneumonia , and streptococcus pyogenes . to our knowledge
, there is limited information on the prevalence of various bacterial pathogens and their antibiotic resistance patterns in tamilnadu , india .
hence , the aim of this study was to determine the prevalence and antimicrobial susceptibility rates of bacterial strains isolated from thoat specimens obtained from a rural health centre of rajah muthiah medical college and hospital ( rmmc and h ) , annamalai university , chidambaram , tamilnadu , india .
the field practice area of the rural health centre of rajah muthiah medical college and hospital ( rmmc and h ) , annamalai university , chidambaram , forms the study area .
the rural health centre is located at south pichavaram , 12 kms to the east of the town of chidambaram and 4 - 6 kms from the sea coast ( bay of bengal ) .
the villages covered by the service area are : 1 ) south pichavaram , 2 ) kanagarapattu , 3 ) t. s. pettai , 4 ) north pichavaram , 5 ) keezhaperambai and 6 ) senjicherry .
these villages form part of the chidambaram taluk in the cuddalore district of the state of tamil nadu , south india .
subjects coming to the general medicine outpatient department were included in the study . in total , 250 patients took part in the study , of which the bacterial infection was confirmed only in 228 patients . out of 228 patients , 108 were males and 120 were females .
the study subjects were aged 10 - 60 years with a mean age of 35 years .
all the subjects were informed of the aim of the study and were assured that all information supplied by them will remain confidential . their verbal consent and cooperation to participate in the study
the criteria for inclusion in the study were : the patients must be suffering from urtis , referred to clinics or hospitals , and must not have been taking antibiotics of any kind at least for the two weeks preceding the study .
the subjects presented urtis , including sore thoat , otitis media , runny nose , sinusitis and cough . the large majority ( 78% ) of them had runny nose and sore thoat while sinusitis was the least noted .
the field practice area of the rural health centre of rajah muthiah medical college and hospital ( rmmc and h ) , annamalai university , chidambaram , forms the study area .
the rural health centre is located at south pichavaram , 12 kms to the east of the town of chidambaram and 4 - 6 kms from the sea coast ( bay of bengal ) .
the villages covered by the service area are : 1 ) south pichavaram , 2 ) kanagarapattu , 3 ) t. s. pettai , 4 ) north pichavaram , 5 ) keezhaperambai and 6 ) senjicherry .
these villages form part of the chidambaram taluk in the cuddalore district of the state of tamil nadu , south india .
subjects coming to the general medicine outpatient department were included in the study . in total , 250 patients took part in the study , of which the bacterial infection was confirmed only in 228 patients . out of 228 patients , 108 were males and 120 were females .
the study subjects were aged 10 - 60 years with a mean age of 35 years .
all the subjects were informed of the aim of the study and were assured that all information supplied by them will remain confidential . their verbal consent and cooperation to participate in the study
the criteria for inclusion in the study were : the patients must be suffering from urtis , referred to clinics or hospitals , and must not have been taking antibiotics of any kind at least for the two weeks preceding the study .
the subjects presented urtis , including sore thoat , otitis media , runny nose , sinusitis and cough . the large majority ( 78% ) of them had runny nose and sore thoat while sinusitis was the least noted .
the specimens were transported to the microbiology laboratory of the department of microbiology , annamalai university , chidambaram for analysis .
a loopful of each sample was inoculated onto blood agar , chocolate agar , and eosin methylene blue agar .
the inoculated plates were incubated at 37c for 24 - 48 h aerobically , except for chocolate agar , in which the plates were incubated for 24 - 48 h at 37c in an atmosphere of 5 - 10% co2 .
after incubation , macroscopic and microscopic examinations of colonies on plates were carried out , and suspect colonies were subcultured on appropriate solid culture media for purification .
they were later sub cultured on appropriate slants and stored at 4c for further analysis .
pure cultures were presumptively identifiedbased on their cultural and morphological characteristics on selective and differential media .
a sterile cotton swab was dipped into the standardized solution of bacterial cultures and used for evenly inoculating mueller - hinton plates ( himedia , mumbai ) and allowed to dry .
thereafter , antibiotic discs with the following drug contents - ampicillin ( 10 g ) , ceftriaxone ( 5 g ) , chloramphenicol ( 10 g ) , co - trimoxazole ( 25 g ) , erythromycin ( 10 g ) , gentamicin ( 10 g ) , amikacin ( 30 g ) , ciprofloxacin ( 5 g ) , cefixime ( 5 g ) , ceftazidime ( 30 g ) and penicillin ( 10 g ) were placed on the plates , spacing them well to prevent the overlapping of inhibition zones .
the plates were incubated at 37c for 24 h , and the diameters were measured .
a sterile cotton swab was dipped into the standardized solution of bacterial cultures and used for evenly inoculating mueller - hinton plates ( himedia , mumbai ) and allowed to dry .
thereafter , antibiotic discs with the following drug contents - ampicillin ( 10 g ) , ceftriaxone ( 5 g ) , chloramphenicol ( 10 g ) , co - trimoxazole ( 25 g ) , erythromycin ( 10 g ) , gentamicin ( 10 g ) , amikacin ( 30 g ) , ciprofloxacin ( 5 g ) , cefixime ( 5 g ) , ceftazidime ( 30 g ) and penicillin ( 10 g ) were placed on the plates , spacing them well to prevent the overlapping of inhibition zones .
the plates were incubated at 37c for 24 h , and the diameters were measured .
one hundred and four isolates ( 45.61% ) were identified as s. aureus , 52 ( 22.81% ) as , hemolytic streptococci , 34 ( 14.91% ) as klehsiella pneumonia , 19 ( 8.33% ) as pseudomonas aeruginosa , 11 ( 4.83% ) as hemolytic streptococci , 5 ( 2.91 % ) as e. coli and 3 ( 1.32% ) as h. influenzae .
of the 228 isolates identified , 53% were recovered from females compared to 47% from males .
the overall resistance rates were generally low for gentamicin , cefixime and ceftazidime respectively . however , penicillin had the highest resistance ( 100% ) .
prevalence and antimicrobial susceptibility ( % ) of bacterial strains recovered from throat swabs the study focused on the prevalence and antibiogram of bacteria causing urtis among people in a rural health centre .
respiratory tract infection is considered as one of the most important infectious diseases in developing countries .
staphylococcus aureus is an increasingly deadly pathogen that is known to cause a variety of nosocomial and community - acquired infections . in our study ,
staphylococcus aureus was the commonest pathogen seen in majority of the population ( 45.61% ) .
staphylococcus aureus has shown a disconcerting propensity to develop resistance to antimicrobial agents and has become an important challenge for the clinicians . hence , before selecting an antibacterial , the clinician must first consider the likely causative organism .
knowledge of the prevalent organisms and their current sensitivity is of great help in choosing an antibacterial . in a study on the epidemiology of respiratory tract bacterial pathogens carried out by varotto et al .
( 2001 ) , p. aeruginosa has been reported as the most prevalent organism ( 24% ) followed by s. pyogenes ( 18% ) , s. aureus ( 17% ) and k. pneumoniae ( 8% ) . in an indian study carried out by kumari et al .
( 2007 ) on bacterial isolates from respiratory tract of icu patients , the percentage isolation rate for p. aeruginosa , klebsiella spp .
have been reported 21.5 , 19 and 8 respectively . as shown in table 1 ,
the bacterial isolates in order of the frequency in our study were s. aureus ( 45.61% ) , hemolytic streptococci ( 22.81% ) and klebsiella pneumoniae ( 14.91% ) , which is not comparable to the two above mentioned studies .
the results of the antibiotic susceptibility study generally revealed that gentamicin and cefixime were the most effective antibiotics against the isolates ( table 1 ) .
s. aureus showed a sensitivity rate of 97.11% to gentamicin while hemolytic streptococci and k. pneumoniae each recorded 96.15% and 97.06% respectively .
it is noteworthy that gentamicin and cefixime is probably less abused than other antibiotics because of its mode of administration ( solely by injection ) and the prohibitive cost of procurement .
these factors may be responsible for the low rate of resistance recorded for these drugs .
the high rate of resistance to penicillin ( 100% ) and co - trimoxazole ( 76.8% ) , which are commonly bought over - the - counter in drug stores , contrasts with the marked levels of susceptibilities of the isolates to gentamicin , cefixime , and ceftazidime , which are less - frequently used , thus suggesting a relationship between antibiotic use and the level of drug resistance encountered in this study as previously suggested in another study .
the increasing frequency of antibiotic resistance has been reported first in infections at sites where penetration of the antimicrobial agent is restricted and the level of therapeutic concentrations is consequently more difficult to be achieved .
it could also hinder the eradication of infections in respiratory tract infections treated using standard antibiotic therapy regimens .
accurate information on local epidemiology and antimicrobial resistance patterns of pathogens is essential to select a clinically effective antibiotic therapy for the infections .
there are many possible reasons for this alarming phenomenon , including inappropriate and incorrect administration of antimicrobial agents in empiric therapies and lack of appropriate infection control strategies .
surveillance of bacterial infections and monitoring their antimicrobial susceptibility pattern must be carried out not only in tertiary hospitals , but also in small hospital settings .
changing the antibiotic of main prescription periodically ( every 6months/ 1 year ) akin to crop rotation to increase soil fertility , could keep resistant organisms at bay in the wards .
further study should be carried out for inpatients in surgery , medicine , wounds and post operative cases for best results .
these types of data will help in designing and validating the accuracy of guidelines for empirical treatment of upper respiratory tract infections . | the study was aimed at determining bacterial agents of the upper respiratory tract and the susceptibility patterns of isolates to antibiotics .
the throat swab samples from 250 patients suspected of upper respiratory tract infection ( urti ) were obtained from the general medicine outpatient department of a rural health centre of rajah muthiah medical college and hospital ( rmmc and h ) , annamalai university , chidambaram , tamilnadu , india and inoculated in the culture medium .
the bacterial infection was confirmed only in 228 patients .
the organisms isolated on medium were identified by their cultural , morphological and biochemical characteristics .
staphylocccus aureus was identified as the most prevalent bacterial isolate ( 45.61% ) followed by hemolytic streptococci ( 22.51% ) .
thirty four strains ( 14.91% ) were identified as klebsiella penumoniae , 19 ( 8.33% ) as pseudomonas aeruginosa and the rest belonged to a hemolytic streptococci , escherichia coli and haemophitus influenzae .
all staphylococcus spp , were resistant to penicillin . ,
ampicillin and co - trimoxazole .
all the isolates were resistant to at least one antibiotic .
the overall resistance rates were generally low for gentaruicin , cefixine and ceftazidime respectively . |
high malaria morbidity and mortality have persisted because of failed transactions between those at risk of malaria infection and available effective antimalarial drugs and preventive measures .
artemisinin - based combination therapies ( acts ) , the most effective drugs to treat plasmodium falciparum , malaria were recommended by world health organization ( who ) in 2001 for malaria endemic countries following resistance to the cheap and the long used antimalarials such as chloroquine , sulfadoxine - pyrimethamine , and amodiaquine and the resultant increasing burden of malaria . since the introduction
nigeria adopted act in 2005 and like the situation in other endemic countries access to acts by most people at risk of malaria has been a challenge .
the acts are not widely used because of their high price , unreliable public sector supply , limited availability in the private sector , and patient self - medication with less expensive monotherapies . in response to the low act access and the threat of artemisinin resistance developing in malaria parasite , the institute of medicine released a report in 2004 recommending the creation of a global subsidy ( by reducing the price of the drugs at the manufacturer level ) and that acts
be made available through both the public and private sectors at the same price as chloroquine and other common therapies . according to the institute
, the subsidy is expected to expand access to acts , crowd out monotherapies , decrease resistance to acts , and therefore decrease the burden of malaria .
this concept was later developed by the roll back malaria partnership into a global mechanism known as the affordable medicines facility - malaria .
affordable medicine facility - malaria - acts distribution channels involve both the public and private sectors .
the private sector was included because studies done previously indicated that the majority of patients in many malaria endemic countries seek treatment outside of the formal public sector , with a large proportion accessing treatment through the private sector instead .
private sector treatment sources vary considerably between and within countries , ranging from private hospitals and clinics to one - room drug shops , general stores , and medicine peddlers .
studies revealed that patients seek malaria treatment in the private sector because of long distance to , long waiting time at , and stock out of drugs in the public sector as well as shortage of skilled providers .
nigeria started the distribution of amfm - acts in 2011 firstly in the private sector then later in public sector .
society for family health ( sfh ) , through the global fund malaria and prepackaged therapy ( ppt ) projects , is working to ensure broad access to acts in nigeria . through the global fund under the amfm - act initiative , sfh is providing a subsidized form of artemether - lumefantrine ( al ) known as actm .
these medicines are distributed through private health care providers including proprietary patent medicine vendors ( ppmvs ) ( local drug shopkeepers ) and community - based pharmacies .
sfh works closely with the federal ministry of health on technical matters , education , training , and coordinating policy formulation and research to move the nation forward on both prevention and treatment issues in her battle against malaria .
while some effort at studying the adherence of drug distributors to use of amfm - acts has been documented , its use at the community level is yet to be studied .
information on awareness of amfm - malaria and how it has affected its use is lacking .
several past studies have concentrated on provider 's knowledge on the drug and availability of the drugs at facilities where they are distributed .
hence , this study aimed at determining awareness , utilization , and factors influencing utilization of amfm - acts in communities in ibadan north - west local government .
the study was conducted in ibadan north - west local government area which is one of the eleven lgas that constitute ibadan metropolitan area .
ibadan is the capital of oyo state , one of the 36 states of the federal republic of nigeria .
the state is centrally situated in the southwestern part of the country and is 128 km north - east of lagos and 345 km south - west of abuja , the federal capital territory . for administrative purposes the city is divided into 11 local government areas ( lgas ) , five of which are urban .
the inhabitants of ibadan north - west lga are mostly yoruba while the main occupations of the people are trading and working in the public service .
there are 11 political wards and 6 primary health centres ( government owned ) in the local government .
treatment of malaria takes place in health facilities and at other healthcare providers ' places such as private patent medicine shops ( ppmvs ) , pharmacy shops and appointed community medicine distributors . however , home management of malaria using drugs bought from ppmvs and pharmacy is a common practice among caregivers in nigeria including those in this study lga .
this was a cross - sectional study and cluster sampling technique was used in selecting caregivers who participated in the study .
women caregivers in their reproductive age ( 1549 years ) whose children had fever within two weeks prior to the survey were included in the study .
it was estimated that a minimum sample size of 430 caregivers will be required to estimate the true proportion of those using amfm - acts within 5% precision and assuming a 50% prevalence of utilization .
four research assistants with ordinary national diploma ( ond ) were trained for two days to assist in data collection .
research assistants were given packets of common antimalarial drugs including that of amfm - acts ( also known as antimalarial packet with the green leaf symbol ) to show caregivers how to ascertain use of amfm - acts .
data were collected over a period of one month . a cluster sampling of five enumeration areas was done .
based on the information from national population commission , ibadan north - west has 509 enumeration areas .
it was estimated that an enumeration area consists of at least five hundred people and women in reproductive age group represent 22 percent of the total population .
a total of 110 women were estimated to be found in each enumeration area . from the list of the enumeration areas 5 enumeration areas
this household served as the starting point and following a predetermined direction , women of reproductive age in consecutive households in each of the cluster were studied till the allocated sample size of 110 was achieved .
questionnaire was adapted from act watch household survey questionnaires used in seven african countries and questions from the literatures that assessed the utilization of artemisinin - based combination therapy were also added .
questionnaire administration commenced after obtaining approval to conduct the study from oyo state ethical review committee and after informed consent was obtained from respondents .
information was collected on sociodemographic characteristics , knowledge on symptoms of malaria , treatment seeking for malaria , awareness of amfm programme , utilization of amfm - acts , and factors influencing utilization of amfm - acts .
data were entered , cleaned , and analyzed using statistical package for social sciences version 15 software .
the chi - square test was used to determine associations between outcome and categorical explanatory variables .
use of amfm - acts was defined as use of amfm - acts within 2448 hours of onset of fever .
information on awareness of amfm - acts was ascertained by asking if the caregivers have heard about amfm programme , if they have seen the packet of antimalarial drug with the green leaf symbol , sources where amfm - acts was seen or heard , and if they knew what the symbol on the amfm - acts packet meant . concerning the meaning of the symbol on the drug packet ,
eight questions were asked and the maximum score that the respondents could get in this domain was 8 .
respondents who scored above the mean were taken to have good knowledge and others having scores below the mean were taken to have poor knowledge .
factors influencing use of amfm - acts were considered sociodemographic characteristics , socioeconomic status , awareness of amfm - acts , access to amfm - acts , and knowledge on symptoms of malaria .
access was defined in terms of geographical accessibility to drug shops where amfm - acts were being sold and also availability of amfm - acts .
geographical access was considered good if it takes 30 minutes or less ( irrespective of mode of transportation ) for someone to reach the source of the drug .
respondents were thereafter classified into low and high socioeconomic class using $ 2 per day as a cut - off .
people of low socioeconomic status were defined as people living below $ 2 per day [ 11 , 12 ] . respondents were categorized as having good knowledge on symptoms of malaria if they have scores above the mean scores , with the total obtainable score based on the number of questions asked and score of the response being 10 marks .
four hundred seventy - eight caregivers participated in the study and their characteristics are shown in table 1 .
many of the caregivers 287 ( 60.3% ) were between 25 and 34 years , 427 ( 89.3% ) were married , and 287 ( 60.6% ) were christians .
more than half 293 ( 61.7% ) of the respondents had secondary education while 267 ( 55.9% ) were traders and less than half 141 ( 32.2% ) were of high socioeconomic status .
majority of the caregivers 415 ( 87.6% ) had good knowledge of the symptoms of malaria in under - five children .
symptoms mentioned were fever 456 ( 95.6% ) , poor appetite 236 ( 49.5% ) , vomiting 132 ( 27.6% ) , and headache 83 ( 17.4% ) .
chloroquine was the most known antimalarial drug 465 ( 97.3% ) and 312 ( 65.3% ) knew about acts .
this was followed by chloroquine 142 ( 29.7% ) , sulphadoxine - pyrimethamine 103 ( 21.5% ) , amodiaquine 19 ( 4.0% ) , and quinine 6 ( 1.3% ) .
one hundred seventy - three ( 36.2% ) knew that act was the antimalarial drug recommended by the government for the treatment of malaria .
most of the respondents 420 ( 88.8% ) gave their febrile child antimalarial drug , 27 ( 5.6% ) gave herbs , and 17 ( 3.6% ) considered the fever not serious enough for the child to be given antimalarial drug .
out of those that gave their child antimalarial drug , 332 ( 79.2% ) did so within 24 hours of onset of fever while 86 ( 20.6% ) responded within 48 hours of onset of fever .
respondents who were aware of amfm programme were 43 ( 9.1% ) and less than half 204 ( 42.9% ) have seen the antimalarial packet that has the symbol of the affordable medicine facility - malaria .
those who were aware of the programme got the information from diverse sources and the commonest source of information was through the radio 13 ( 31.7% ) . for those who have seen the antimalarial packet that has the amfm symbol , the commonest place where they saw it was at the pharmacy 125 ( 62.8% ) . out of those who have seen the symbol , only 97 ( 47.5% ) had a good knowledge of what the symbol meant .
most of the respondents who mentioned they have seen the amfm symbol 183 ( 89.7% ) when shown the packet felt it meant nothing , 23 ( 11.3% ) knew it meant subsidized antimalarial drug , and 17 ( 8.3% ) knew it meant act .
overall , 123 ( 29.2% ) used amfm - act as their first line antimalarial drug ( table 2 ) .
the mean price of the drug was 242.0 90.39 which is equivalent to $ 1.5 ( 0.6 ) .
forty - eight ( 39.7% ) bought it from ppmvs which constituted the commonest source of amfm - acts .
about 43 ( 35% ) mentioned that health workers prescribed amfm - acts , 63 ( 51.2% ) administered the drug based on self - medication , and 17 ( 13.8% ) did so based on recommendation by neighbours , friends , and relations .
more than half 67 ( 54.5% ) cited effectiveness of the drug as the main reason for choosing it while 42 ( 34.1% ) mentioned recommendation by people as a basis for buying the drug .
majority of the respondents 101 ( 82.1% ) reported availability of the antimalarial whenever they go to buy .
with regard to geographical access , 93 ( 89.4% ) had good access to the drugs .
significant factors at 5% level of significance were put in the logistic regression model . age group ( 2534 years ) ( p < 0.05 ) , public servants ( p < 0.001 ) , respondents with tertiary education ( p < 0.001 ) , respondents with high socioeconomic status ( p < 0.001 ) , respondents with poor knowledge of symptoms of malaria in under - five children ( p < 0.05 ) , awareness of amfm - acts ( p < 0.001 ) , availability of amfm - acts ( p < 0.001 ) , and sources of drug being private clinic ( p < 0.001 ) were significantly associated with the use of amfm - acts .
on logistic regression only people who were aware of amfm - acts predicted its use ( aor : 0.073 ; ci : 0.0320.166 ) ( table 3 ) .
the success of malaria control programme hinges on people 's awareness of its existence and utilization of interventions provided .
many studies on amfm have examined the affordability and availability of the drug from the providers ' point of view .
this study focused basically on the consumers / end users to see if they are aware , if they use the drug and factors influencing their use of the drug .
findings from this study showed that a high proportion of caregivers of children less than five were not aware of the initiative among caregivers of children less than five years .
this finding was consistent with a study done in jos which found that majority of the caregivers were not aware of artemisinin - based combination therapy when it was adopted as the treatment of first choice for uncomplicated malaria .
a literature on knowledge , attitudes , and practices about malaria treatment and prevention in uganda also found radio as the commonest source of information on malaria .
the mean price of amfm - acts was $ 1.5 ( 0.6 ) . this price is more than the stipulated price for children 's dose .
studies have shown that the price at which amfm - acts are sold in some countries that has adopted the initiative was less than the unsubsidized acts but more than the government recommended retail price [ 3 , 15 ] .
for example , a study done in nigeria and ghana found the adult dose of amfm - acts to be cheaper than non - amfm drugs but more than the anticipated price by government .
this suggests that the objective of amfm to increase affordability of the drug has been partly achieved .
although prices are much lower for amfm - acts , research should still be carried out to explore why the drugs are being sold at prices higher than the stipulated government price by drug sellers and other healthcare providers .
an assessment of the use of antimalarial drugs 2448 hours of onset of fever gave intriguing result .
the study showed that a similar proportion of caregivers used chloroquine and amfm - acts as their first line drugs .
this is contrary to findings from nationally representative outlet surveys in six sub - saharan countries in 2011 ( benin , the democratic republic of congo , madagascar , nigeria , uganda , and zambia ) which found that nonartemisinin monotherapies were the most commonly acquired antimalarial drugs for children less than five years of age .
utilization of chloroquine has been fuelled by its comparatively low price , familiarity of these older products , past successful treatment experiences , and excessive pill burden of artemether - lumefantrine .
however , use of amfm - acts can be increased by scaling - up awareness campaign at community level by the government and , non - government organizations involved .
there is a possibility of amfm - acts knocking out cheap and long used antimalarials if this is done .
this possibility is assumed based on the success of subsidized acts that were introduced in kenya and uganda .
increased sensitisation about the treatment failure rates and the additional costs that could be incurred due to treatment failure both among health professionals and the community could also help to increase utilization of subsidised acts and caregivers desist from using chloroquine and sp .
majority of the caregivers in this study got their drugs from ppmvs . the literature on treatment seeking pattern in sub - saharan including nigeria revealed that medicine sellers are widely used as sources of drug for malaria and fever [ 19 , 20 ] .
majority of the caregivers reported availability of the drug whenever they go to buy it .
this finding is in consonance with a study done in seven african countries in 2012 ( ghana , kenya , madagascar , niger , nigeria , uganda , and tanzania ) which found out that there was increase in availability and market share of quality assured acts ( qaact ) at the ppmvs except for niger and madagascar .
another study carried out to determine artemisinin - based combination therapy availability and use in the private sector of five amfm phase 1 countries found that market penetration of one or more amfm qaact was high both in rural and urban areas . in this study
this was similar to a study on insecticides treated net where awareness predicted the ownership and use of the nets .
similarly , in a study in kenya , community awareness activities were reported to have been instrumental to a substantial increase in act availability and coverage .
the main limitation of this study was that caregiver 's pattern of use of amfm - acts was estimated using caregiver 's self - report .
recall bias may likely occur from this , but it was minimized by asking for their pattern of use in the last two weeks prior to the survey .
findings from this study revealed that awareness of amfm - acts was low and this translated to low utilization of amfm - acts .
this may hinder achieving the objectives of amfm which are to expand access to acts . scaling - up of programmes to increase awareness is needed to increase coverage . | introduction .
distribution of affordable medicine facility - malaria artemisinin combination therapies ( amfm - acts ) started in nigeria in 2011 , but its use at community level has not been documented .
methods .
four hundred seventy - eight caregivers whose under - five children had fever within two weeks prior to the survey were selected using cluster sampling technique .
information on sociodemographic characteristics , treatment seeking for malaria , and awareness and use of amfm - acts was collected using an interviewer administered questionnaire .
result .
more than half of the respondents ( 51.2% ) bought amfm - acts without prescription .
awareness of amfm was low as only 9.1% has heard about the programme .
overall , 29.2% used amfm - acts as their first line choice of antimalarial drug .
on bivariate analysis age , group ( 2534 years ) , public servants , respondents with tertiary education , respondents with high socioeconomic status , respondents with poor knowledge of symptoms of malaria , awareness of amfm - acts , availability of amfm - acts , and sources of drug were significantly associated with utilization of amfm - acts ( p < 0.05 ) .
logistic regression demonstrated that only people who were aware of amfm - acts predicted its use ( aor : 0.073 ; ci : 0.0320.166 ; p < 0.001 ) . conclusion .
interventions which targeted at raising awareness of amfm - acts among people at risk of malaria are advocated for implementation . |
thrombocytopenia is a well known complication in intensive care unit
( icu ) patients .
the incidence of thrombocytopenia of less than
100 10platelets / l in icu patients has been reported to vary
from 23 to 41% , but lower frequencies ( 1017% ) have been reported for counts
lower than 50 10 platelets / l .
mortality rates as high as 3854%
have been observed , and have been reported to be proportional to the nadir of
the platelet count .
previous studies have not clearly demonstrated that
thrombocytopenia results in increased mortality or increased transfusion
requirements , however .
two independent factors have made this important and
seemingly straightforward issue difficult to resolve .
numerous studies have
demonstrated that severe underlying illness predisposes to the development of
thrombocytopenia in icu patients , and the influence of thrombocytopenia on
mortality is therefore difficult to assess .
although thrombocytopenia in the
critically ill is more often a symptom than a disease process per se ,
it might increase mortality in several ways .
thrombocytopenia can result in a
mild , moderate , or severe haemorrhagic disorder , which could enhance the risk
of morbidity and mortality in critically ill surgical patients .
the adverse effects of anaemia in such patients have also
recently been discussed .
apart from its haemostatic effect , thrombocytopenia also increases the
susceptibility to and severity of certain infections .
although several studies have found a relationship
between thrombocytopenia and the likelihood of death , especially in septic
patients ,
thrombocytopenia has rarely been identified as an independent predictive factor
of death using multiple logistic regression .
nevertheless , these previous
studies failed to reach any definite conclusions regarding whether
thrombocytopenia per se was responsible for the poorer prognosis , or
whether this higher mortality simply reflected more severe underlying
illness .
second , the threshold value for severe thrombocytopenia that is
supposed to jeopardize the prognosis is difficult to determine .
we and others
have suggested that a platelet count less than 50 10/l is
associated with a poor outcome .
moreover , guidelines for platelet transfusion
have proposed that the threshold value of
50
one of the commonest methods to evaluate excess mortality is to
perform a case - control study in which confounding variables ( eg severity of
underlying illness , reason for hospitalization , and so forth ) are carefully
matched in the two populations .
to date , however , no case - control studies that
have evaluated morbidity and mortality associated with thrombocytopenia in icu
patients have been published in which these important variables have been
carefully matched .
we therefore designed a case - control study to determine to what extent
severe thrombocytopenia ( defined as < 50 10 platelets / l )
increases mortality and blood product requirements in surgical icu
patients .
we performed a matched cohort study , with a matched control patient
without thrombocytopenia for each thrombocytopenic patient ( 1:1 matching ) .
the
study was conducted in the service de ranimation chirurgicale of tenon
university hospital in paris .
this 8-bed icu admits patients from all surgical
departments and operating rooms of the hospital .
the study period ran from
january 1 , 1996 to december 31 , 1996 , during which time 298 patients were
admitted to the icu and thrombocytopenic patients were prospectively
identified .
patients who
experienced even a single episode of thrombocytopenia of less than
50 10platelets / l during the icu stay were classified as
' cases ' .
platelet transfusions were administered to actively
bleeding patients and patients scheduled for emergency surgery if their
platelet count fell below 50 10/l . likewise , platelet
transfusions were administered to patients at risk for bleeding complications
( eg postoperative patients , or after gastrointestinal bleeding ) when their
platelet count fell below 20 10/l .
none of patients with a history of platelet disorders , haematologic
malignancies or chemotherapy , splenectomy , mechanical heart valves , or patients
undergoing cardiopulmonary bypass surgery were included in the patient
population .
control patients had to have had no evidence of severe
thrombocytopenia ( < 50 10
a computer - generated list of eligible control
patients was obtained from a database that included 695 patients hospitalized
between november , 1995 and march , 1998 .
control patients were selected
according to the following matching criteria : age ( 5 years ) , acute
physiology and chronic health evaluation ( apache ) ii score calculated on the
first day of icu admission ( 5 points ) , primary diagnosis and duration of
stay in the icu ( 5 days ) .
the list of potential control patients was
reviewed for the best possible match , giving highest priority to primary
diagnosis , duration of stay in the icu , apache ii score and age . in the case of
multiple acceptable control patients , the one with the date of icu admission
closest to that of the patient was chosen .
the following information was recorded : primary diagnosis ; age ; sex ;
dates of admission and discharge from the icu ; previous health status ; severity
of underlying medical conditions stratified according to the criteria of mccabe
and jackson as fatal , ultimately fatal and
nonfatal ; first - day organ dysfunction and/or infection ( odin ) score , based on the presence or absence of cardiac , respiratory ,
renal , hepatic , neurologic , haematologic dysfunctions and/or infection ; red
blood cells ( rbcs ) ; fresh frozen plasma and platelet transfusion requirements ;
and full blood count and coagulation studies ( factor v assay and d - dimer
detection were performed only when prothrombin time was abnormal ) .
the severity
of illness was evaluated with the first - day apache ii score and the first - day new simplified acute physiology score
( saps ) ii .
shock was defined as a decrease in systolic blood
pressure ( < 90 mmhg ) despite adequate vascular filling or the need for
vasoactive drugs ( dopamine > 5
the definition of disseminated intravascular coagulation ( dic )
required the following four criteria : a platelet count less than
150 10/l ; a decrease in prothrombin level activity to less
than 50% ; a decrease in the level of factor v to less than 50% ; and the
presence of fibrin degradation products ( d - dimers ) .
analysis of prior data from our unit indicated that the odds ratio
for death was 3.9 for cases and that the mortality rate for control patients
was 23% .
on the basis of these results , we estimated
that 28 patients in each group ( control and case ) would be required to test the
null hypothesis at the 0.05 significance level with a power of 0.7 .
the attributable mortality due to severe thrombocytopenia was
defined as the crude mortality rate of the control patients subtracted from
that of the cases .
the point estimate of the attributable mortality and 95%
confidence intervals ( cis ) were calculated as previously described .
mcnemar 's test with continuity correction and exact
binomial probabilities were used to determine whether the crude mortality rates
of the cases and control patients were significantly different .
because the study involved a matched cohort , the odds
ratio was used as a measure of relative risk . because incidence of outcome is
more than 10% , correction of the odds ratio
was made according to the method of
zhang and yu in order to represent better the true
relative risk .
the 95% ci of the odds ratio was calculated using
miettinen 's test - based approach .
differences in medians were tested using wilcoxon 's test for
continuous variables and the test for categorical variables ,
with yates ' correction when appropriate .
we performed a matched cohort study , with a matched control patient
without thrombocytopenia for each thrombocytopenic patient ( 1:1 matching ) .
the
study was conducted in the service de ranimation chirurgicale of tenon
university hospital in paris .
this 8-bed icu admits patients from all surgical
departments and operating rooms of the hospital .
the study period ran from
january 1 , 1996 to december 31 , 1996 , during which time 298 patients were
admitted to the icu and thrombocytopenic patients were prospectively
identified .
patients who
experienced even a single episode of thrombocytopenia of less than
50 10platelets / l during the icu stay were classified as
' cases ' .
platelet transfusions were administered to actively
bleeding patients and patients scheduled for emergency surgery if their
platelet count fell below 50 10/l . likewise , platelet
transfusions were administered to patients at risk for bleeding complications
( eg postoperative patients , or after gastrointestinal bleeding ) when their
platelet count fell below 20 10/l .
none of patients with a history of platelet disorders , haematologic
malignancies or chemotherapy , splenectomy , mechanical heart valves , or patients
undergoing cardiopulmonary bypass surgery were included in the patient
population .
control patients had to have had no evidence of severe
thrombocytopenia ( < 50 10 platelets / l ) at any time during
hospitalization in the icu .
a computer - generated list of eligible control
patients was obtained from a database that included 695 patients hospitalized
between november , 1995 and march , 1998 .
control patients were selected
according to the following matching criteria : age ( 5 years ) , acute
physiology and chronic health evaluation ( apache ) ii score calculated on the
first day of icu admission ( 5 points ) , primary diagnosis and duration of
stay in the icu ( 5 days ) .
the list of potential control patients was
reviewed for the best possible match , giving highest priority to primary
diagnosis , duration of stay in the icu , apache ii score and age . in the case of
multiple acceptable control patients ,
the one with the date of icu admission
closest to that of the patient was chosen .
the following information was recorded : primary diagnosis ; age ; sex ;
dates of admission and discharge from the icu ; previous health status ; severity
of underlying medical conditions stratified according to the criteria of mccabe
and jackson as fatal , ultimately fatal and
nonfatal ; first - day organ dysfunction and/or infection ( odin ) score , based on the presence or absence of cardiac , respiratory ,
renal , hepatic , neurologic , haematologic dysfunctions and/or infection ; red
blood cells ( rbcs ) ; fresh frozen plasma and platelet transfusion requirements ;
and full blood count and coagulation studies ( factor v assay and d - dimer
detection were performed only when prothrombin time was abnormal ) .
the severity
of illness was evaluated with the first - day apache ii score and the first - day new simplified acute physiology score
( saps ) ii .
shock was defined as a decrease in systolic blood
pressure ( < 90 mmhg ) despite adequate vascular filling or the need for
vasoactive drugs ( dopamine > 5
the definition of disseminated intravascular coagulation ( dic )
required the following four criteria : a platelet count less than
150 10/l ; a decrease in prothrombin level activity to less
than 50% ; a decrease in the level of factor v to less than 50% ; and the
presence of fibrin degradation products ( d - dimers ) .
analysis of prior data from our unit indicated that the odds ratio
for death was 3.9 for cases and that the mortality rate for control patients
was 23% .
on the basis of these results , we estimated
that 28 patients in each group ( control and case ) would be required to test the
null hypothesis at the 0.05 significance level with a power of 0.7 .
the attributable mortality due to severe thrombocytopenia was
defined as the crude mortality rate of the control patients subtracted from
that of the cases .
the point estimate of the attributable mortality and 95%
confidence intervals ( cis ) were calculated as previously described .
mcnemar 's test with continuity correction and exact
binomial probabilities were used to determine whether the crude mortality rates
of the cases and control patients were significantly different .
because the study involved a matched cohort , the odds
ratio was used as a measure of relative risk . because incidence of outcome is
more than 10% , correction of the odds ratio
was made according to the method of
zhang and yu in order to represent better the true
relative risk .
the 95% ci of the odds ratio was calculated using
miettinen 's test - based approach .
differences in medians were tested using wilcoxon 's test for
continuous variables and the test for categorical variables ,
with yates ' correction when appropriate .
during the study period , 36 of the 298 studied icu patients
developed severe thrombocytopenia of less than 50 10
platelets / l , producing a global incidence rate of 12 episodes of
thrombocytopenia per 100 admissions .
thrombocytopenia occurred 3.2 4.8
days ( median 2 days , interquartile range 2.5 days , range 026 days ) after icu
admission , for a mean duration of 3.4 2.9 days ( median 2 days ,
interquartile range 4 days , range 113 days ) .
thrombocytopenia was related to
sepsis in eight patients ; sepsis and dic in 11 patients ; bleeding in nine
patients ; bleeding and dic in six patients ; undetermined shock and dic in one
patient ; and haemolysis - elevated liver enzymes - low platelets syndrome with dic
in one patient .
the mean platelet count was 127
75 10/l ( median 116 ) versus 199
123 10/l ( median 161 ) in cases and control patients ,
respectively ( not significant ) . during icu stay , mean nadir platelet count was
28 14 10/l ( median 29.5 ) versus 184
142 10/l ( median 114 ) ; mean nadir haemoglobin value was
6.9 1.7g / dl ( median 7.2 ) versus 8.7 2.3g / dl ( median 8 ;
p = 0.009 ) ; and mean nadir leucocyte count was 6.2
4.1 10/l versus 10.2 4.9 10/l
( p < 0.0004 ) in cases and control patients , respectively .
nineteen
cases ( 52.7% ) and eight control patients ( 22.2% ) had evidence of dic
( p < 0.01 ) .
the median age of the cases was 65 years ( range 27104 years , mean
60.7 years ) versus 65 years ( range 2783 years , mean 60.6 years ) for control
patients .
twenty - one ( 58% ) case - control pairs differed by no more than 3 years ,
and 27 ( 75% ) by no more than 5 years ( table 1 ) .
the
median apache ii score of cases was 21 ( range 541 , mean 20.8 ) versus 21 for
control patients ( range 437 , mean 22.7 ) .
the median duration of icu stay of
cases was 5 days ( range 135 days , mean 8.3 days ) versus 4 days for control
patients ( range 126 days , mean 6.4 days ) .
thirty - five ( 97% ) of the pairs were
matched for primary diagnosis .
overall , matching was successful for 243 out of
288 ( 84.3% ) variables . because the study end point mortality is strongly related to
severity of illness and/or initial respiratory status , we controlled matching by
comparing another seven criteria that evaluated the severity of disease in the
two groups of patients ( table 2 ) .
no significant
differences in these indices were observed between cases and control patients .
this was particularly the case when comparing the four variables that were
individually predictive of survival for all patients : mean saps ii score , mean
number of organ - system failures , number of patients with cardiac failure on
admission , and number of patients with renal failure on admission .
finally , the dates of admission of
cases and control patients differed by less than 1 year in 27 pairs ( 75% ) .
seventeen ( 47% ) cases died , versus 10 ( 28% ) control patients ( table 3 ) .
twenty - seven matched pairs had a concordant outcome
( 18 lived and nine died ) .
nine pairs had a discordant outcome , and in eight of
these pairs the case died ( exact binomial probabilities : 0.037 ) .
the estimated
attributable mortality was 19.5% ( 95% ci 3.235.8 ) , and the estimated odds
ratio was 2.7 ( 95% ci 1.027.10 ) .
primary diagnosis in the nine discordant case - control pairs was
septic shock in six pairs , acute pancreatitis in one pair , undetermined shock
in one pair and haemorrhagic shock in one pair .
causes of mortalities included
refractory septic shock in eight cases ( 47% ) and four control patients ( 40% ) ;
multiple organ failure - related sepsis in five cases ( 29% ) and three control
patients ( 30% ) ; uncontrolled bleeding in four cases ( 24% ) and two control
patients ( 20% ) ; and one undetermined shock in one contol ( 10% ) .
thus , 17 cases experienced a thrombocytopenia
below 50 10 platelets / l for more than 3 days .
mortality rate
was 47% , versus 53 in patients without a prolonged thrombocytopenia ( not
significant ) .
likewise , among the 16 cases transfused with platelets , eight
died ( 50% ) compared with nine ( 45% ) cases who were not transfused ( not
significant ) .
a last sensitivity analysis was done on the timing variable . in
15 cases , thrombocytopenia occurred more than 2 days after icu admission .
mortality rate was 65% in these patients compared with 35% in cases in whom
thrombocytopenia occurred 2 days or less after icu admission ( p
= 0.008 ) .
two time - point analyses of illness severity scores in the nine
discordant pairs showed that the apache ii , saps ii and odin model scores were
significantly higher for cases than for control patients on the day of
thrombocytopenia ( table 4 ) .
moreover , cases had
comparable values of severity of illness scores between day of admission and
day of thrombocytopenia ; this was contrary to the situation with control
patients , who had a statistically significant decrease in severity of illness
scores during the same period ( table 4 ) .
thirty ( 83% ) cases required transfusion of blood products , versus 21
( 58% ) control patients ( table 5 ) .
thirteen pairs had
discordant transfusion requirements , and in 11 of these pairs the cases were
transfused ( paired 4.92 , p < 0.04 ) .
the estimated
attributable transfusion requirement was 25% ( 95% ci 5.444.6 ) , and the
estimated odds ratio was 1.52 ( 95% ci 1.052.20 ) .
units of rbcs transfused were 8.0 7.0 ( median 7.5 ) versus
2.6 4.3 ( median 1.5 ; p < 0.0001 ) , and units of fresh frozen
plasma transfused were 5.0 7.0 ( median 1.0 ) versus 1.0 2.0 ( median
0 ; p = 0.05 ) in cases and control patients , respectively .
sixteen cases
( 44.4% ) received platelet transfusion ( 5.3 7.7 units of platelets
transfused ) , versus no control patients . in eight patients ( one died ) , platelet
transfusions resulted in a rise of platelet count to greater than
50 10/l , with a correction of thrombocytopenia a few days
later . in eight patients ( seven died ) , only a transient rise of platelet count
was noted and thrombocytopenia persisted during the entire icu stay , despite
platelet transfusion .
after onset of thrombocytopenia , 4.4 7.2 units of
rbcs and 2.5 5.4 units of fresh frozen plasma were transfused .
during the study period , 36 of the 298 studied icu patients
developed severe thrombocytopenia of less than 50 10
platelets / l , producing a global incidence rate of 12 episodes of
thrombocytopenia per 100 admissions .
thrombocytopenia occurred 3.2 4.8
days ( median 2 days , interquartile range 2.5 days , range 026 days ) after icu
admission , for a mean duration of 3.4 2.9 days ( median 2 days ,
interquartile range 4 days , range 113 days ) .
thrombocytopenia was related to
sepsis in eight patients ; sepsis and dic in 11 patients ; bleeding in nine
patients ; bleeding and dic in six patients ; undetermined shock and dic in one
patient ; and haemolysis - elevated liver enzymes - low platelets syndrome with dic
in one patient .
the mean platelet count was 127
75 10/l ( median 116 ) versus 199
123 10/l ( median 161 ) in cases and control patients ,
respectively ( not significant ) . during icu stay , mean nadir platelet count was
28 14 10/l ( median 29.5 ) versus 184
142 10/l ( median 114 ) ; mean nadir haemoglobin value was
6.9 1.7g / dl ( median 7.2 ) versus 8.7 2.3g / dl ( median 8 ;
p = 0.009 ) ; and mean nadir leucocyte count was 6.2
4.1 10/l versus 10.2 4.9 10/l
( p < 0.0004 ) in cases and control patients , respectively .
nineteen
cases ( 52.7% ) and eight control patients ( 22.2% ) had evidence of dic
( p < 0.01 ) .
the median age of the cases was 65 years ( range 27104 years , mean
60.7 years ) versus 65 years ( range 2783 years , mean 60.6 years ) for control
patients .
twenty - one ( 58% ) case - control pairs differed by no more than 3 years ,
and 27 ( 75% ) by no more than 5 years ( table 1 ) .
the
median apache ii score of cases was 21 ( range 541 , mean 20.8 ) versus 21 for
control patients ( range 437 , mean 22.7 ) .
the median duration of icu stay of
cases was 5 days ( range 135 days , mean 8.3 days ) versus 4 days for control
patients ( range 126 days , mean 6.4 days ) .
thirty - five ( 97% ) of the pairs were
matched for primary diagnosis .
overall , matching was successful for 243 out of
288 ( 84.3% ) variables . because the study end point mortality is strongly related to
severity of illness and/or initial respiratory status , we controlled matching by
comparing another seven criteria that evaluated the severity of disease in the
two groups of patients ( table 2 ) .
no significant
differences in these indices were observed between cases and control patients .
this was particularly the case when comparing the four variables that were
individually predictive of survival for all patients : mean saps ii score , mean
number of organ - system failures , number of patients with cardiac failure on
admission , and number of patients with renal failure on admission .
finally , the dates of admission of
cases and control patients differed by less than 1 year in 27 pairs ( 75% ) .
seventeen ( 47% ) cases died , versus 10 ( 28% ) control patients ( table 3 ) .
twenty - seven matched pairs had a concordant outcome
( 18 lived and nine died ) .
nine pairs had a discordant outcome , and in eight of
these pairs the case died ( exact binomial probabilities : 0.037 ) .
the estimated
attributable mortality was 19.5% ( 95% ci 3.235.8 ) , and the estimated odds
ratio was 2.7 ( 95% ci 1.027.10 ) .
primary diagnosis in the nine discordant case - control pairs was
septic shock in six pairs , acute pancreatitis in one pair , undetermined shock
in one pair and haemorrhagic shock in one pair .
causes of mortalities included
refractory septic shock in eight cases ( 47% ) and four control patients ( 40% ) ;
multiple organ failure - related sepsis in five cases ( 29% ) and three control
patients ( 30% ) ; uncontrolled bleeding in four cases ( 24% ) and two control
patients ( 20% ) ; and one undetermined shock in one contol ( 10% ) .
thus , 17 cases experienced a thrombocytopenia
below 50 10 platelets / l for more than 3 days .
mortality rate
was 47% , versus 53 in patients without a prolonged thrombocytopenia ( not
significant ) .
likewise , among the 16 cases transfused with platelets , eight
died ( 50% ) compared with nine ( 45% ) cases who were not transfused ( not
significant ) .
a last sensitivity analysis was done on the timing variable . in
15 cases ,
mortality rate was 65% in these patients compared with 35% in cases in whom
thrombocytopenia occurred 2 days or less after icu admission ( p
= 0.008 ) .
two time - point analyses of illness severity scores in the nine
discordant pairs showed that the apache ii , saps ii and odin model scores were
significantly higher for cases than for control patients on the day of
thrombocytopenia ( table 4 ) .
moreover , cases had
comparable values of severity of illness scores between day of admission and
day of thrombocytopenia ; this was contrary to the situation with control
patients , who had a statistically significant decrease in severity of illness
scores during the same period ( table 4 ) .
thirty ( 83% ) cases required transfusion of blood products , versus 21
( 58% ) control patients ( table 5 ) .
thirteen pairs had
discordant transfusion requirements , and in 11 of these pairs the cases were
transfused ( paired 4.92 , p < 0.04 ) .
the estimated
attributable transfusion requirement was 25% ( 95% ci 5.444.6 ) , and the
estimated odds ratio was 1.52 ( 95% ci 1.052.20 ) .
units of rbcs transfused were 8.0 7.0 ( median 7.5 ) versus
2.6 4.3 ( median 1.5 ; p < 0.0001 ) , and units of fresh frozen
plasma transfused were 5.0 7.0 ( median 1.0 ) versus 1.0 2.0 ( median
0 ; p = 0.05 ) in cases and control patients , respectively .
sixteen cases
( 44.4% ) received platelet transfusion ( 5.3 7.7 units of platelets
transfused ) , versus no control patients . in eight patients ( one died ) , platelet
transfusions resulted in a rise of platelet count to greater than
50 10/l , with a correction of thrombocytopenia a few days
later . in eight patients ( seven died ) , only a transient rise of platelet count
was noted and thrombocytopenia persisted during the entire icu stay , despite
platelet transfusion .
after onset of thrombocytopenia , 4.4 7.2 units of
rbcs and 2.5 5.4 units of fresh frozen plasma were transfused .
the present results suggest that thrombocytopenia of less than
5010platelets / l in icu patients is associated with increased
mortality , with a relative risk of 2.7 ( 95%ci 1.02 - 7.10 ) , and with excess blood
product consumption , with a relative risk of 1.52 ( 95%ci 1.05 - 2.20 ) .
the incidence rate of thrombocytopenia reported in the present study
( 12% surgical icu patients ) is in the range reported in previous studies .
dic , immune mechanisms , and
haemophagocytic histiocytes are the commonest mechanisms of increased platelet
destruction during bacterial infection . in critically ill surgical patients ,
blood loss with subsequent volume replacement using crystalloids or colloids ,
when sufficiently severe , can significantly decrease the platelet count .
likewise , a fall in platelet count has been noted after
blood transfusions , and the incidence of thrombocytopenia has been specifically
reported to be directly proportional to the number of rbc transfusions .
patients with more severe underlying illness are more likely to
develop thrombocytopenia , and the severity of underlying illness has an important
impact on the outcome of icu patients with severe thrombocytopenia . as previously suggested , mortality
appears to be proportional to the nadir platelet count .
relatively
old studies reported that thrombocytopenia was associated with an
increased mortality rate in septic patients .
recent studies using multivariate
analysis have reported conflicting results , however . sprung et al
, in a large prospective study , identified
thrombocytopenia ( < 100 10
platelets / l ) as an independent
predictor of poor prognosis in septic patients , corresponding to a relative
risk of death of 1.66 ( 95%ci 1.062.60 ) . in a recent multicenter prospective
study , brun - buisson
et al also reported a
relative risk of late mortality of 1.5 ( 95% ci 1.22.0 ) in severely ill septic
thrombocytopenic patients ( platelet count < 50 10/l ) .
another smaller study showed comparable results in
septic patients . on the other hand , pittet
et al did not identify thrombocytopenia as an independent
predictor of mortality at the onset of sepsis in icu patients , like
brun - buisson et al did for early
mortality . finally ,
when the entire cohort of icu patients was studied ,
thrombocytopenia was not identified as a variable that was independently
associated with death in two studies .
these discrepencies may be attributable to differences in
the variables and analytical methods used , and could also result from the
complex interactions noted in multivariate analysis .
casecontrol studies require a considerably smaller sample size than
the corresponding longitudinal studies , and is as effective as the cohort study
as a basis for causal inference .
critical to the
validity of this study is the success in matching cases and control patients
for important confounding variables , especially the severity of underlying
illness .
we used three variables that are strongly correlated with outcome in
patients admitted to icus to match cases and control patients : age , a severity of disease
scoring system ( apache ii ) , and specific diagnostic categories according to the
one main reason for admission .
matching was 84.3% successful for 288 possible
variables . to verify the adequacy of matching for severity of underlying
illness and primary diagnosis independently , we compared the cases and control
patients with respect to another seven potentially confounding variables .
no
statistically significant differences in these indices were observed between
cases and control patients . it may be argued that we matched patients at the
time of admission .
we realize that this does not reflect the severity of
illness at the time of thrombocytopenia , and that matching on variables
recorded at the time or just before the diagnosis of thrombocytopenia may have
yielded different results .
the reliability of systems such as apache ii , saps
ii and odin is not sufficiently defined to study the daily probability of
death , however .
moreover , thrombocytopenia is linked to admission
characteristics and/or diagnosis , because nearly 60% of patients with
thrombocytopenia occurred during the first 2 days after icu admission .
we found that severe thrombocytopenia is associated with increased
mortality , mainly in septic patients , thereby confirming the results of
previous cohort studies . in the icu
setting , however , thrombocytopenia is a
symptom that has been associated with several acute diseases .
it is therefore difficult to distinguish causes from
consequences of severe thrombocytopenia clearly in critically ill patients . for
example
, it is difficult to state that bleeding not represent the results of
thrombocytopenia rather than the cause .
although matched case - control studies
are attractive because matching assumes comparability for certain variables ,
the finding of a higher mortality in cases compared with in control patients
only implies an association between mortality and thrombocytopenia .
thus , the
present analysis identified an increased difference in severity of illness
scores between cases and control patients between the day of admission and the
day of thrombocytopenia .
the finding that illness severity decreased over time
in control patients , but remained constant in the cases , supports the notion
that severe thrombocytopenia probably largely reflects the unfavourable course
of the underlying disease and possibly the aggravating role of thrombocytopenia
itself .
thrombocytopenia lasting for less than 3 days confers a similar risk to
that of thrombocytopenia for many days .
as the present results show , platelet
transfusion failed to restore a normal platelet count , but normalization of
platelet count , when it occured , was associated with better prognosis , as
previously reported .
patients who require intensive platelet support frequently have
complex medical problems , including dic , clinical bleeding and uncontrolled
sepsis .
patients in this setting receive multiple drugs and frequent blood
product transfusions . patients with thrombocytopenia secondary to sepsis often
require platelet transfusions , although their response is often suboptimal
because of continued platelet destruction , reflecting the persistance of the
underlying disease .
likewise , in patients receiving massive transfusion ,
thrombocytopenia is due both to consumption of platelets and to dilution by
transfused blood and colloid fluid resuscitation .
these patients may also have
platelet function defects due to dic , hypothermia and ' stunned '
recently transfused platelets .
patients may remain midly thrombocytopenic for
several days before having a rebound thrombocytosis . of interest ,
the mortality impact of thrombocytopenia
occurred during the first 2 days after icu admission , which is quite different
from late - onset icu - acquired thrombocytopenia .
this result is in agreement with
a previous analysis that indicated that late
mortality is essentially associated with the characteristics of the underlying
disease .
the mechanism of increased mortality directly related to
thrombocytopenia is difficult to assess , but several points should be
considered .
first , the present results show that severely thrombocytopenic
patients required more blood product transfusions .
in addition to the possible
volume overload , higher postoperative infection rates have been reported in
patients who received blood transfusions .
for example , in the absence of
neutropenia , thrombocytopenia has been positively correlated with an increased
incidence and severity of pneumonia in elderly patients .
two recent studies have shown that patients with a cardiovascular diagnosis and a
high degree of acute illness appear to be at increased risk of death from a
moderate degree of anaemia .
finally , severe thrombocytopenia is an important
risk factor for spontaneous intracerebral haemorrhage associated with a high
mortality in critically ill patients .
the present report shows that thrombocytopenia of less than
5010platelets / l is associated with excess mortality that is
independent of the patient 's age and initial severity of illness , and
leads to excess blood product consumption , thus imposing a significant economic
burden .
thrombocytopenia appears to be mostly a marker of severity of
underlying processes , rather than causally related to death .
thus , the exact
relationship between thrombocytopenia and mortality has yet to be elucidated ,
especially in septic patients .
further studies of the specific role of
thrombocytopenia in shock and infections are necessary .
moreover , the risks and
benefits of the various strategies for the management of thrombocytopenic icu
patients should be reevaluated in a variety of clinical settings .
the authors thank prof jean - yves fagon for his critical review of
the manuscript .
results of matching of cases to control patients using four major
criteria apache , acute physiology and chronic health evaluation ; ica ,
intensive care unit . matching criteria and clinical characteristics of the study
population
* miscellaneous included two cases of nosocomial
pneumonia , one case of acute pancreatitis , and one case of undetermined shock .
apache , acute physiology and chronic health evaluation ;
odin , organ dysfunstion and/or infection ; icu , intensive care unit ; sap ,
simplified acute physiology score .
crude mortality , attributable mortality , and odds ratio of death due
to severe thrombocytopenia in intensive care unit patients ci , confidence interval .
comparison of illness severity scores between cases and control
patients on the day of admission and on the day of onset of thrombocytopenia in
the nine discordant pairs * p < 0.05 by wilcoxon 's signed - rank
test ( score on day of admission versus score on day of thrombocytopenia in
control group ) . calculated from the equation proposed by le
gall et al . . crude transfusion requirements , attributable transfusion
requirements , and odds ratio of transfusion requirements due to severe
thrombocytopenia in intensive care unit patients ci , confidence interval | background : that thrombocytopenia results in increased mortality or
transfusion requirements has not been confirmed by previous studies .
we
performed a case - control study in which 36 patients who developed severe
thrombocytopenia of less than 50109
platelets / l were carefully
matched for the severity of underlying disease and other important
variables.results:seventeen ( 47% ) thrombocytopenic patients died , versus 10 ( 28% )
matched control patients who were not thrombocytopenic.nine pairs had a
discordant outcome , and in eight of these pairs the thrombocytopenic patient
died ( exact binomial probability 0.037 ) .
the estimated attributable mortality
was 19.5% ( 95% confidence interval 3.235.8 ) , and the estimated odds ratio was
2.7 ( 95% confidence interval 1.027.10 ) .
thrombocytopenic patients had
comparable values for severity of illness scores between day of admission and
day of thrombocytopenia , in contrast with control patients who had a
statistically significant decrease in severity of illness scores during the
same period .
thirty ( 83% ) of the thrombocytopenic patients required transfusion
of blood products , versus 21 ( 58% ) control patients ( paired 2
test 4.92 , p < 0.04 ) .
the estimated attributable transfusion
requirement was 25% ( 95% confidence interval 5.444.6 ) , and the estimated odds
ratio was 1.52 ( 95 confidence interval 1.052.20).conclusion : the present study suggests that thrombocytopenia of less than
50 109 platelets / l may be a marker for more severe illness and
increased risk of death , rather than causative , because a true causal
relationship is not established .
thrombocytopenia also leads to an excess of
blood product consumption . |
most almond ( prunus amygdalus batsch ) cultivars are self - incompatible ( si ; socias i company , 1990 ) .
si in the prunus species shows the gametophytic self - incompatibility ( gsi ) system , controlled by a single polymorphic locus containing at least two linked genes , one specifically expressed in the pistil and the other in the pollen ( kao and tsukamoto , 2004 ) .
pollen tube growth is arrested in the style whenever the single s allele expressed in the haploid pollen matches one of the two s haplotypes expressed in the diploid pistil tissue . the pistil component of si in rosaceae , solanaceae , and plantaginaceae has been determined to be an s - rnase ( mcclure et al . , 1989 ) .
the prunus
s - rnase is of the t2-type ( igic and kohn , 2001 ) , with five conserved domains ( c1 , c2 , c3 , rc4 , and c5 ) and one hyper - variable region ( sassa et al . , 1997 ) .
the candidate gene for the pollen component in almond has been identified to be an sfb by ushijima et al .
( 2003 ) , showing a tight association with the s - rnase gene ( ikeda et al . , 2005 ) . in spite of the knowledge on the genetic structure of the female and male determinants in si , the nature of their interactions remains unclear .
the s - rnases are proteins and , as such , are built from sequences of amino acid residues , encoded by the corresponding gene . the linked amino acid residues bond in space to form a 3d structure .
the knowledge of the 3d structure has been useful in order to understand how some proteins work and which molecular mechanisms underpin their function .
thus , the 3d structure of the s - rnase proteins involved in si may shed light on elucidating the recognition mechanism of gsi in the rosaceous species at the molecular levels to understand how these proteins mediate the gsi function to fulfill their biological roles .
protein structure can be determined experimentally using x - ray crystallography , nuclear magnetic resonance spectroscopy , and cryoelectron microscopy , but these approaches are time - consuming ( ida et al . , 2001 ) .
molecular modeling may be defined as the science and/or art that defines molecular structure and function and that yields a 3d model through computation .
protein structures are guided by two sets of principles operating on vastly different time scales .
the first set of principles is defined by the laws of physics , while the second set is directed by the theory of evolution .
each of these two sets of principles has led to the development of predictive methods to build 3d protein models ( hrmova and fincher , 2009 ) .
currently , one of the most popular comparative modeling programs is modeller ( sali and blundell , 1993 ) . it is a computer program that models 3d structures of proteins and their assemblies by satisfaction of spatial restraints .
the user provides an alignment of a sequence to be modeled with related 3d structures already known and modeller will calculate a new 3d model of a target protein .
the array of 1050 models typically produced by modeller can be evaluated to assess the stereo - chemical quality and the energy profiles of protein models .
thus , after selecting the best model , the structure needs to be put in perspective with a biological function and tested to see if the model is helpful in proposing a useful hypothesis in biology .
consequently , our objective was to identify the 3d structures of the almond s - rnases and sfbs through molecular modeling tools and to investigate a link between their 3d structures and the si mechanism .
three different s - rnases from two almond cultivars were modeled because their sequences and physiological activity were available ( fernndez i mart et al . , 2009 ) .
the s - rnase sequences have been deposited in the embl / ddbj / genbank under ab467371 ( sf - rnase from blanquerna ) , ab481108 ( s8-rnase from blanquerna ) , and ab488496 ( s23-rnase from vivot ) .
the modeling procedure started with the alignment of the sequence to be modeled ( target ) with related known 3d structures ( template ) derived from the protein data bank ( pdb ) using fasta and blast ( embl nucleotide database ) . in this procedure , the template to be selected among all possibilities must show the highest identity with the target , at least higher than 35% .
once the best candidate template was selected , the sequence adjustment between the s - rnase sequences and the template was performed manually to minimize the number of gaps and insertions / deletions .
the four models with the lowest value of the modeller objective function were chosen for further refinement .
this program detects errors in protein structures and thus serves to indicate their quality . on the other hand , stereo - chemical quality and the overall g - factors of the protein models
this software compares the residue - by - residue geometry of a set of closely related structures .
the models with lower number of amino acid residues in disallowed regions were selected as the most suitable models . a ramachandran plot ( also known as a ramachandran map or a ramachandran diagram ) outputted by procheck
visualizes dihedral angles against of amino acid residues in the protein structure , thus showing the possible conformation of and angles for a polypeptide ( ramachandran et al . , 1963 ) .
during a further modeling , the loop refinement protocol was used to generate 40 new models from the previously best model .
the same steps as described above were followed , selecting the best four models according to their lowest values of the modeller objective function , and then selecting the
finally , the molecular graphics were generated with pymol , which visualizes protein structures ( http://www.pymol.org ) .
the 3d models of the sf- , s23- , and s8-rnases were compared with the related known 3d structures derived from pdb . the best candidate template selected was the rnase mc1 mutant with accession 1j1 g ( numata et al . , 2003 ) , because the identity between this template and the target sequences was 42% . on the other hand , the sfbf , sfb8 , and sfb23 models could not be generated because sequence identity higher than 30% was not found in pdb .
protein structures represent combinations of secondary structural elements , -helices and -strands that are inter - connected by loops .
these structural elements form the core regions ( the inside of the molecule ) and are connected by loop regions on the protein surface with surface - exposed -helices and -strands the structure of the s - rnases belonged to the and class , with six -helices and six -strands connected by loops .
the folding topologies of its main chains were very similar to the topologies of the rnase t2 family enzymes .
ramachandran plot statistics for the s - rnases showed than 97% amino acid residues were positioned in the allowed regions .
in fact , when structures place 9597% or more of the amino acid residues in the allowed positions , they are considered to be reliable in modeling experiments , and this indicates how well the structures fits with the expected main chain length and torsion angle distributions ( laskowski et al . , 1993 ; kleywegt and jones , 1996 ) .
the best four models of the sf- , s23- , and s8-rnases were selected for further modeling .
as shown in figure 1 , in the model sfi1_bl00040001 ( figure 1a ) , all residues were positioned in the allowed region ( red arrow ) , whereas in the model sfi1_bl00010001 ( figure 1b ) , 1.6% of the residues were in a disallowed region ( green arrow ) .
higher numbers of residues in the disallowed region reflect a distorted geometry in the models , because there are higher proportions of residues falling outside the limits of main chain bond length and torsion angles derived from the small molecule library ( engh and huber , 1991 ) .
these results indicate that our models were optimal . the ramachandran plot of sfi1_bl00040001 ( a ) , and sfi1_bl00010001 ( b ) .
when the three s - rnases were superpositioned , the sf - rnase structure contained an additional extended loop , which was not present in the s8 or s23 models .
this loop , shown in figures 2 and 3 contained the amino acid residues ckg npq rqa ksq pkn rgk sqp ksq att qfl , which were placed between the conserved domains rc4 and c5 . through the software pymol , it has been possible to visualize which amino acid residues comprised -helices , -strands , and loops .
it has been suggested that loops in 3d structures serve to interconnect -helices and -strands , and also that longer surface - exposed loops could be susceptible to proteolytic degradation ( branden and tooze , 1998 ) . as the main structural difference found between the sf- , s8- , and s23-rnases resides in the presence of this extended looping region , this long loop could be prone to degradation and , as a consequence , this s - rnase could be less stable . as a result of this possible degradation , the pollen tube could grow through its own pistil giving rise to sc .
ribbon diagram ( a ) and surface representation ( b ) of modeled structure of almond sf - rnase ( magenta ) , s8-rnase ( cyan ) , and s23-rnase ( red ) , showing secondary structural elements and surfaces .
multiple sequence alignment of the sf , s8 , and s23 sequences of rnases , indicating the amino acid residues that belong to the extended loop in the sf 3d structure ( marked in blue ) .
additionally , the 3d models of the sf- , s8- , and s23-rnases were compared with that of another rosaceous species , the pyrus pyrifolia
s3-rnase ( matsuura et al . , 2001 ) .
the structure of the pear s3-rnase was consistent with the models of the almond s8 and s23 rnase .
the fact that both the pear and almond s - rnases confer si , and that their models did not contain this extended loop , the main structural differences between the si and the sc rnases could reside in the presence of the loop .
therefore , the amino acid residues that form the extended loop positioned at the surface of the sf - rnase ( figure 2 ) , between the conserved domains rc4 and c5 , could be responsible for the differences in function of rnases .
further studies are required to ascertain how this loop mediates si and to reveal if it indeed is involved in the si mechanism in plants .
the molecular nature of the s - locus has been widely studied in many species . in spite of the knowledge on the genetic structure of the female ( rnase ) and male ( sfb ) determinants of si ,
the presenceof a loop in the predicted structure of sf is a remarkable difference between this s - rnase allele and the other two analyzed in this work , thus we may suggest that this extended loop may be involved in the manifestation of self-(in)compatibility in almond .
however , further details and approaches are strongly needed in order to better understand the role of this loop in the si complex .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | gametophytic self - incompatibility ( gsi ) is a mechanism in flowering plants , to prevent inbreeding and promote outcrossing .
gsi is under the control of a specific locus , known as the s - locus , which contains at least two genes , the rnase and the sfb .
active s - rnases in the style are essential for rejection of haploid pollen , when the pollen s - allele matches one of two s - alleles of the diploid pistil .
however , the nature of their mutual interactions at genetic and biochemical levels remain unclear .
thus , detailed understanding of the protein structure involved in gsi may help in discovering how the proteins involved in gsi may function and how they fulfill their biological roles . to this end ,
3d models of the sc ( sf ) and two si ( s8 and s23 ) s - rnases of almond were constructed , using comparative modeling tools .
the modeled structures consisted of mixed and folds , with six helices and six -strands .
however , the self - compatible ( sf ) rnase contained an additional extended loop between the conserved domains rc4 and c5 , which may be involved in the manifestation of self - compatibility in almond . |
the ras association domain family 1 gene ( rassf1 ) was identified on chromosome 3p21.3 , a region frequently deleted in cancer .
there are two major transcripts of rassf1 , termed rassf1a and rassf1c , which are transcribed from different cpg island promoters . the promoter of rassf1a is often hypermethylated in cancer , whereas the promoter region of rassf1c is never methylated [ 2 , 3 ] .
both isoforms encode a ras association domain in the c - terminus , an atm - kinase phosphorylation site , a sarah protein interaction domain , and the n - terminal sequence of rassf1a harbors a diacyl glycerol binding domain [ 1 , 4 ] .
it has been demonstrated that rassf1a encodes a tumor suppressor gene , which reduces tumor growth in vivo and in vitro [ 1 , 58 ] .
it has been reported that rassf1a binds to microtubules and protects cells from microtubule destabilizing agents [ 7 , 1215 ] .
rassf1a is regulated by the binding of ras and the novel ras effector 1 ( nore1 ) and mediates proapoptotic signals through binding of the mammalian sterile 20-like kinase 1 and 2 ( mst1 and mst2 ) [ 1619 ] . moreover ,
an association of rassf1a with the bh3-like protein modulator of apoptosis was observed and this interaction regulates conformational change of bax and apoptosis [ 20 , 21 ] .
additionally , it was reported that rassf1a inhibits the anaphase promoting complex ( apc ) through its binding to cdc20 and induces mitotic arrest by stabilizing mitotic cyclins and it was further shown that the aurora mitotic kinases are involved . however , we were not able to verify the interaction between rassf1a and cdc20 .
in the c - terminal part of rassf1a and rassf1c , a protein - protein interaction domain called sarah ( sav / rassf / hpo ) has been determined .
the sarah domain is a key feature of the hippo signaling pathway components , by which the interaction of sav , rassf , and hpo is accomplished . in the drosophila hippo pathway , salvador ( sav ,
the human homologue is named ww45 ) acts as a scaffold protein that interacts with the proapoptotic kinase hippo ( hpo , human homologue mst ) [ 2729 ] .
hpo is able to phosphorylate the kinase warts ( human homologue lats ) , which in drosophila leads to cell cycle arrest and apoptosis [ 2830 ] .
it was shown that the single drosophila orthologue of the human rassf proteins restricts hpo activity by competing with sav for binding to hpo .
previously showed that human rassf1a interacts with mst1 through the c - terminus and more precisely through the sarah domain .
mst1 has two caspase 3 cleavage sites and both mst1 and mst2 play a role in processes of apoptosis both before and after caspase cleavage
. the cleaved form of mst1 translocates in the nucleus and phosphorylates histone h2b at ser14 [ 34 , 35 ] .
h2b phosphorylation correlates with apoptotic chromatin condensation and nuclear fragmentation in mammalians and yeast [ 35 , 36 ] .
following death receptor activation , mst1 ( homologue of hpo ) is known to become activated through caspase - dependent cleavage .
the cleaved fragment then localizes from the cytoplasm to the nucleus , where it induces apoptosis by chromatin condensation through activation of the c - jun n - terminal kinase pathway .
both rassf1a and ww45 activate mst2 by promoting its autophosphorylation . moreover , rassf1a stabilizes mst1/2 activation by preventing the dephosphorylation of these kinases . activated mst1/2 phosphorylates different targets including lats kinases , which in turn activate the transcription coactivator yap1 [ 40 , 41 ] .
to gain new insights into the tumor suppressor function of rassf1a , we deleted its sarah domain and analyzed its altered function .
deletion of the sarah domain resulted in a decreased colony formation of tumor cells . during mitosis , abnormal spindle formation was observed .
we demonstrate that the interaction of rassf1a and rassf1c with mst1 and mst2 depends on the sarah domain .
deregulation of the sarah domain may contribute to altered proapoptotic and mitotic signaling of rassf1a .
the localization experiments and the protein expression experiments were performed in hek293 and cos7 cells ( atcc , manassas , virginia , usa ) . for stable transfection , the lung cancer cell line a549 ( atcc ) was used .
the matchmaker two - hybrid system ( clontech , mountain view , usa ) was utilized .
the genes mst1 and mst2 were cloned after amplification of the fragments from est - clones irakp961c0282q and irakp961i0613q ( rzpd , berlin , germany ) , respectively . rassf1asarah and
rassf1a were cloned into the vector pgadt7 , rassf1c and rassf1csarah into the vector pas2 - 1 , and mst1 and mst2 into pgbkt7 [ 25 , 45 ] .
mutant forms of rassf1 were generated with the quickchange xl site - directed mutagenesis kit ( stratagene , la jolla , usa ) and forward primer ( 5-aggaaaatgactctgggccccttgggtgacctct ) and the complementary reverse primer .
the yeast strain pj69 - 4a was cotransformed with 0.1 g of each plasmid using the peg / liac method .
the interaction analysis was carried out on sd minimal medium plates without adenine and histidine and the transformation efficiency was controlled on sd plates with adenine and histidine .
the strength of interaction was investigated by quantification of the expression of the -galactosidase reporter gene with o - nitrophenyl--d - galactopyranoside ( onpg ) as substrate at 420 nm .
mst1 and mst2 were cloned into the vector pcmv - tag1 ( stratagene , la jolla , usa ) and/or in the vector pebg . to investigate the interaction of specific rassf1 forms , mst1 and mst2 , cotransfections ( lipofectamine 2000 , invitrogen , carlsbad , usa )
plasmids ( pebg and pcmv - tag 1 ) were used , that express gst - flag - rassf1a , gst - flag - rassf1c , gst - flag - rassf1asarah , gst - flag - rassf1csarah , and gst or flag - mst1 , flag - mst2 , and flag - ww45 .
the gst - fused proteins were precipitated with glutathione - sepharose ( amersham biosciences , freiburg , germany ) .
the interaction was determined with anti - flag - antibodies ( f3165 , sigma , steinheim , germany ) and anti - gst antibodies ( santa cruz , santa cruz , usa ) .
a549 were treated with 3 m staurosporine for 3 h or transfected with 10 g of constructs with turbofect for 36 h ( fermentas , st .
total protein was isolated using flag - lysis buffer , samples were denatured with laemmli - buffer , separated in 10% sds - page , and blotted onto pvdf membranes .
first antibodies are : anti - gapdh ( fl332 santa cruz , usa ) , anti - p - mst1 ( thr183)/mst2 ( thr180 ) ( # 3681 cell signaling , frankfurt , germany ) , anti - flag ( f3165 sigma , steinheim , germany ) , and secondary antibodies are hrp coupled ( sc2004/5 santa cruz , usa ) .
ecl ( wbkls0100 millipore , schwalbach , germany ) was used for detection with versadoc ( bio - rad , mnchen , germany ) .
rassf1a , rassf1asarah , and rassf1c were cloned into the vector pcmv - tag1 ( stratagene , la jolla , usa ) .
the lung cancer cell line a549 was transfected using lipofectamine 2000 ( invitrogen , carlsbad , usa ) .
colonies were selected under 1 mg / ml geneticin ( gibco , karlsruhe , germany ) in dmem and clones were picked after 4 weeks .
expression of flag - rassf1 was confirmed by rt - pcr using the flag - specific primer ( 5-tggattacaaggatgacgacg ) and rassf1-specific primer l27111 ( 5-tcctgcaaggagggtggcttc ) .
growth curves of stable transfected clones were analyzed by seeding 150,000 cells in triplicates in 6-well plates .
every 24 hours , cells were counted using a neubauer counting chamber . in order to investigate the proliferation in soft agar ,
experiments were performed in duplicates with 5,000 cells per plate under selection with 1 mg / ml geneticin .
colony size was measured after 4 weeks with a microscope ( leica dmirb , wetzlar , germany ) .
therefore , colonies were stained with 400 l of 5 mg / ml int and the size of 25 colonies was determined with metavue ( molecular devices gmbh , ismaning , mnchen ) .
rassf1a and rassf1c were cloned into the fluorescence vector peyfp - c2 ( clontech , mountain view , usa ) .
the deletion of the sarah domain of rassf1a and rassf1c was accomplished by the quickchange xl site - directed mutagenesis kit ( stratagene , la jolla , usa ) with the upper sarah deletion primer 5-aggaaaatgactctgggccccttgggtgacctct and the complementary lower primer .
after transient transfection into hek293 cells with lipofectamine 2000 ( invitrogen , carlsbad , usa ) , the localization of yfp - rassf1a , yfp - rassf1as , yfp - rassf1c , and the vector control were investigated with a fluorescence microscope .
cells were co - stained with anti--tubulin ( molecular probes , invitrogen , carlsbad , usa ) antibodies and alexa fluor goat anti - mouse ( molecular probes , invitrogen , carlsbad , usa ) antibodies to show the colocalization with the microtubules and spindle poles , respectively .
nuclei of the cells were visualized by staining with dapi ( 0,1 g / ml in pbs ) .
cells with highly condensed chromosomes and spindle structures were classified as mitotic cells . for microtubule stability analysis , the cells were treated one day after transfection for one hour with 20 m nocodazole , fixed , and stained with dapi and anti--tubulin antibodies .
the lung cancer cell line a549 was transiently transfected with different rassf1a constructs tagged with yellow fluorescence ( peyfp - c2 ) using lipofectamine 2000 ( invitrogen , carlsbad , usa ) . after two days ,
after fixation with formaldehyde , a tunel staining was performed with the in situ cell death detection kit , tmr red ( roche , mannheim , germany ) .
yellow fluorescence expressing cells ( 500 ) were counted and the rate of apoptotic cells ( red fluorescence ) was calculated .
all statistical evaluations were performed using the spss 12.0 software ( spss science , chicago , il ) . a probability of p < 0.05 was considered as significant .
in silico analysis of rassf1a ( 340 aa ) and rassf1c ( 270 aa ) by prosite ( www.expasy.org ) revealed the presence of a sav - rassf - hpo ( sarah ) domain at their c - terminus ( 290 to 337 and 220 to 267 , resp . )
( figure 1(a ) ) . to gain insight into the function of the sarah domain of rassf1 , the interaction of rassf1a , rassf1c , rassf1asarah , and rassf1csarah with mst1 and mst2
was investigated in the yeast two - hybrid system ( figures 1(b ) and 1(c ) ) . both rassf1 isoforms ( rassf1a and rassf1c )
interacted with mst1 and mst2 , but when the sarah domain was deleted the proteins were not able to interact anymore .
these interactions were quantified in an o - nitrophenyl--d - galactopyranoside ( onpg ) assay ( figure 1(c ) ) .
the interaction of rassf1a and rassf1c with mst1 and mst2 was verified by coprecipitation experiments ( figure 1(d ) and data not shown ) .
the interaction between rassf1a and mst1 and mst2 was confirmed ( figure 1(d ) ) .
there was also an interaction between rassf1c and mst1 and mst2 ( data not shown ) .
interaction of rassf1a and rassf1c with mst1 and mst2 was abolished , when the sarah domain was deleted ( figure 1(d ) and data not shown ) .
growth effects of the sarah domain of rassf1 were investigated in stable transfected lung cancer cells .
the lung cancer cell line a549 was transfected with rassf1a , rassf1c , and rassf1as , and the control vector ( pcmv - tag1 ) and the growth of these cells were evaluated ( figure 2 ) .
we picked stable transfected colonies and expression of rassf1-specific forms was confirmed by rt - pcr ( figure 2(a ) ) .
proliferation of rassf1a expressing cells was significantly reduced at 96 h compared to rassf1c expressing cells and control cells ( p = 0.022 and p = 0.007 , resp . ) .
rassf1as clone1 and clone2 had a significant reduction of growth at 96 h compared to rassf1c ( p = 0.027 and 0.042 , resp . ) and controls ( p = 0.018 and 0.029 , resp . ) .
subsequently , the anchorage independent growth was determined in soft agar experiments ( figures 2(c ) and 2(d ) ) . in these experiments , cells expressing rassf1as ( clone1 ) exhibited a significantly reduced colony growth ( average colony size : 22 m ) compared to rassf1a ( average : 36 m ; p < 0.01 , welch 's test ) and control ( average : 66 m ; p < 0.01 , welch 's test ) .
we transfected yellow fluorescent protein - tagged rassf1a and rassf1asarah ( rassf1as ) into hek293 and cos7 cells and the localization was determined by fluorescence microscopy ( figure 3(a ) ) .
rassf1a and rassf1as are both localized at the tubulin - containing cytoskeleton during interphase . in mitotic cells , rassf1a and rassf1as were detected at spindles and centrosomes ( figure 3(a ) ) . in the rassf1as expressing hek293 cells , multipolar spindles and unequal alignment of the chromosomes between poles were observed ( figure 3(a ) ) . in cos7 cells ,
interestingly , mitotic rate of rassf1as expressing cells was significantly ( p = 0.001 ) reduced to 3.4% compared to 8.6% in rassf1a transfected hek293 cells ( figure 3(b ) ) .
the majority ( 74.3% ; p < 0.001 ) of mitoses in rassf1as expressing cells were abnormal ( multi- or monopolar ) compared to rassf1a transfected cells , where only 0.6% of abnormal mitosis were counted ( figure 3(c ) ) . to determine
if the aberrant spindle formation in rassf1as expressing cells is due to an altered microtubule stability of these cells , transiently transfected cells were treated with 20 m nocodazole for one hour ( figure 4(a ) ) . in yfp control cells ,
rassf1a , rassf1as , and rassf1c overexpressing cells were able to stabilize microtubules from depolymerization by nocodazole ( figure 4(a ) and data not shown ) . to analyze the effect of the deletion of the sarah domain on the proapoptotic function of rassf1a , a transient transfection into the lung cancer cell line a549
was performed and the rate of transfected and apoptotic cells was calculated after 1 to 2 days ( figure 4(b ) ) .
apoptotic cells were stained using red fluorescence tunel - kit and yfp was used as a control .
the rate of apoptosis was significantly ( p = 0.001 ) higher , when the cells expressed rassf1a
( 28% ) in comparison to rassf1c ( 16% ) and the yfp control ( 14% , figure 4(b ) ) .
the deletion of the sarah domain ( rassf1as ) resulted in a significantly increased apoptotic rate of 39% compared to rassf1c ( p < 0.001 ) and rassf1a ( p = 0.006 , figure 4(b ) ) . in summary ,
our results show that expression of rassf1a with a deletion of the sarah domain deregulates normal mitotic progression and enhances apoptosis .
subsequently , rassf1a - induced autophosphorylation of mst1 and mst2 was analyzed in a549 lung cancer cells ( figure 5 ) .
for this propose , we have utilized an antibody that detects endogenous mst1 and mst2 only when phosphorylated at thr183 and thr180 , respectively .
treatment of a549 cells with 3 m staurosporine induced phosphorylation of mst1 and mst2 ( figure 5 ) , as described previously [ 34 , 46 ] . however , when a549 cells were transfected with rassf1a and rassf1asarah , phosphorylation of mst1 and mst2 was not detected ( figure 5 ) .
. showed that the mst1 kinase autoactivation through phosphorylation is inhibited by coexpression of rassf1a and rassf1c .
rassf1a is a tumor suppressor gene , which is involved in several signaling pathway including apoptosis , microtubule stability , proliferation , and mitotic regulation [ 2 , 3 ] . in our study , we have analyzed the function of the sav - rassf1-hpo ( sarah ) domain of rassf1a . here
, we report that the sarah domain regulates several pathways , which are frequently altered in tumors .
the sarah domain is involved in apoptosis and growth - suppressing functions of rassf1a like anchorage - independent proliferation . moreover , the sarah domain is important for mitotic progression and spindle formation .
it has been previously reported that rassf1 interacts through its c - terminal domain with mst1 and thereby regulates mst1-mediated apoptosis [ 16 , 18 , 19 ] .
this complex may regulate several pivotal signaling pathway including apoptosis and phosphorylation of warts / lats serine threonine kinases that regulate mitotic progression .
it has been reported that rassf1a regulates a proapoptotic pathway through its interaction with the proto - oncogene ras and the novel ras effector 1 ( nore1 ) [ 18 , 32 ] .
rassf1a and nore1 interact with the proapoptotic ste20 protein kinase mst1 via the c - terminus [ 16 , 19 ] through the sarah domain .
the homologue of rassf1 , nore1 forms a complex with mst1 that mediates a proapoptotic pathway induced by ras .
early on , it was reported that mst1 is a serine / threonine protein kinase that could autophosphorylate itself and later praskova et al .
demonstrated that mst1 phosphorylates and activates itself , whereas this autophosphorylation is inhibited when mst1 is bound to rassf1a and rassf1c .
we show that the interaction of rassf1a and rassf1c with mst1 and mst2 depends on the c - terminal sarah domain .
since the sarah domain binds the proapoptotic kinases mst1 and mst2 , a deregulation of these kinases may contribute to the apoptotic rate in the cells with truncated rassf1a .
however , we could not observe an autophosphorylation of mst1/2 after transfection of rassf1a and rassf1asarah .
this indicates that rassf1a - induced apoptosis observed in a549 cells occurs mst independent , possibly through the n - terminus of rassf1a , that associates with mdm2 and death - domain - associated protein ( daxx ) and contributes to p53 activation in response to dna damage .
alternatively , rassf1a was further linked to apoptosis through interacting with the microtubule - associated protein c19orf5 [ 48 , 49 ] .
furthermore , donninger et al . described rassf1a to interact with the potential tumor suppressor salvador to promote apoptosis independently of hippo signaling by modulating p73 .
it was demonstrated that rassf1a colocalizes with the microtubule network during interphase and is found at the spindles and centrosomes during mitosis [ 14 , 23 ] .
rassf1a binds to tubulin , thereby stabilizing microtubules [ 7 , 1214 ] and regulating the mitotic progression .
rassf1a overexpression leads to a mitotic arrest at metaphase , to a g1 arrest , to a g2/m arrest , to a g1 and g2/m arrest , and a prometaphase arrest .
the domain required for both microtubule association and stabilization was mapped to an amino - acid fragment from 120 to 288 .
thus , the microtubule binding site and the sarah domain are different [ 12 , 14 , 53 ] and this is consistent with our observation .
showed that the microtubule binding was lost upon mutation of the phosphorylation site 203 in rassf1a .
however , we and others did not observe an altered microtubule binding using phosphomimicking or nonphosphorylatable mutants of rassf1a [ 15 , 24 ] .
rassf1a was also reported to interact with map1b ( microtubule - associated protein 1b ) and c19orf5 ( chromosome 19 open reading frames 5 ) , both microtubule - associated proteins [ 13 , 49 , 53 ] .
c19orf5 is a hyperstabilized microtubule - specific binding protein of which accumulation causes mitochondrial aggregation and cell death .
regarding c19orf5 , it was demonstrated that its knockdown led to mitotic abnormalities , that c19orf5 localizes to centrosomes , and it was stated that c19orf5 is required for the recruitment of rassf1a to the spindle poles [ 53 , 55 ] .
liu et al . reported that rassf1a caused hyperstabilization of microtubules and the accumulation of c19orf5 on them .
the complex lats1/mst2/ww45 is found together with rassf1a at the centrosome , and it was shown that defects in this pathway may lead to abnormal mitosis caused by cytokinesis failure .
thus , rassf1a may mediate organization of mitotic spindle poles through the recruitment of mst and lats to the centrosomes .
in summary , our data indicate that rassf1a is important for several signals , which are frequently altered in tumorigenesis , including apoptosis , mitotic spindle organization , and proliferation .
our data suggest that other domains ( e.g. , microtubule association domain ) than sarah also significantly contribute to the proapoptotic and antiproliferative function of rassf1a .
specific interaction of rassf1a with mst / lats and other binding partners ( e.g. , ras , mdm2 , daxx , c19orf5 , and salvador ) might be important in the regulation of proliferation and apoptosis and in the formation of normal mitotic spindles and processes of dividing chromosomes by rassf1a . | the ras association domain family 1a ( rassf1a ) tumor suppressor encodes a sav - rassf - hpo domain ( sarah ) , which is an interaction domain characterized by hww45 ( dsav ) and mst1/2 ( dhpo ) . in our study , the interaction between rassf1a and rassf1c with mst1 and mst2 was demonstrated and it was shown that this interaction depends on the sarah domain .
sarah domain - deleted rassf1a had a similar growth - reducing effect as full - length rassf1a and inhibited anchorage independent growth of the lung cancer cell lines a549 significantly . in cancer cells expressing the sarah
deleted form of rassf1a , reduced mitotic rates ( p = 0.001 ) with abnormal metaphases ( p < 0.001 ) were observed and a significantly increased rate of apoptosis was found ( p = 0.006 ) compared to full - length rassf1a . although the association with microtubules and their stabilization was unaffected , mitotic spindle formation was altered by deletion of the sarah domain of rassf1a . in summary ,
our results suggest that the sarah domain plays an important role in regulating the function of rassf1a . |
ureteral rupture is a urological emergency usually presenting with acute pain and requiring urgent surgical intervention .
we report an unusual case where this condition was discovered incidentally on imaging , in the absence of any specific symptoms , to investigate chronic urinary retention caused by massive prostatic enlargement .
a 77-year - old caucasian gentleman was admitted with acute kidney injury discovered on blood tests .
he was known to have high - grade prostate intraepithelial neoplasia and atypical small acinar proliferation from prostate biopsies performed for a raised prostate - specific antigen ( psa ) , with a bone scan negative for potential metastases and was on psa surveillance with the latest reading of 43 pre - admission .
prior to admission , he had lower urinary tract symptoms in the form of frequency , nocturia , intermittent urinary stream , terminal dribbling and a feeling of incomplete emptying .
there was no significant past medical history . on examination , he was haemodynamically stable and afebrile .
there was a palpable non - tender bladder with dullness to percussion and no renal angle tenderness .
blood tests revealed a stage 3 acute kidney injury ( baseline renal function was normal ) with raised inflammatory markers and elevated psa compared with baseline ( table 1 ) . a mid - stream urine specimen sent for microscopy
a bedside bladder scan revealed a bladder volume of > 999 ml , necessitating the insertion of a urethral catheter with a residual volume of 2.4 l , and intravenous antibiotics were commenced . an ultrasound scan ( uss ) of the urinary tract
was performed to evaluate for the presence of hydronephrosis ; but while this demonstrated a minor dilatation of the left renal pelvis , it also found a shallow fluid collection around the left kidney extending into the left lateral retroperitoneum , separate from the left psoas muscle ( fig . 1 ) .
in the urinary bladder , there was an irregular 4 cm mass on the left posterolateral aspect .
in view of the findings , an urgent flexible cystoscopy was performed , revealing a massive median lobe of the prostate protruding up and back into the bladder ; the ureteric orifices were not visualized because of this , and the bladder mucosa was normal .
the patient subsequently underwent a computed tomography ( ct ) urogram , which showed bilateral fullness in the pelvicalyceal systems and confirmed a 4.8 4.4 2.8 cm fluid collection adjacent to the lower pole of the left kidney as detected on the uss , with perinephric stranding .
the fluid collection was demonstrated to be extravasated urine on the delayed post - contrast images arising from a defect in the left proximal ureter , and extending down the left paracolic gutter ( figs 2 and 3 ) .
mmol / l potassium4.6 mmol / l urea38.7 mmol / l creatinine305 mmol / l estimate glomerular filtration rate17 ml / min/1.73 m prostate - specific antigen71.8 ng / ml c - reactive protein295 mg / l
figure 1:uss demonstrating shallow fluid collection around left kidney .
figure 2:axial section ct image demonstrating defect in left proximal ureter from which contrast is extravasating , with perinephric fluid collection and fat stranding .
figure 3:coronal section ct image demonstrating fluid collection extending down left paracolic gutter . also visible
axial section ct image demonstrating defect in left proximal ureter from which contrast is extravasating , with perinephric fluid collection and fat stranding .
management options were discussed with the patient in the form of conservative treatment with antibiotics and catheterization , or insertion of a nephrostomy as retrograde ureteral stenting would have not been possible due to prostatic size .
he opted for the conservative approach which was successful with resolution of renal function and inflammatory markers to normal parameters .
he was offered a millins ' prostatectomy but declined and was happy to be managed with a long - term catheter and repeat psa measurement in 3 months ' time .
non - traumatic ureteral rupture has been reported more frequently in the recent literature despite its unusual occurrence .
physical obstruction distally within the urinary tract , most commonly by a ureteral calculus , is thought to transmit increased intraluminal pressures proximally into the ureter .
other reported causes of such obstruction include gravid uterus , urinary tract malignancy and even iatrogenic surgical ligation of the ureter [ 24 ] .
urinary retention has previously been described as a cause of ureteral rupture , but this was in the presence of abdominal pain , reported also in other cases when urine has leaked either into the retroperitoneal space or the peritoneal cavity .
the absence of specific abdominal symptoms in this particular case was unusual and therefore meant that the incidental discovery of a ruptured ureter on imaging was unexpected , albeit probably not surprising given the prostatic size .
severe intractable abdominal pain similar to that attributed to renal colic is the most observed presentation of ureteral rupture [ 1 , 6 ] , and its presence is confirmed on imaging , which may be performed to investigate other causes of pain .
ct scan of the urinary tract with a delayed excretory phase of contrast is likely to be the most used means and can determine the presence of extravasation , likely location of rupture and extent of urinoma or abscess formation , as well as the nature of the obstructing lesion .
if retrograde ureteral stent placement is to be performed , then pyelography can be performed at the same time to confirm ct findings .
ultrasonography has a limited role for detecting ureteral pathologies but was key here in detecting a perinephric fluid collection and possible bladder mass , which led to further investigations .
many reported cases of non - traumatic ureteral rupture have involved acute surgical management in the form of percutaneous drainage of urinomas or fluid collections , and urinary diversion via insertion of percutaneous nephrostomies and placement of ureteral stents [ 2 , 4 , 6 ] . in our case , however , factors that obviated this were the absence of any clinical signs or symptoms of ureteral rupture , the patient 's improving clinical condition and the massive median lobe of prostate preventing visualization of the ureteric orifices at flexible cystoscopy and thus making potential retrograde ureteral stent insertion difficult and complicated . there is documented evidence of successful conservative treatment with subsequent reabsorption of urine , and our case is an additional demonstration of this .
this case also highlights what is now documented to be a rare but serious complication of high - pressure chronic urinary retention .
although there is a previous report of ureteral rupture caused by neurogenic urinary retention , to our knowledge , this is the first case in a male patient with bladder outflow obstruction from prostatic enlargement . in a situation of chronic retention where high pressures are suspected present as indicated by acute kidney injury ,
when performing an uss one must be open to the possibility of ureteral rupture occurring even in the absence of symptoms .
| non - traumatic ureteral rupture has been reported more frequently , resulting from increased intraluminal pressures from distal urinary tract obstruction .
we report the case of a 77-year - old man presenting with chronic urinary retention secondary to massive prostatic enlargement through acute kidney injury .
ultrasound scan detected a shallow left perinephric fluid collection with a possible bladder mass , demonstrated on flexible cystoscopy to be a massive median lobe of prostate .
computed tomography confirmed extravasation of urine from the left proximal ureter . in the absence of specific symptoms , the patient had successful conservative management with antibiotics and urinary catheterization for his acute episode , although declined further surgical intervention . |
chronic pelvic pain is a common complaint among women of reproductive age often due to infection , endometriosis , or adhesive disease .
however , parasitic infection with resultant pain is usually not considered in the differential diagnosis .
we report a case of chronic right lower quadrant abdominal and pelvic pain associated with appendiceal enterobius vermicularis infestation that resolved after appendectomy and antiparasitic therapy .
a 35 year - old , white , nulligravid female presented to the university of south florida complaining of 2-years duration of chronic pelvic pain , particularly on the right side .
the pain was not associated with her menses and was said to be intermittent in nature .
she denied any weight loss , gastrointestinal complaints , or irritation of the vulva , vagina , or anus .
she reported no recent travel outside of the country and had no pets at home .
her gynecologic history was significant for laparoscopy 2 years prior for a left ovarian cyst .
a left salpingo - oophorectomy was performed at that time , and there was no note of endometriosis .
prior treatment with oral contraceptive pills and nonsteroidal antiinflammatory drugs provided no relief . on physical examination , her abdomen was soft and nontender .
her pelvic examination revealed normal external genitalia with tenderness in the right adnexa , but no appreciable masses or nodularity .
a laparoscopy was then performed and right pelvic side wall adhesions involving the appendix were discovered ( figure 1 ) .
a normal right tube and ovary were appreciated , and there was still no evidence of endometriosis .
a nematode was discovered in the appendiceal lumen consistent with e. vermicularis
( figure 2 ) .
the patient was treated with a single tablet of mebendazole ( vermox ) 100 mg orally , with a second dose given 2 weeks later .
postoperative follow - up at 2 weeks and at 4 months posttreatment revealed complete resolution of her pain .
e. vermicularis or pinworm is a common parasitic infection that is found mostly in the pediatric population .
the life cycle of this parasite starts with the ingestion of the eggs , which then hatch in the small intestine where the adult worm resides and mates .
the female e. vermicularis then migrates down to and out of the rectum and deposits her eggs on the perianal skin .
the majority of adult patients are asymptomatic ; however , symptoms of e. vermicularis infestation can include perineal puritis , weight loss , vulvovaginitis , and chronic salpingitis .
e. vermicularis has been documented on pap smears and throughout the genital tract including endometrium , fallopian tube , as well as in granulomas involving the ovary and pelvic peritoneum .
presumably , in these uncommon circumstances , the female worm travels from the anus to the vagina and ascends the genital tract all the way into the pelvic cavity . in 1939 , botsford reported a small series of patients presenting with complaints of chronic right lower quadrant abdominal pain , similar to our case , who were found to have appendiceal enterobius infestation on pathological examination of the appendix . in 2 more recent surgical series , performed outside of the united states , the incidence of enterobius infestation was low and commonly associated with an appendix that appeared normal .
less commonly , the appendix specimen showed acute and chronic inflammatory changes and peritoneal granulomas and periappendiceal adhesive disease , as in our case .
although these studies demonstrated the importance of pathological identification of nematodes and their oocytes , to make a specific diagnosis , they did not emphasize the need for any systemic therapy , as they considered appendectomy as curative .
in a recent report from the gynecological literature , the pathological specimen from a hysterectomy case for chronic pelvic pain demonstrated the surprise finding of acute and chronic salpingitis due to e. vermicularis infestation of the fallopian tube .
this same patient had an appendectomy 5 years earlier for e. vermicularis - related appendicitis .
it was not clear in this case whether the tubal infestation was caused by local spread from the infested appendiceal stump at the time of surgery or had later spread via a transperineal route to her upper genital tract .
this case clearly demonstrates the importance of eradicating the entire reservoir of e. vermicularis from the gastrointestinal tract , even after appendectomy .
the treatment of e. vermicularis infection involves one dose of pyrantel pamoate 11 mg / kg , or mebendazole 100 mg orally with a second dose in 2 weeks to treat possible reinfection .
the differential diagnosis for chronic pelvic pain includes pelvic infection , endometriosis , adhesive disease , or gastrointestinal pathology .
nematode infestation of the appendix as a cause of chronic right - sided pelvic pain is an uncommon diagnosis , which should be considered when the more common etiologies have been ruled out .
this particular patient had no identifiable risk factors for parasitic infection , the diagnosis being made only after microscopic examination of the appendix .
appendiceal nematode infestation manifested by gynecologic complaints , although rare , can occur and can be easily treated . | parasitic infestation is an uncommon cause of chronic pelvic pain among women of reproductive age . a case of chronic right - sided pelvic pain associated with appendiceal enterobius vermicularis infestation
was managed with appendectomy and antiparasitic therapy resulting in a complete resolution of symptoms . |
parkinson disease ( pd ) , which is the most common neurodegenerative parkinsonian syndrome , is characterized by the degeneration of both dopaminergic and nondopaminergic neurons with neuronal intracytoplasmic inclusions known as lewy bodies 1 .
although the clinical diagnosis of pd is often straightforward and robust in the cases with the typical cardinal symptoms and characteristic signs , the correct diagnosis can be challenging , especially in clinically mild or uncertain cases . furthermore , it remains difficult to accurately differentiate pd from other neurodegenerative parkinsonian syndromes ( non - pd ) , such as multiple system atrophy ( msa ) , progressive supranuclear palsy ( psp ) , and corticobasal degeneration .
an accurate and early diagnosis is the key to correct management and prognostication . in recent years
, molecular imaging techniques using pet or single - photon emission computed tomography ( spect ) have offered a variety of tests that can improve the diagnostic accuracy of pd and non - pd .
dopamine transporter ( dat ) ligands 24 and the sympathetic innervation of the heart 1,5 have become very popular and are used widely in clinical practice .
for example , imaging of dat binding with [ i]-2-carbomethoxy-3-(4-iodophenyl)-n-(3-fluoropropyl)-nortropane ( fp - cit ) successfully visualizes the presynaptic dopaminergic degeneration of the nigrostriatal tract 4,6 .
dat spect abnormalities can help to support a diagnosis of degenerative parkinsonian syndromes by excluding patients with essential tremor 7 , and psychogenic 8 and vascular parkinsonism 9 who have a normal nigrostriatal function . however , dat imaging as a stand - alone tool can not differentiate between the various types of degenerative parkinsonian syndromes with sufficient accuracy 10,11 .
similarly , several studies have shown the utility of meta-[i]-iodobenzylguanidine ( mibg ) myocardial scintigraphy in assessing the sympathetic cardiac nerve terminals in pd patients with a decreased cardiac mibg uptake in comparison with other parkinsonian syndrome patients in whom the mibg uptake is normal 12 . because these molecular imaging tools are limited in terms of their test accuracy ( ta ) , the use of either dat spect or mibg scintigraphy alone is insufficient for distinguishing pd from non - pd 13 . in this retrospective study , we compared the results of spect studies of striatal dat receptors and myocardial mibg scintigraphy with the clinical diagnoses in 34 patients with suspected parkinsonian syndromes to evaluate the diagnostic accuracy and usefulness of combined spect imaging in the same patient population .
thirty - four patients ( male / female : 19/15 , mean age : 62.113.7 years , age range : 3181 years ) , who underwent both [ i]-mibg scintigraphy and dat spect within a 4-month period for a differential diagnosis between pd and other parkinsonian syndromes were consecutively enrolled in this retrospective study , which was carried out from april 2014 until june 2016 . because many patients underwent two tests during a period ranging from 3 to 4 months at our hospital , we established a 4-month interval between the tests .
the study was approved by the institutional ethics committee and all procedures were in accordance with the ethical standards on human investigation and with the principles of the declaration of helsinki .
all of the patients underwent molecular in - vivo brain and cardiac diagnostic examinations , which consisted of presynaptic striatal dat scintigraphy with fp - cit and the assessment of myocardial sympathetic innervation with mibg .
the final diagnosis was made by the neurologists of tokushima university hospital , department of neurology , who carefully assessed the medical history and symptoms , the possible response to dopaminergic treatment , and the diagnostic mri findings .
furthermore , five patients were diagnosed with probable msa - p according to the established criteria 15,16 and two patients fulfilled the national institute of neurologic disorders and stroke criteria for probable psp 17 .
the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 .
after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . ,
tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators .
the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 .
the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis , irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side .
although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with
dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd .
for this study , we adopted the average value of the right and left sbrs as the sbr .
after the patient had rested for 15 min in the supine position , 111 mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously .
early and delayed spect were performed at 15 min and 3 h after the injection , respectively .
planar imaging for 5 min in the anterior projection was performed automatically during spect .
planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) .
the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 .
the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 .
the values were expressed as meansd and were analyzed using student s t - test and -test .
p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups .
the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively .
the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) .
the normality of the data distribution was assessed using the shapiro wilk test . the mann
whitney u - test was used for the comparison of non - normally distributed data .
differences with a p value of less than 0.05 were considered to be biologically significant .
the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program .
the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis .
we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination .
the brain spect scans were acquired according to the european association of nuclear medicine procedure guidelines for brain neurotransmission spect using [ i]-labeled dat ligands ( fp - cit , datscan ; nihon medi - physics co. ltd , tokyo , japan ) 18 .
after an injection of 167 mbq of datscan , the projection data were obtained in a 128128 matrix on a ecam , toshiba ( toshiba medical systems corp . , tochigi , japan ) ( 15 april 201414 april 2015 ) , or symbia t16 , siemens ( siemens healthcare , erlangen , germany ) ( 15 april 201530 july 2016 ) mounted with low - energy to medium - energy general - purpose collimators .
the data were reconstructed using the filtered back projection method without attenuation and scatter correction from 15 april 2014 to 14 april 2015 .
the data were reconstructed by the ordered subset expectation maximization method ( iteration 12 , subset 6 ) using a syngo miva10c with the symbia net software program ( siemens ag , munich , germany ) and corrected for attenuation by computed tomography and the triple energy window scatter correction method from 15 april 2015 to 30 july 2016 . the specific binding ratio ( sbr ) was calculated semiquantitatively using the dat view software program ( nihon medi - physics , tokyo , japan ) on the basis of bolt s method as described in detail elsewhere 19 . for the quantitative analysis ,
irregular regions of interest ( rois ) were drawn on single - slice views in areas corresponding to the left and right striata on either side .
although dat spect in pd typically shows an asymmetric loss of dat binding ( most prominent in the putamen ) , the tracer uptake is more symmetric in msa 20 and psp 21 . in one report of pathology - confirmed pd and msa cases with a similar disease duration 22,23 , visual inspection and a semiquantitative analysis with
dat spect detected a bilateral reduction in striatal dat binding in all patients , but the trend toward greater asymmetric binding was greater in msa than in pd .
for this study , we adopted the average value of the right and left sbrs as the sbr .
after the patient had rested for 15 min in the supine position , 111 mbq of [ i]-mibg ( myomibg ; fuji ri pharma , tokyo , japan ) was injected intravenously .
early and delayed spect were performed at 15 min and 3 h after the injection , respectively .
planar imaging for 5 min in the anterior projection was performed automatically during spect .
planar scanning and spect were performed using a dual - head gamma camera equipped with a low - energy to medium - energy general purpose parallel - hole collimator ( toshiba ecam ) .
the heart to mediastinum ( h / m ) ratio was calculated by dividing the count density of the left ventricular roi by that of the mediastinal roi , according to the standard method , which has been described previously 23 , 24 .
the h / m ratios calculated from the roi counts obtained by delayed spect were used for the comparison study because delayed scans show the neuronal uptake of mibg more explicitly 24 .
the values were expressed as meansd and were analyzed using student s t - test and -test .
p values of less than 0.05 were considered to indicate a statistically significant difference between pd and non - pd groups .
the sensitivity and specificity of the respective diagnostic index ( h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for differentiation between pd and non - pd were assessed using a receiver - operating characteristic ( roc ) analysis . for the combined use of dat spect and mibg myocardial scintigraphy , the cutoff delayed - phase h / m and sbr values were 2.745 and 3.24 , respectively .
the statistical analyses were carried out using the spss software program ( ibm spss statistics , version 23 ; ibm corp . , armonk , new york , usa ) .
the mann whitney u - test was used for the comparison of non - normally distributed data .
differences with a p value of less than 0.05 were considered to be biologically significant .
the analysis of the descriptive statistics and basic comparisons were carried out using the spss software program .
the sensitivity and specificity of the respective diagnostic index ( the h / m ratios of the mibg uptake in the delayed phase , sbr on dat spect , and combined dat spect and mibg myocardial scintigraphy ) for the differentiation between pd and non - pd were assessed in a roc analysis .
we also calculated the specificity , sensitivity , positive predictive value , negative predictive value , and ta of the combination of the two methods using the cutoff value for their combination .
although there was no significant difference in the age of the patients in the two groups , a statistically significant difference was observed in the male - to - female ratio .
the demographic and clinical features of the pd and non - pd patients the mean h / m ratio of the mibg uptake in the delayed phase ( 2.190.55 vs. 2.440.80 , cutoff=2.745 , p=0.025 ) was significantly lower in the pd patients than in the non - pd patients ; however , the mean sbr ( 2.460.87 vs. 2.941.40 , cutoff=3.24 , p=0.08 ) did not differ to a statistically significant extent ( table 1 and fig .
roc analyses were used to set the cutoff sbr value ( in dat spect ) and the h / m ratio ( in delayed scanning ) in mibg scintigraphy for differentiating pd from non - pd ( fig .
2 ) . in the comparison with the separate use of the sbr and the delayed h / m ratio , the combined use of the two semiquantitative analyses using cutoff values of less than 3.24 and less than 2.745 , respectively , allowed for more accurate differentiation between pd and non - pd .
the sensitivity and specificity of sbr were 86.7 and 52.6% , respectively ( table 2 ) .
the sensitivity and specificity of the delayed h / m ratio on mibg scintigraphy were 93 and 47% , respectively .
the accuracy of these analyses in differentiating between pd and non - pd was the same using each of the molecular methods ( dat spect , 67.6% ; mibg scintigraphy , 67.6% ) . in comparison with the sbr and delayed h / m ratio , the combined use of two semiquantitative analyses enabled pd and non - pd to be differentiated more accurately ( cutoff values : sbr<3.24 ; delayed h / m ratio<2.745 ) .
these indices distinguished pd from non - pd with a sensitivity of 86.7% , a specificity of 73.7% , an accuracy of 79.4% , a positive predictive value of 72.2% , and a negative predictive value of 87.5% ( table 2 ) .
box plots of the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients .
the box plots show the striatal fp - cit and cardiac delayed mibg accumulation in the pd and non - pd patients .
whiskers represent the minimum and maximum values . there was a statistically significant difference in the delayed h / m ratios of the pd and non - pd groups .
the sbr values of the two groups did not differ to a statistically significant extent .
- cit , n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ; h / m , heart to mediastinum ratio ; mibg , meta-[i]-iodobenzylguanidine ; non - pd , degenerative parkinsonian syndrome other than pd ; pd , parkinson s disease ; sbr , specific binding ratio . the receiver - operating characteristic curves of the delayed h / m ratio and sbr . delayed h / m ratio : auc=0.698 ; cutoff value=2.745 ; sensitivity=0.93 ; specificity=0.47 .
auc , area under the curve ; h / m , heart to mediastinum ratio ; sbr , specific binding ratio .
the statistical parameters that were evaluated when investigating the combined use of fp - cit spect and mibg scintigraphy in differentiating pd from non - pd
this retrospective study investigated the extent to which the combined use of mibg scintigraphy and dat spect may improve diagnostic accuracy in differentiation between pd and non - pd .
the analysis showed that the individual molecular approaches had a mild ta in differentiating between pd and non - pd ( mibg scintigraphy , 62% ; fp - cit spect , 69% ) . in agreement with previous studies 25,26
, we confirmed that the combined use of dat spect and mibg scintigraphy was more useful for differentiating between pd from non - pd in comparison with the use of either method alone .
dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 .
according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 .
the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd .
the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 . in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively .
the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 .
the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 .
there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 ,
[ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp .
the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding .
in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 .
the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 .
some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 .
a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study
, we therefore explored the extent to which the combination of these two tracers would improve the ta .
our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% .
first , this study was carried out in a single university hospital ; thus , the study population was small .
second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases .
third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs by considering their biological half - lives .
dat spect is therefore very popular in the clinical setting and enables highly accurate differentiation between degenerative parkinsonian syndromes and movement disorders that are not associated with a dopamine deficit 79 .
according to plotkin , the resulting accuracy , sensitivity , and specificity of fp - cit spect in the detection of pd and non - pd were 94 , 93 , and 100% , respectively 28 .
however , the loss of dopaminergic neurons in the substantia nigra and the reduction of striatal dopamine projections are histopathological characteristics that are common to the degenerative parkinsonian syndromes and the low accumulation of striatal dat shows a moderate ta in differentiating pd from non - pd .
the sensitivity ( 86.7% ) and specificity ( 52.6% ) that were observed in the present study were consistent with previous studies 10,20,29,30 .
in our study , the sensitivity and specificity in differentiating pd from non - pd using the h / m ratio of the mibg uptake were 93 and 47% , respectively .
the usefulness of mibg myocardial scintigraphy in the diagnosis of dlb was suggested recently in a meta - analysis . the sensitivity and specificity of mibg scintigraphy in the differential diagnosis between pd and other parkinsonian syndromes ranged from 71 to 100% and from 50 to 100% , respectively , with pooled estimates of 88% ( 95% confidence interval : 8690% ) and 85% ( 95% confidence interval : 8188% ) 5 .
the specificity observed in the present study was lower than expected . in terms of the diagnostic performance , mibg scintigraphy is usually a sensitive , but not specific , test for pd 5 .
there might be several reasons for this . as the myocardial [ i]-mibg uptake in patients with non - pd ( especially in patients with msa and psp ) is often found to be slightly reduced in comparison with healthy controls 31 ,
[ i]-mibg scintigraphy may yield some false - positive results in patients with msa and psp .
the myocardial sympathetic nervous system of msa patients typically shows mild degeneration 32 ; thus , false - positive results may account for this scintigraphic finding .
in addition to lbd - related postganglionic sympathetic degeneration , false - positive results on [ i]-mibg scintigraphy may also occur because of age - related degeneration because the myocardial [ i]-mibg uptake has been reported to decrease significantly with age 30,33 .
the combination of dopaminergic and sympathetic scintigraphic imaging tests for differentiating between pd and non - pd has been explored by other authors 25,26,28,30,34 .
some studies showed the complementary role of [ i]-mibg scintigraphy and dat spect in the differential diagnosis between pd and non - pd 25,34 . a recent study showed that [ i]-fp - cit spect presents high sensitivity in the diagnosis of lewy body disease ; thus , [ i]-mibg scintigraphy may play a complementary role in the differential diagnosis between pd and other parkinsonism 30 . in the present study
, we therefore explored the extent to which the combination of these two tracers would improve the ta .
our data show that by combining fp - cit spect and mibg scintigraphy , the ta can be increased to 79.4% .
first , this study was carried out in a single university hospital ; thus , the study population was small .
second , the clinical diagnosis was used as the gold standard in our study because of the absence of histopathological confirmation ; thus , the clinical diagnosis may have been incorrect in some cases .
third , before a molecular examination is performed , it is necessary to establish an appropriate withdrawal period in which to cease the administration of interfering drugs by considering their biological half - lives .
the combination of dat spect and mibg myocardial scintigraphy may improve the sensitivity of the diagnosis of pd .
in particular , this approach is based on commercially available radiotracers and can therefore be used in clinical practice without short - lived pet tracers .
the sample size of the present study was small and the study was carried out in a single center .
the results should therefore be confirmed in a multicenter study with a large population of pathologically diagnosed patients .
| objectivemolecular imaging of nigrostriatal dopamine transporters ( dat ) and sympathetic cardiac innervation with single - photon emission computed tomography ( spect ) are useful tools for differentiating idiopathic parkinson disease ( pd ) from other degenerative parkinsonian syndromes ( non - pd ) . nevertheless , these modalities are often insufficient for achieving a definite diagnosis .
the aims of this study were to evaluate the diagnostic accuracy of the combination of these tools.materials and methodsthe spect radiotracers [ 123i]-n--fluoropropyl-2-carbomethoxy-3-(4-iodophenyl)-nortropane ( fp - cit ) and meta-[123i]-iodobenzylguanidine ( mibg ) were used to research presynaptic dopaminergic projections ( dat spect ) and myocardial adrenergic innervation ( mibg scintigraphy ) , respectively .
pd patients ( n=15 ; age : 61.513.6 years ) and non - pd patients ( n=19 ; age : 62.614.2 years ) who underwent both tests were enrolled in this study .
receiver - operating characteristic analyses were used to set the cutoff values of the specific binding ratio in dat spect and the heart to mediastinum ratio in delayed scan in mibg scintigraphy for differentiating pd from non - pd .
we calculated the sensitivity , specificity , and test accuracy of the individual methods and also the combination of these two modalities.resultswhen dat spect and mibg scintigraphy were used individually , they showed mild accuracy in differentiating pd from non - pd ( dat , 67.6% ; mibg , 67.6% ) .
the combination of the two approaches using cutoff values of less than 3.24 for the specific binding ratio and less than 2.745 for the delayed heart to mediastinum ratio enabled more accurate differentiation between pd and non - pd .
the accuracy of these indices in distinguishing pd from non - pd was 79.4%.conclusionthese results suggested that the combination of dat spect and mibg scintigraphy may improve the diagnostic accuracy in differentiating pd from non - pd . |
nitric oxide ( no ) is an essential radical molecule that is involved in various physiological processes , ranging from signalling processes to cytotoxic activities . in mammals
it is produced by a class of enzymes , the no synthases ( noss ) , through a two - step reaction . in the first step ,
l - arginine is oxidised to n - hydroxyarginine ( noha ) ; in the second step , this is oxidised to citrulline and no . among the various reaction intermediates , the ferrous nitrosyl complex { feno } plays a dominant role .
its formation in the course of noha oxidation and its oxidation determine the ability of nos to release no .
moreover , it is the starting point of a futile cycle in which the reactivity of the { feno } species controls the balance between no release and production of reactive nitrogen and oxygen species ( rnos ) .
the interaction of no with haemoproteins ( e.g. , globin , cytochrome c oxidase , soluble guanylate cyclase ) is crucial in the homeostasis of no and for its biological impact.[5d , 9 ] as noss are the source and the first target of no , characterisation of the structural and electronic properties of the nos { feno } complex appear necessary to understand the biology of no and rnos .
however , only a few spectroscopic characterisations have been performed so far on this species .
epr is one of the most suitable techniques to describe its electronic properties and to investigate its reactivity .
reports have described the epr fingerprint of nos { feno } complexes and the influence of the haem distal environment on its geometry.[10a , d ] although the existence of a thermal equilibrium between a rhombic ( r ) and an axial ( a ) form has been described for several haemoproteins ( including myoglobins , haemoglobins and neuroglobins ) , the possible existence of such conformers and their respective structures have never been addressed for noss or other haemothiolate proteins ; despite intensive investigations , the nature of the equilibrium and the identification of the low temperature ( a ) form remain under debate ( see refs .
, we decided to investigate the temperature dependence of the nos { feno } complex .
the changes were followed by epr spectroscopy on two different noss : the mammalian inducible nos ( inos ) and the nos - like protein from bacillus subtilis ( bsnos ) , which have been shown to enhance and abolish , respectively , the damaging oxidative reactivity of the potent rnos peroxynitrite .
we analysed the influence of the haem distal environment by varying the nature of the substrate .
our results allow a better analysis of the effect of temperature on the different features of the epr spectra , and are a novel contribution to the on - going debate over the properties of haem { feno}.
the ferrous nitrosyl complex for the mammalian inos was prepared in the presence of substrate ( l - arginine ) and cofactor ( tetrahydrobiopterin , h4b ) . the epr spectrum obtained at low temperature for this sample
similarly to what has been observed for such complexes in other haem proteins , the spectrum is a mixture of a rhombic powder pattern ( r ) and a more axial one ( a ) . the r form is characteristic of a six - coordinated species , with hyperfine coupling because of the nitrogen nucleus ( n , i=1 ) of no , well resolved on the central g value . the simulated spectrum for the rhombic pattern is shown along the experimental spectrum in figure 1 , and the parameters are reported in table 1 . the differences between experimental and simulated spectra above 340 mt might be due to excess no in solution ( figure 1 , top spectrum ) .
the a form has a dominant contribution at g=2.040 and is very difficult to characterise , as was the case in previous studies of ferrous nitrosyl complexes.[11d ] this form has been routinely observed in the six - coordinated { feno } complex , both in proteins and in model complexes ( refs .
it is noteworthy that this form is clearly distinct from the ferrous hemin nitrosyl complex in solution ( figure 1 ) , and is also different from the five - coordinated { feno } complex within nos , which displays a characteristic epr spectrum.[10b , d ] these observations have also been reported for the other family of haemothiolate proteins , cytochromes p450 ( cyps ) .
as observed for other ferrous haem nitrosyl complexes in proteins , the epr spectrum was found to be highly dependent on temperature .
the spectrum recorded on the same sample at 250 k ( along with a simulated spectrum ; figure 1 , bottom spectra ) remains rhombic , but its g anisotropy is dramatically reduced , with loss of the hyperfine coupling resolution .
epr spectra of no in buffer , and of the ferrous nitrosyl complexes of hemin and inos ( + h4b/+arg ) at 4 k and 250 k. spectra in grey are simulations ( parameters in table 1 ) .
experimental conditions : microwave frequency 9.49 ghz ( no buffer ) , 9.42 ghz ( inos no 4 k , hemin no ) and 9.38 ghz ( inos no 250 k ) ; microwave power 1 mw ( no buffer , hemin no ) , 0.2 w ( inos no 4 k ) and 0.1 mw ( inos no 250 k ) ; modulation amplitude 0.5 mt ( no buffer , hemin - no ) , 0.4 mt ( inos no 4 k ) and 0.2 mt ( inos no 250 k ) ; modulation frequency 100 khz ; temperature 10 k ( no buffer , hemin no ) , 4 k ( inos no 4 k ) and 250 k ( inos no 250 k ) .
epr parameters obtained from the simulation of the experimental spectra for the nitrosyl complexes of inos and bsnos at low and high temperature in the presence of substrate and cofactor in order to understand the changes observed in these spectra , we recorded epr spectra of the nitrosyl complex as a function of temperature . selected spectra
because of the large g anisotropy ( compared to systems with a proximal histidine , such as myoglobin ) , it is evident that the g values of the r form here change with temperatures .
clear changes in the spectrum start at around 70 k , with gmax ( r form ) decreasing and gmid and gmin increasing with increasing temperature .
in addition , these features became significantly broadened as the temperature was increased , thus making the principal g values difficult to determine at 140 k and above .
however , we note that the low field edge can still be followed clearly enough , even at higher temperatures . in parallel , the additional feature at g=2.040 ( dash / dotted line ) broadens at 70 k and then seems to split into two at 140 k ( and above ) .
experimental epr spectra for inos ( + h4b/+arg ) nitrosyl complex at the indicated temperatures . experimental conditions : microwave frequency 9.41 ghz , microwave power 0.2 w ( 10 k ) , 3.2 w ( 40 k ) and 50.0 w ( 100200 k ) ; modulation amplitude 0.4 mt ; modulation frequency 100 khz .
the dotted lines indicate the field positions corresponding to the three principal g values of the lowest temperature spectrum .
the dash - dotted line indicates the field position of the additional epr feature at g=2.040 at 10 k. several models have been proposed to explain the temperature - related evolution of the ferrous nitrosyl complexes of haem proteins , in particular for myoglobin and haemoglobin .
equilibrium between an axial and a rhombic form has been suggested , with the rhombic form dominant at low temperatures.[11c a second proposition involves two axial forms ( in addition to the rhombic one ) , with one at low temperatures and the other at high temperatures .
finally , multifrequency epr studies led to the conclusion that the g values of both axial and rhombic forms were also temperature dependent.[11a , e ] overall , a consensus seems to have emerged , with a model based on equilibrium between rhombic and axial forms and the g values of both changing with temperature.[11d , f , g ] such an equilibrium was also suggested in the case of cyp450scc in the presence of cholesterol .
in contrast , the temperature dependence observed for the five - coordinated { feno } complex did not display interconversion between the two forms in the case of soluble guanylate cyclase.[8a ] in the present case , { feno } complex of inos in the presence of the cofactor and substrate l - arginine , it is clear from the dependence of the g values that the rhombic form ( dominant at low temperatures ) evolves towards a more axial species at high temperatures .
in contrast , the evolution of the resonance at g=2.040 at 10 k ( attributed to an axial form ) is not obvious and is more difficult to follow .
indeed , the observed changes in the g=2.040 region might arise from the appearance of a new powder pattern or from the superimposition of the r and a ( low - temperature ) forms . as a result , assessment of the ratio between r and a contributions
, it is not clear whether or not the two forms interconvert . in a previous study on the proximal environment of a nos - like protein of the bacterium b. subtilis
, we reported epr spectra of the { feno } complex.[10b in that case , the contribution of the g=2.040 form was found to be negligible at low temperatures .
we therefore performed a temperature study of the observed epr spectrum , and compared the results with those for mammalian inos .
the ferrous nitrosyl complex of the nos - like protein from b. subtilis was prepared in the presence of substrate ( l - arginine ) and cofactor .
the epr spectrum obtained at low temperature ( figure 3 , top spectrum ) exhibits a single rhombic pattern with three well - resolved g values characteristic of a six - coordinated complex .
in addition , and in contrast to what was observed for inos , the hyperfine coupling with the nitrogen nucleus of no was also resolved at all three turning points .
the parameters of the simulated spectrum are reported in table 1 . the experimental spectrum recorded at 10 k
could be well reproduced by using a single powder pattern , thus showing that a single { feno } species is highly dominant in solution .
it is noteworthy that the contribution of the axial form ( responsible for the resonance at g=2.040 ) is negligible here .
experimental ( black ) and calculated ( grey ) epr spectra for bsnos ( + h4b/+arg ) nitrosyl complex at 10 k ( top ) and 220 k ( bottom ) .
experimental conditions : microwave frequency 9.41 ghz ; microwave power 1.0 w ( 10 k ) and 1.6 mw ( 220 k ) , modulation amplitude 0.5 mt ( 10 k ) and 0.25 mt ( 220 k ) ; modulation frequency 100 khz .
the high - temperature spectrum , recorded on the same sample at 220 k , is also shown in figure 3 along with its simulated spectrum ( bottom spectra ) . at this temperature ,
the spectrum remains rhombic , with nitrogen hyperfine coupling still well resolved on the central g value resonance , but the line width is broader and the g anisotropy is significantly lower than at 10 k. selected spectra recorded at different temperatures are displayed in figure 4 .
similarly to inos , the gmax value decreases while the gmid and gmin values increase continuously as the temperature is increased . within the temperature range 1060 k ,
the changes in g values are not very significant , and the hyperfine coupling of the nitrosyl nitrogen remains resolved at all three turning points .
changes become more important above 60 k where clear shifts in the gmax and gmin values are evident . in addition , above 140 k , the hyperfine coupling resolution is lost for the gmax and gmin features , as the whole spectrum keeps broadening .
experimental epr spectra for bsnos ( + h4b/+arg ) ferrous nitrosyl complex at selected temperatures .
experimental conditions : microwave frequency 9.41 ghz ; microwave power 1 w ( 10 k ) , 0.4 mw ( 60 k and 100 k ) and 1.6 mw ( 140220 k ) ; modulation amplitude 0.5 mt ( 10 k ) and 0.25 mt ; modulation frequency 100 khz .
the vertical dotted lines indicate the field positions corresponding to the three principal g values of the lowest temperature spectrum .
similarly to what has been observed in the case of peroxidases,[11h the epr spectrum of the { feno } complex of bsnos in the presence of cofactor and substrate l - arginine can be considered as a single rhombic powder pattern : specifically , the axial contribution ( resonance at g=2.040 ) was not present in this sample .
there was therefore neither equilibrium nor a conversion between rhombic and axial forms . only decrease in g anisotropy of the rhombic pattern and some line broadening were observed with increasing temperature .
this effect can be attributed , at least in part , to fast relaxation of the transition metal as well as dynamics due in particular to the rotation of the no unit .
these observations on bsnos do not support the existence of equilibrium between the r and a forms , and suggest that the a form could be a degraded version of the nitrosyl complex .
the temperature - dependence patterns of the { feno } epr spectra clearly differ between inos ( figure 2 ) and bsnos ( figure 4 ) . moreover , line broadening with increasing temperature is less pronounced in bsnos and occurs at higher temperatures .
. however , significant differences between their overall 3d structures remain . in particular , the dimer interface is truncated in the bsnos protein . furthermore , the conformational equilibrium between the loose and tight forms and the parameters that control it vary among nos and nos - like proteins .
these differences in the quaternary structure of nos proteins could partly explain the nos - dependent effect of temperature on conformational equilibrium of the { feno } species . additionally , despite an identical catalytic site
, the interaction of a distal ligand ( no , co or o2 ) with the l - arginine substrate seems different between bsnos and inos.[10a , d , 20 ] finally , a conserved valine residue in mammalian nos on the roof of the haem pocket ( modulating haem no binding kinetics ) is a bulkier isoleucine in bsnos.[5b , 21 ] these differences might also explain the distinct sensitivities of the { feno } geometries of inos and bsnos to temperature .
thus , we investigated the influence of the nature of the substrate on the { feno } epr spectrum .
we prepared samples of inos with no substrate or with its second natural substrate , noha .
the corresponding spectra at low , intermediate and high temperatures are shown in figure 5 . in the absence of substrate ( top spectra )
, the lowest temperature spectrum exhibits the characteristic rhombic powder pattern of a six - coordinated nitrosyl complex .
the additional feature at 2.040 is also present , although it is less pronounced in this particular sample compared to what was observed in the presence of l - arginine .
when the temperature was increased , a similar trend as that seen for inos with l - arginine was observed .
when noha was the bound substrate , { feno } displayed a rhombic epr signal at 4 k , somewhat different to those observed in the presence or absence of l - arginine : the anisotropy was reduced , and the gmax resonance did not exhibit the characteristic triplet shape because of the nitrogen hyperfine coupling . when the temperature was raised , the same trend was observed , but this is harder to follow because of the lower anisotropy : both features rapidly merge over a temperature range similar to that observed with l - arginine as substrate .
experimental epr spectra for inos + h4b / no substrate ( top ) and inos + h4b/+noha ( bottom ) ferrous nitrosyl complex at 4 k , 120 k and 230 k. experimental conditions : microwave frequency 9.42 ghz ( 4 k ) and 9.38 ghz ( 120 and 230 k ) ; microwave power 0.2 w ( 4 k ) and 0.13 mw ( 120 and 230 k ) ; modulation amplitude 0.4 mt ( 4 k ) and 0.2 mt ( 120 and 230 k ) ; modulation frequency 100 khz .
the observed changes in the { feno } epr spectrum with increasing temperature depend on the presence and the nature of the substrate : the proportion of the low - temperature ( a ) form was highest in the presence of l - arginine , and the emergence of the r form occurred at a lower temperature ( below 120 k ) in the presence of substrate .
this suggests that the substrate and the distal h - bond network control the geometry of the { feno } species .
indeed , the a form of the { feno } species seems favoured by the h - bond interaction between no and the nitrogen of the distal histidine.[11f , g ] besides , the evolution of the spectrum is observed at higher temperature , when this interaction is suppressed.[11f in this regard , l - arginine ( and to a lesser extent noha ) could play a similar role in nos to that in neuro- and haemoglobin ( ascribed to the distal histidine ) . in the case of haemothiolate cyp450s ,
various situations were encountered , as the proportions of r and a forms depended on the nature of the cyp450 , and the substrate could also influence the different components in the epr spectra.[14 , 16 ] as the occurrence of rhombic and axial forms is also observed in model complexes,[13 , 22 ] it is not clear what the real cause for the existence of each form is .
however , the observations made on cyp450s and noss clearly indicate important roles for the distal pocket and the substrate on the structural and electronic properties of the { feno } complex . in the case of nos
, this should be important with respect to the catalytic cycle and the structure function relationship , as the ferrous nitrosyl moiety is a reaction intermediate during the second step of catalysis .
the ferrous nitrosyl complex for the mammalian inos was prepared in the presence of substrate ( l - arginine ) and cofactor ( tetrahydrobiopterin , h4b ) . the epr spectrum obtained at low temperature for this sample
similarly to what has been observed for such complexes in other haem proteins , the spectrum is a mixture of a rhombic powder pattern ( r ) and a more axial one ( a ) . the r form is characteristic of a six - coordinated species , with hyperfine coupling because of the nitrogen nucleus ( n , i=1 ) of no , well resolved on the central g value . the simulated spectrum for the rhombic pattern is shown along the experimental spectrum in figure 1 , and the parameters are reported in table 1 . the differences between experimental and simulated spectra above 340 mt might be due to excess no in solution ( figure 1 , top spectrum ) .
the a form has a dominant contribution at g=2.040 and is very difficult to characterise , as was the case in previous studies of ferrous nitrosyl complexes.[11d ] this form has been routinely observed in the six - coordinated { feno } complex , both in proteins and in model complexes ( refs .
it is noteworthy that this form is clearly distinct from the ferrous hemin nitrosyl complex in solution ( figure 1 ) , and is also different from the five - coordinated { feno } complex within nos , which displays a characteristic epr spectrum.[10b , d ] these observations have also been reported for the other family of haemothiolate proteins , cytochromes p450 ( cyps ) .
as observed for other ferrous haem nitrosyl complexes in proteins , the epr spectrum was found to be highly dependent on temperature .
the spectrum recorded on the same sample at 250 k ( along with a simulated spectrum ; figure 1 , bottom spectra ) remains rhombic , but its g anisotropy is dramatically reduced , with loss of the hyperfine coupling resolution .
epr spectra of no in buffer , and of the ferrous nitrosyl complexes of hemin and inos ( + h4b/+arg ) at 4 k and 250 k. spectra in grey are simulations ( parameters in table 1 ) .
experimental conditions : microwave frequency 9.49 ghz ( no buffer ) , 9.42 ghz ( inos no 4 k , hemin no ) and 9.38 ghz ( inos no 250 k ) ; microwave power 1 mw ( no buffer , hemin no ) , 0.2 w ( inos no 4 k ) and 0.1 mw ( inos no 250 k ) ; modulation amplitude 0.5 mt ( no buffer , hemin - no ) , 0.4 mt ( inos no 4 k ) and 0.2 mt ( inos no 250 k ) ; modulation frequency 100 khz ; temperature 10 k ( no buffer , hemin no ) , 4 k ( inos no 4 k ) and 250 k ( inos no 250 k ) .
epr parameters obtained from the simulation of the experimental spectra for the nitrosyl complexes of inos and bsnos at low and high temperature in the presence of substrate and cofactor in order to understand the changes observed in these spectra , we recorded epr spectra of the nitrosyl complex as a function of temperature . selected spectra
because of the large g anisotropy ( compared to systems with a proximal histidine , such as myoglobin ) , it is evident that the g values of the r form here change with temperatures .
clear changes in the spectrum start at around 70 k , with gmax ( r form ) decreasing and gmid and gmin increasing with increasing temperature .
in addition , these features became significantly broadened as the temperature was increased , thus making the principal g values difficult to determine at 140 k and above .
however , we note that the low field edge can still be followed clearly enough , even at higher temperatures . in parallel , the additional feature at g=2.040 ( dash / dotted line ) broadens at 70 k and then seems to split into two at 140 k ( and above ) .
experimental epr spectra for inos ( + h4b/+arg ) nitrosyl complex at the indicated temperatures . experimental conditions : microwave frequency 9.41 ghz , microwave power 0.2 w ( 10 k ) , 3.2 w ( 40 k ) and 50.0 w ( 100200 k ) ; modulation amplitude 0.4 mt ; modulation frequency 100 khz .
the dotted lines indicate the field positions corresponding to the three principal g values of the lowest temperature spectrum .
the dash - dotted line indicates the field position of the additional epr feature at g=2.040 at 10 k. several models have been proposed to explain the temperature - related evolution of the ferrous nitrosyl complexes of haem proteins , in particular for myoglobin and haemoglobin .
equilibrium between an axial and a rhombic form has been suggested , with the rhombic form dominant at low temperatures.[11c a second proposition involves two axial forms ( in addition to the rhombic one ) , with one at low temperatures and the other at high temperatures .
finally , multifrequency epr studies led to the conclusion that the g values of both axial and rhombic forms were also temperature dependent.[11a , e ] overall , a consensus seems to have emerged , with a model based on equilibrium between rhombic and axial forms and the g values of both changing with temperature.[11d , f , g ] such an equilibrium was also suggested in the case of cyp450scc in the presence of cholesterol .
in contrast , the temperature dependence observed for the five - coordinated { feno } complex did not display interconversion between the two forms in the case of soluble guanylate cyclase.[8a ] in the present case , { feno } complex of inos in the presence of the cofactor and substrate l - arginine , it is clear from the dependence of the g values that the rhombic form ( dominant at low temperatures ) evolves towards a more axial species at high temperatures .
in contrast , the evolution of the resonance at g=2.040 at 10 k ( attributed to an axial form ) is not obvious and is more difficult to follow .
indeed , the observed changes in the g=2.040 region might arise from the appearance of a new powder pattern or from the superimposition of the r and a ( low - temperature ) forms . as a result , assessment of the ratio between r and a contributions
, it is not clear whether or not the two forms interconvert . in a previous study on the proximal environment of a nos - like protein of the bacterium b. subtilis
, we reported epr spectra of the { feno } complex.[10b in that case , the contribution of the g=2.040 form was found to be negligible at low temperatures .
we therefore performed a temperature study of the observed epr spectrum , and compared the results with those for mammalian inos .
the ferrous nitrosyl complex of the nos - like protein from b. subtilis was prepared in the presence of substrate ( l - arginine ) and cofactor .
the epr spectrum obtained at low temperature ( figure 3 , top spectrum ) exhibits a single rhombic pattern with three well - resolved g values characteristic of a six - coordinated complex .
in addition , and in contrast to what was observed for inos , the hyperfine coupling with the nitrogen nucleus of no was also resolved at all three turning points .
the parameters of the simulated spectrum are reported in table 1 . the experimental spectrum recorded at 10 k
could be well reproduced by using a single powder pattern , thus showing that a single { feno } species is highly dominant in solution .
it is noteworthy that the contribution of the axial form ( responsible for the resonance at g=2.040 ) is negligible here .
experimental ( black ) and calculated ( grey ) epr spectra for bsnos ( + h4b/+arg ) nitrosyl complex at 10 k ( top ) and 220 k ( bottom ) .
experimental conditions : microwave frequency 9.41 ghz ; microwave power 1.0 w ( 10 k ) and 1.6 mw ( 220 k ) , modulation amplitude 0.5 mt ( 10 k ) and 0.25 mt ( 220 k ) ; modulation frequency 100 khz .
the high - temperature spectrum , recorded on the same sample at 220 k , is also shown in figure 3 along with its simulated spectrum ( bottom spectra ) . at this temperature ,
the spectrum remains rhombic , with nitrogen hyperfine coupling still well resolved on the central g value resonance , but the line width is broader and the g anisotropy is significantly lower than at 10 k. selected spectra recorded at different temperatures are displayed in figure 4 .
similarly to inos , the gmax value decreases while the gmid and gmin values increase continuously as the temperature is increased . within the temperature range 1060 k , the changes in g values are not very significant , and the hyperfine coupling of the nitrosyl nitrogen remains resolved at all three turning points .
changes become more important above 60 k where clear shifts in the gmax and gmin values are evident . in addition , above 140 k , the hyperfine coupling resolution is lost for the gmax and gmin features , as the whole spectrum keeps broadening .
experimental epr spectra for bsnos ( + h4b/+arg ) ferrous nitrosyl complex at selected temperatures .
experimental conditions : microwave frequency 9.41 ghz ; microwave power 1 w ( 10 k ) , 0.4 mw ( 60 k and 100 k ) and 1.6 mw ( 140220 k ) ; modulation amplitude 0.5 mt ( 10 k ) and 0.25 mt ; modulation frequency 100 khz .
the vertical dotted lines indicate the field positions corresponding to the three principal g values of the lowest temperature spectrum .
similarly to what has been observed in the case of peroxidases,[11h the epr spectrum of the { feno } complex of bsnos in the presence of cofactor and substrate l - arginine can be considered as a single rhombic powder pattern : specifically , the axial contribution ( resonance at g=2.040 ) was not present in this sample .
there was therefore neither equilibrium nor a conversion between rhombic and axial forms . only decrease in g anisotropy of the rhombic pattern and some line broadening were observed with increasing temperature .
this effect can be attributed , at least in part , to fast relaxation of the transition metal as well as dynamics due in particular to the rotation of the no unit .
these observations on bsnos do not support the existence of equilibrium between the r and a forms , and suggest that the a form could be a degraded version of the nitrosyl complex .
the temperature - dependence patterns of the { feno } epr spectra clearly differ between inos ( figure 2 ) and bsnos ( figure 4 ) .
moreover , line broadening with increasing temperature is less pronounced in bsnos and occurs at higher temperatures .
the haem - binding sites in inos and bsnos have high structural homology . however , significant differences between their overall 3d structures remain . in particular ,
furthermore , the conformational equilibrium between the loose and tight forms and the parameters that control it vary among nos and nos - like proteins .
these differences in the quaternary structure of nos proteins could partly explain the nos - dependent effect of temperature on conformational equilibrium of the { feno } species .
additionally , despite an identical catalytic site , the interaction of a distal ligand ( no , co or o2 ) with the l - arginine substrate seems different between bsnos and inos.[10a , d , 20 ] finally , a conserved valine residue in mammalian nos on the roof of the haem pocket ( modulating haem no binding kinetics ) is a bulkier isoleucine in bsnos.[5b , 21 ] these differences might also explain the distinct sensitivities of the { feno } geometries of inos and bsnos to temperature .
thus , we investigated the influence of the nature of the substrate on the { feno } epr spectrum .
we prepared samples of inos with no substrate or with its second natural substrate , noha .
the corresponding spectra at low , intermediate and high temperatures are shown in figure 5 . in the absence of substrate ( top spectra )
, the lowest temperature spectrum exhibits the characteristic rhombic powder pattern of a six - coordinated nitrosyl complex .
the additional feature at 2.040 is also present , although it is less pronounced in this particular sample compared to what was observed in the presence of l - arginine .
when the temperature was increased , a similar trend as that seen for inos with l - arginine was observed .
when noha was the bound substrate , { feno } displayed a rhombic epr signal at 4 k , somewhat different to those observed in the presence or absence of l - arginine : the anisotropy was reduced , and the gmax resonance did not exhibit the characteristic triplet shape because of the nitrogen hyperfine coupling . when the temperature was raised , the same trend was observed , but this is harder to follow because of the lower anisotropy : both features rapidly merge over a temperature range similar to that observed with l - arginine as substrate . experimental epr spectra for inos + h4b / no substrate ( top ) and inos + h4b/+noha ( bottom ) ferrous nitrosyl complex at 4 k , 120 k and 230 k. experimental conditions : microwave frequency 9.42 ghz ( 4 k ) and 9.38 ghz ( 120 and 230 k ) ; microwave power 0.2 w ( 4 k ) and 0.13 mw ( 120 and 230 k ) ; modulation amplitude 0.4 mt ( 4 k ) and 0.2 mt ( 120 and 230 k ) ; modulation frequency 100 khz .
the observed changes in the { feno } epr spectrum with increasing temperature depend on the presence and the nature of the substrate : the proportion of the low - temperature ( a ) form was highest in the presence of l - arginine , and the emergence of the r form occurred at a lower temperature ( below 120 k ) in the presence of substrate .
this suggests that the substrate and the distal h - bond network control the geometry of the { feno } species .
indeed , the a form of the { feno } species seems favoured by the h - bond interaction between no and the nitrogen of the distal histidine.[11f , g ] besides , the evolution of the spectrum is observed at higher temperature , when this interaction is suppressed.[11f in this regard , l - arginine ( and to a lesser extent noha ) could play a similar role in nos to that in neuro- and haemoglobin ( ascribed to the distal histidine ) . in the case of haemothiolate cyp450s ,
various situations were encountered , as the proportions of r and a forms depended on the nature of the cyp450 , and the substrate could also influence the different components in the epr spectra.[14 , 16 ] as the occurrence of rhombic and axial forms is also observed in model complexes,[13 , 22 ] it is not clear what the real cause for the existence of each form is .
however , the observations made on cyp450s and noss clearly indicate important roles for the distal pocket and the substrate on the structural and electronic properties of the { feno } complex . in the case of nos
, this should be important with respect to the catalytic cycle and the structure function relationship , as the ferrous nitrosyl moiety is a reaction intermediate during the second step of catalysis .
we investigated the effects of temperature on the { feno } epr spectra of no synthases .
the larger g anisotropy of nos { feno } signals and the comparative study of two isozymes made the analysis of the evolution of the various forms with temperature easier and allowed assessment of different models .
we confirmed that different forms coexist over a large range of temperatures and that the r form experiences a continuous change in its geometry as the temperature increases . in a similar way to
that reported for globins , we observed that these two phenomena depend on the interaction of no with the distal environment and , in this case , with the substrate .
however , interconversion of the r and a forms over temperature is not straightforward , and the exact nature of the a form remains in doubt .
comparative analysis of various nos isoforms by advanced epr techniques ( eseem , high - field epr ) appears to be a promising approach to definitively resolve these questions in the case of nos .
enzyme preparation : the wild - type inos oxygenase domain ( inos ) containing a his6 tag at its c terminus was expressed in escherichia coli bl21 by using the pcwori vector as previously described .
the bacterial nos - like protein from b. subtilis ( bsnos ) was overexpressed in e. coli bl21 as previously reported .
all proteins were purified in the absence of h4b and arg , by using ni - nitrilotriacetate affinity chromatography as described previously .
ferrous nitrosyl complex preparation : inos or bsnos was washed and conditioned in hepes buffer ( 100 mm , ph 7.4 ) in the absence or presence of cofactor h4b ( 100 m ) and substrate arginine ( 5 mm ) or n - hydroxy - arginine ( noha , 5 mm ) , depending on the experiment , by three consecutive cycles of dilution / concentration of enzyme ( 10 ) at 4 c in a centricon 30 kda filters ( merck millipore ) .
special care was given to the preparation of fresh sodium dithionite solutions to avoid , as much as possible , the presence of degradation products ( with consequent loss of dithionite reductive potency ) . fresh sodium dithionite ( 15 mg ) was conditioned in a small glass bottle under an inert atmosphere before being dissolved in freshly degassed hepes ( 2 ml ; 100 mm , ph 7.4 ) , added with an anaerobic hamilton syringe . to determine the true dithionite concentration , this freshly prepared solution was diluted ( 500 ) in hepes ( 100 mm , ph 7.4 ) containing cytochrome c. the difference in absorption ( 550=21 000 m cm ) before and after dithionite addition allowed determination of concentration ( generally , 2025 mm ) . theoretically , the concentration of such a solution was expected to be 40 mm . to prepare no solution , hepes ( 2 ml ; 100 mm , ph 7.4 ) was degassed in a small hermetically sealed glass bottle .
no gas , previously washed koh ( 0.1 n ) to eliminate nitrated no derivatives , was allowed to bubble in the solution for 5 min .
no concentration was verified by reaction with a solution of oxyhaemoglobin in hepes ( 100 mm , ph 7.4 ) . the difference in absorption at 577 and 590 nm ( for oxyhaemoglobin and methaemoglobin ,
respectively ) , recorded before and after reaction with no , allowed determination of the concentration of freshly prepared no - saturated solution ( 577590=10 300 m cm ) ; this was found to be 3.3 mm , in accordance with the literature .
inos and bsnos fe samples were reduced directly in an anaerobic epr tube with the freshly prepared dithionite ( stoichiometry 1:2.4 ) .
after equilibration of the sample ( 10 min ) no ( 3 equiv ) was added from the saturated no solution to allow formation of the ferrous nitrosyl complexes .
care was taken to perform this step very slowly , as local no excess can lead to protein degradation with loss of the proximal thiolate bond . to avoid fe contamination in the sample and spectral analysis complication due to a mixture of epr active species , sodium dithionite ( 1 equiv . )
was finally added to the sample before it was frozen ( 198 k ) in an ethanol / dry ice bath and transferred to in liquid nitrogen ( 77 k ) .
epr spectroscopy : epr spectra were recorded on an elexsys 500 x - band spectrometer ( bruker ) equipped with a continuous - flow esr 900 cryostat and an itc504 temperature controller ( oxford instruments , abingdon , uk ) . | nitric oxide is produced in mammals by a class of enzymes called no synthases ( noss ) .
it plays a central role in cellular signalling but also has deleterious effects , as it leads to the production of reactive oxygen and nitrogen species .
no forms a relatively stable adduct with ferrous haem proteins , which , in the case of nos , is also a key catalytic intermediate . despite extensive studies on the ferrous nitrosyl complex of other haem proteins ( in particular myoglobin ) , little
characterisation has been performed in the case of nos .
we report here a temperature - dependent epr study of the ferrous nitrosyl complex of the inducible mammalian nos and the bacterial nos - like protein from bacillus subtilis .
the results show that the overall behaviours are similar to those observed for other haem proteins , but with distinct ratios between axial and rhombic forms in the case of the two nos proteins .
the distal environment appears to control the existence of the axial form and the evolution of the rhombic form . |
a 13-year - old girl had a history of partial atrioventricular septal defect ( pavsd ) total correction at the age of 4 years .
however , a right ventricular assist device was applied at the operation because of the akinetic right ventricle ( rv ) .
two years later , based on our echocardiography results , the patient was transferred due to severe tricuspid regurgitation and rv chamber enlargement with rv dysfunction .
she underwent tricuspid septal commissuroplasty , de vega - type tricuspid annuloplasty , right atrial reduction plasty , and isthmus ablation .
further , we performed one - and - a - half ventricle repair . in spite of the operation ,
six years after the one - and - a - half ventricle repair , cardiac magnetic resonance imaging was performed to evaluate her cardiac function and measure the left ventricle ( lv ) volume .
the patient s rv end diastolic volume index and rv ejection fraction were 500.4 ml / m , and 13.2% , respectively .
the lv stroke volume index and ejection fraction were 38.2 ml / m and 28% , respectively .
echocardiographic evaluation showed global rv akinesia and lv ejection fraction of 25% with paradoxical interventricular septal motion .
we suspected that the rv enlargement affected both the rv and the lv function and it resulted in decreased lv contractility .
she underwent extracardiac conduit fontan operation with polytetrafluoroethylene ( ptfe , gore - tex ; wl gore & associates , flagstaff , az , usa ) 24-mm tube graft , rv exclusion , atrial septectomy , and permanent pacemaker implantation .
rv exclusion procedures include tricuspid valve obliteration ( from the rv side ; 5 - 0 prolene double layer , reinforcement suture from the ra side ; 4 - 0 polyester ptfe pledget - supported interrupted mattress suture ) and pulmonary valve obliteration ( 6 - 0 prolene running suture ) to reduce the rv volume with no flow connection , thrombin soaked gel - foam packing to the rv , and rv free - wall wide resection , and it was performed under the condition of cardiac arrest ( fig .
permanent pacemaker bipolar leads were implanted at the lv apex , rv apex , left atrial roof , and ra free wall owing to a history of frequent atrial flutter and junctional rhythm .
we did not perform arrhythmia surgery because the patient underwent an electrophysiology study and radiofrequency catheter ablation for supraventricular arrhythmia before the operation .
the cardiopulmonary bypass time was 308 minutes , and the aortic cross clamp time was 146 minutes .
we performed the computed tomographic angiography not cardiac magnetic resonance imaging to evaluate the patient s postoperative cardiac function and chamber size , because she was implanted with a permanent pacemaker .
the rv volume was markedly reduced , and the rv was occluded with thrombosis ( fig .
further , the stroke volume index and the ejection fraction of lv were increased to 48.03 ml / m and 33% , respectively ( table 1 ) .
the patient s vital signs were stable with a central venous pressure of 18 to 21 mmhg .
she was extubated in 14 hours , and she stayed in the intensive care unit for 90 hours .
we removed the chest tubes 5 days later , and she was discharged on postoperative day 21 .
she has been followed up for 21 months without any complaint of dyspnea and palpitation .
3 ) , and in the latest echocardiography , the ejection fraction of the lv had increased to 54% .
recently , the importance of rv failure has been noted , in light of the incomplete understanding of the rv failure mechanism and a poorer prognosis than lv failure has .
further , it has been reported that an increased rv volume and decreased ejection fraction are associated with lower survival rates in patients with congestive heart failure .
the rv volume overload is one of the causes of right ventricle failure ( rvf ) .
further , it is known that the rv volume over load leads to the leftward displacement of the interventricular septum and changes in the lv geometry , thus resulting in decreased lv contractility .
, several reports have shown the successful outcome of the original and the modified rv exclusion procedures in the case of ebstein s anomaly .
. she could have lived in a biventricular state if she had undergone the total correction of pavsd at the appropriate time . however , this case is meaningful in determining how to manage patients with rvf .
we performed an rv exclusion to normalize the motion of the interventricular septum by reducing the rv volume .
further , we demonstrated that both the end diastolic volume index and the stroke volume index increased after the operation .
another beneficial effect of rv exclusion is lung expansion , because an enlarged heart can compress the lungs .
in addition , adequate lung expansion helps to reduce pulmonary vascular resistance , and decreased pulmonary vascular resistance is a good prognostic factor among functional univentricular patients . in conclusion ,
the rv exclusion procedure in selected patients with severe rvf might be a safe and beneficial option to improve lv function . | a 13-year - old girl , who had undergone the total correction of partial atrioventricular septal defect at the age of 4 years , was admitted with severe tricuspid regurgitation in echocardiography .
she had received one - and - a - half ventricle repair during follow - up .
her right ventricle showed global akinesia , and the ejection fraction of the left ventricle was 25% with paradoxical interventricular septal motion .
we performed right ventricular exclusion adjunct to the fontan procedure .
she is doing well two years after the operation without complications . |
while superficial non - ampullary duodenal tumors are relatively rare , the frequency of their detection has recently increased due to the widespread use of endoscopy and continual improvements in endoscopic techniques ( 1,2 ) .
although there are no established indications or treatments for superficial non - ampullary duodenal tumors ( 2,3 ) , appropriate treatment is necessary for lesions that are histopathologically diagnosed to be high - grade dysplasia or which measure at least 20 mm in diameter , as these present a high risk of malignancy ( 2,4 ) .
endoscopic treatment for superficial non - ampullary duodenal tumors is technically challenging because of the anatomical features of the duodenum and a high incidence of adverse events , such as perforation ( 2 - 5 ) .
for many years , surgical repair has been the standard treatment for iatrogenic gastrointestinal perforations .
however , surgical procedures in the duodenum are relatively invasive , and they are also associated with significant morbidity and mortality . for these reasons , endoscopic closure of gastrointestinal perforation has recently been introduced as an attractive alternative ( 6 - 8 ) .
the over - the - scope - clipping system ( otsc system , ovesco endoscopy ag , tbingen , germany ) is an endoscopic device used to mechanically compress gastrointestinal tract tissue ( 9 ) .
the otsc system is composed of an applicator cap with a mounted otsc clip , a thread fitted to the otsc clip to assist clip release , and a hand wheel for clip release .
the otsc clip is deployed by rotating the hand wheel to pull the thread connected to the otsc clip .
the otsc twin grasper with two jaws , which can be separately opened as an assistive device to approximate tissue , is also available ( 10 ) ( fig .
the otsc system has made the closure of gastrointestinal tract defects , such as perforations , anastomotic insufficiency , and fistula , as well as hemostasis in difficult - to - treat non - variceal bleeding , possible ( 6 - 10 ) .
it also helps to achieve a more reliable closure , as its wide mouth allows it to hold a greater amount of tissue than the conventional through - the - scope clip ( ttsc ) ( 8,10 ) .
we herein describe a patient in whom the otsc system was useful for the treatment of a duodenal perforation caused by endoscopic submucosal dissection ( esd ) .
the otsc system is composed of an applicator cap with a mounted otsc clip , a thread fitted to the otsc clip to assist clip release , and a hand wheel for clip release .
the otsc twin grasper with two jaws , which can be separately opened as an assistive device to approximate tissue , is available ( reproduced with the permission of ovesco endoscopy ag , tbingen , germany ) .
a 39-year - old male with no clinical history was referred to our hospital for close examination and treatment of a duodenal tumor detected by screening radiography of the stomach . on upper gastrointestinal endoscopy
performed at our hospital , a reddened , sessile , elevated lesion measuring approximately 30 mm in diameter was detected in the anterior wall of the duodenal bulb , and a whitish lower elevated area spread around the lesion ( fig .
2a ) . magnifying endoscopy with narrow - band imaging ( olympus gif type h260z , olympus , tokyo , japan ) revealed leaf - like villous structures of various sizes .
the widths of the villi were greater than those in the surrounding normal areas ( fig .
chromoendoscopy with indigo carmine clearly showed the border of the lesion and the unevenness of its surface ( fig .
a reddened , sessile , elevated lesion measuring approximately 30 mm in diameter was observed in the anterior wall of the duodenal bulb , and an area of whitish lower elevation extended around the lesion .
c : magnifying endoscopy with narrow - band imaging combined with water - immersion technique .
spraying of indigo carmine clearly showed the border of the lesion and the unevenness of its surface .
we performed esd to confirm the histopathological diagnosis , because the accuracy of diagnosing duodenal tumor by biopsy is low , and because the risk of malignant transformation is high for tumors measuring at least 20 mm in diameter .
although a mixture of glyceol and hyaluronic acid was locally injected into the submucosa , the elevation of the lesion was insufficient , and submucosal dissection was difficult ( fig .
was changed to snaring with circumferential incision , which caused a full - thickness perforation measuring approximately 18 mm in diameter ( fig .
since the lesion was located in the anterior wall of the duodenal bulb , the resected lesion was not inverted into the peritoneal cavity , and the tumor was not exposed to the peritoneal cavity .
since arterial bleeding was observed from the full thickness resection site , hemostasis by cauterization was performed with hot biopsy forceps ( radial jaw 4 hot biopsy forceps , boston scientific , marlborough , usa ) at the perforated site .
the endoscope ( olympus gif type q260j , olympus ) was immediately withdrawn . after being attached to an 11/6 t otsc system ( fig .
both edges of the perforation were grasped with both jaws of the otsc twin grasper , which can be opened separately , in positions of correct approximation ( fig .
the grasped tissue was pulled into the applicator cap , and the otsc clip was then deployed while also performing continuous suction ( fig . 3f and g , supplementary material ) .
the procedure was completed after the effective closure of the perforation was confirmed by the dilation of the duodenum under insufflation .
transcutaneous needle decompression ( supercath , medikit , tokyo , japan ) was immediately performed for decompression after pneumoperitoneum .
thereafter , the patient fasted , was intravenously administered antibiotics and proton - pump inhibitors , and was intubated by nasogastric suction .
seven days after the procedure , the absence of leakage was confirmed by a gastrografin swallow ( fig .
the tumor size in the resected specimen was 2725 mm , and the pathological diagnosis was tubular adenoma with low - grade dysplasia . on follow - up
upper gastrointestinal endoscopy performed one month later , the perforation was closed by the otsc clip ( fig .
upper gastrointestinal endoscopy performed one year later showed disengagement of the otsc clip and scarring of the resection site ( fig .
a : although a mixture of glyceol and hyaluronic acid was locally injected into the submucosal layer , the elevation of the lesion was poor , and submucosal dissection was difficult .
b : resection of the lesion by snaring with a circumferential incision resulted in a full - thickness perforation measuring approximately 18 mm in diameter and arterial bleeding from the full thickness resection site .
c : the applicator cap with a mounted otsc clip was attached to the tip of the endoscope .
d : the left edge of the perforation was grasped with one jaw of the otsc twin grasper .
e : the other jaw of the otsc twin grasper was opened to grasp the right edge of the perforation while grasping the left edge with one jaw .
f : the grasped tissue was pulled into the applicator cap , with continuous suctioning .
while endoscopic treatment for superficial non - ampullary duodenal tumors is a challenging technique that is often associated with procedural accidents , the otsc system was shown in this patient to be useful for the management of such iatrogenic events .
esd is widely accepted as a treatment for gastrointestinal tumors because of its high en bloc resection rate , which makes accurate histopathological evaluation possible . however , the usage of this technique to treat duodenal lesions remains controversial due to its technical difficulty and its high incidence of procedural accidents ( 1 - 3,5 ) . based on excellent long - term outcomes ,
some endoscopic centers consider that piecemeal resection by endoscopic mucosal resection is acceptable , and refrain from aggressively performing duodenal esd from the viewpoint of safety ( 3 ) . indeed
, the incidence of adverse events in duodenal esd has been reported to range from 6.7 - 36.6% for intraprocedural perforations and 0 - 14.3% for delayed perforations ( 1 - 5 ) , being markedly higher than their incidences in other parts of the gastrointestinal tract .
moreover , emergency operations have been performed in 3.3 - 25.0% of the patients who underwent duodenal esd due to the conversion of esd to surgery necessitated by intraprocedural uncontrollable perforation or delayed perforation ( 1 - 5 ) .
this is due to the anatomical features of the duodenum , such as a narrow lumen , precipitous flexure that makes the endoscope unstable , abundant brunner 's glands in the submucosal layer that inhibit proper mucosal lifting , rich submucosal vasculature , and thin muscle layer associated with a high incidence of perforation ( 1,3,4,11 ) . in this patient ,
the otsc system was shown to be useful for closing an acute iatrogenic duodenal perforation .
conventionally , ttscs have been used for the endoscopic closure of gastrointestinal perforations . however , due to the narrow wingspan of the ttsc , multiple clips were needed for the repair , the leak rate after the repair of large perforations exceeding 1 cm was significant , and the treatment ended in mere surface layer closure rather than in full - thickness closure ( 6,7,10 ) . the otsc system , on the other hand , has higher gripping and holding strengths ( 10,11 ) and it is designed to achieve full - thickness closure of perforations with diameters up to 3 cm ( 6 ) .
it has been reported to be particularly effective for closing defects and leaks without causing fibrosis at the edges ( 8,10 ) . according to a systematic review of the clinical outcomes of endoscopic closure of acute iatrogenic gastrointestinal perforations , the success rate using the otsc system was remarkably high , namely at 87.8% ( 7 ) .
few short- or long - term adverse events associated with otsc placement have been reported ( 8) , thus indicating its safety as an endoscopic device . in duodenal esd
, it is necessary to pay attention not only to intraprocedural but also delayed perforation , due to exposure of the post - esd ulcer to bile and pancreatic juice and to excessive thermal injury to the muscle layer ( 1 - 5,11 ) .
complete closure of a post - esd ulcer is useful for preventing delayed perforation ( 2,3,11 ) .
the usefulness of the otsc system for complete closure of duodenal post - esd ulcers has been reported ( 11 ) .
provided a complete resection can be achieved , the otsc system may therefore become useful not only for closing intraprocedural perforations , but also for preventing delayed perforations ( 2,11 ) . in conclusion ,
the otsc system is considered to be a safe and efficient device for closing gastrointestinal perforations . for endoscopists who perform endoscopic treatments in the duodenum , which are technically difficult and associated with a high incidence of procedural accidents , endoscopic closure with the otsc system
a full - thickness perforation measuring approximately 18 mm in diameter was endoscopically closed with the otsc system .
| endoscopic treatment for superficial non - ampullary duodenal tumors is technically difficult and challenging due to the anatomical characteristics of the duodenum .
it is frequently complicated by procedural accidents , such as perforation .
surgical repair has long been the standard treatment for acute iatrogenic gastrointestinal perforation .
however , endoscopic closure has recently emerged as an attractive alternative . in the patient presented herein , the over - the - scope - clipping system ( otsc system ) was found to be useful for closing a duodenal perforation that had occurred during endoscopic submucosal dissection . for endoscopists who perform endoscopic treatment of the duodenum , endoscopic closure with the otsc system
is considered to be a technique that is necessary to master . |
the aim of this review of the literature is to ignite the discussion related to the oral care practices of nurses for older acute medical hospitalised patients .
this paper explores the literature related to oral health care for older patients admitted to acute medical wards . caring for older patients with complex medical issues is challenging and one fundamental basic care is the provision of oral care which is often overlooked .
literature relevant to this issue was reviewed to confirm our view that oral care was often overlooked and could be improved . as people age ,
their susceptibility to chronic and life - threatening diseases as well as acute infections increases , exacerbated by compromised immune systems .
the number of elderly people in society is increasing and consequently also the number of older people in need of health care and nursing care .
it is projected that by 2025 , the number of elderly people will increase by 146% to 1.25 billion worldwide . over the past two decades in australia ,
the growth in this population of older people is staggering , posing tremendous challenges in caring for this group and their chronic conditions .
the consequences of chronic diseases and conditions are significant , leading to disabilities and reduced quality of life .
individuals with the most prevalent medical problems tend to have the highest rates of oral disease , with an association between poor oral health and adverse medical outcomes such as aspiration pneumonia and cardiovascular disease [ 5 , 6 ] .
attention has been focused on oral care as the evidence accumulates to support an association between the bacteria in the mouth and those respiratory pathogens that cause pneumonia .
the benefits of this literature review can bring to light practice gaps , and areas for practice improvements for nursing care of this vulnerable group , through research , quality improvement activities , and development of practice guidelines within a policy framework .
a search of the literature used the search terms oral health and/or oral hygiene and nursing and medical patients published between 2006 to 2012 in the databases of cinahl and medline .
there were over 600 articles retrieved on oral hygiene / health ; however there was limited literature that specifically focused on oral care for older medical patients in acute care .
the literature in this review was obtained from nursing , medical , dental journals and government publications , and grey literature discussing oral care , hygiene , and inpatients .
the literature excluded from this review was articles discussing oral health for children , and oral surgery .
good oral health is important . having a clean and healthy mouth contributes to a sense of well - being [ 811 ] allows for fluid and nutritional intake , assists with communication and quality of life , and assists with clear speech and communication .
note that maintenance of good oral health is important for patients in hospital , as it contributes to the well - being , recovery , and nutritional needs of the patient , and it requires the involvement of nursing staff .
the literature suggests that oral care is not a highly technical skill or requires huge resources .
it is an individualised and practiced behaviour and is an essential aspect of nursing care [ 9 , 14 , 15 ] . as dickinson et al .
state , when a person is unable to perform their own oral care in hospital it becomes the responsibility of nursing staff .
bissett and preshaw suggest that oral care is like other personal care needs such as bathing and toileting ; it is an essential component of holistic care .
these variances in oral care relate to different factors , such as oral care being neglected [ 1719 ] and oral care being given a low priority when compared to other nursing care elements [ 7 , 8 ] .
oral hygiene is often thought to be underrecognised by nurses for the fundamental impact it can have on a person 's wellbeing and health status . in this context
the delivery of oral care to hospitalised patients is recognised in the nursing literature as an imperative to maintaining health and wellbeing [ 22 , 23 ] particularity in vulnerable groups of patients who can not maintain their own oral health when hospitalised .
there are some particular patient groups known to be at risk of oral health problems or who have existing oral diseases and conditions .
these specific patient groups are cancer patients , palliative patients , patients who are intubated [ 17 , 25 ] , critically ill patients , frail patients , and the elderly [ 9 , 26 ] .
research evidence suggests there are subgroups of older people known to be at risk of poor oral care , in particular people with dementia , and those that have come from residential care [ 20 , 2830 ] .
it is often these subgroups of older people that are a majority of the patients on acute medical wards .
acute medical wards in hospitals are frequently filled with frail people , and this group of patients have been noted to often have comprised oral health ( andersson 1999 and hrn et al .
fitzpatrick noted that older adults due to ageing processes have a loss of soft tissue attachments , which results in loosening of teeth root exposure , and teeth can become more brittle .
due to the ageing process older people have oral care needs that need to be met .
common side effects of poor oral care are pain , difficulty with swallowing , poor or compromised nutritional intake , infection , systemic infection , and impaired communication .
in the acute medical setting , nursing staff are responsible for assisting with oral health care .
admission to hospital is not only a time for the active management of the presenting disease but also an excellent opportunity for the health promotion and screening for undetected pathology .
discuss the importance of nurses performing daily oral care for older people on acute , subacute , and rehabilitation wards ; however these authors recognise that much of the nurses education in this important area has been provided during their early training and regular updates may not occur .
the lack of knowledge about oral health care among nursing staff is also supported by wrdh et al . .
nursing staff know that good nursing includes oral health care , but this knowledge does not always mean that oral health care is administered [ 2 , 32 ] . in a study that was conducted by wardh et al .
where 22 in - depth nursing interviews were administered , it was found that the quality of oral health care is largely dependent upon the cooperation of the elderly patients .
some nurses reacted in a negative way due to the risk of being bitten by elderly patients during oral health care .
patients who wore dentures were not always willing to take them out and cleaning dentures also seemed to be a repulsive activity . some of the nursing staff in this study experienced a lack of time as a factor inhibiting good oral health care .
others did not see lack of time as a problem , but in stressful situations , oral health care could be easily forgotten .
some nursing staff worry about whether it is right or wrong to force oral health care when an elderly patient refuses care .
oral health care is an important part of treatment for all patients , particularly those who require assistance with activities of daily living .
the majority of hospitalised patients within the acute medical units are older over age 65 years .
concerns regarding the nursing care older people receive in the acute care environment are frequently cited in the literature and in particular the link between patient outcomes and nursing care . in this paper
we have focused on oral care ; however other aspects of patient care require similar attention but are beyond the scope of this paper . as the population ages , the likelihood of altered physical ability and presence of disease increases , leading to a reduced ability to perform activities of daily living , for example , oral care .
comorbid conditions most likely to be seen in the older population include , but are not limited to , diabetes , congestive heart failure , renal disease , glaucoma , and cataracts .
these isues can lead to the need for hospitalisation often resulting in a protracted length of stay and the increased chance of deconditioning which in turn prevents older patients from attending to basic care needs such as oral care .
hospitalization can represent the beginning of functional decline and increased dependency that may lead to an individual requiring long - term care [ 3437 ] .
the state of a patient 's oral health can have a significant impact on their health outcomes , most notably psychosocial well - being , respiratory health , and nutritional status .
routine oral care ( tooth brushing , mouth toilets , etc . ) are often the responsibility of the nurse or health assistant without the required knowledge and skill or comprehensive hospital protocols to follow .
this responsibility is related to decreased functional decline leading to ability to attend to adls .
the link between functional decline and the need to assist with adls may not always be apparent to the nurses caring for hospitalised older patients . to overcome this issue the literature on this subject
has identified the need for nurses to routinely assess oral health status and to determine what assistance is required for the patient to maintain good oral health , especially for older patients as debility and frailty can interfere with a patient 's ability to self - manage their oral care .
many patients are often admitted through the emergency department and as such may not come in with the basics toothbrush and toothpaste .
nursing admission assessments do not routinely include assessment of the oral cavity or the patient 's ability to manage self - care , and much of the assessment of oral care needs and self - care abilities of patients is subjective with decision support protocols not routinely available within the acute environment .
anecdotal evidence suggests assessment of patient 's oral health on medical in - patient wards is generally poor .
it is encouraging to note that the literature supports that when nurses are offered education and decision support they respond positively and actively engage [ 3840 ] .
the available literature supports the view that oral health care of hospitalised patients is variable and overlooked and that nurses ' knowledge and practice are variable .
nurses play a key role undertaking oral health care including the identification and evidence to guide the patients at risk for therapy - related oral mucositis , periodontal disease ( a chronic inflammatory condition ) , chronic infection of the tissue surrounding the teeth , and assessment of patients ' ability to independently manage their oral hygiene .
this requires the involvement of the nursing staff , especially in cases where oral care and any necessary dental treatment are vital to ensure medical treatment . in the acute medical setting
, oral health care seems to be separated from other nursing activities and is not discussed when nursing care plans are written , only when oral problems are obvious . to enhance the integration of oral care within routine nursing practice using a patient centred approach ,
some strategies may include education of staff , patient , and carers ; provision and/or increased accessibility of equipment ( toothbrush , toothpaste , and mouth wash ) ; and inclusion of oral health care as a major component of all documentation of nursing care .
policies and practices that support the maintenance of good oral health are needed to lessen the disease burden and promote healthful aging for this growing population .
health care professionals need to reduce the obvious service fragmentation and collaborate , especially since the most severe oral problems are usually found in the older patients . | oral health care is an essential aspect of nursing care .
there are many variances in the quality and frequency of the oral care that is delivered to patients by nursing staff , such as oral care being given a low priority when compared to other nursing care elements , oral care being neglected , and oral care delivery being dependent on the nurse 's knowledge of oral hygiene . additionally , there are some particular patient groups known to be at risk of oral health problems or who have existing oral diseases and conditions . as people age their susceptibility increases to chronic and life - threatening diseases , and they can be at increased risk of acute infections increases compromised by ageing immune systems .
the aim of this literature review was to ignite the discussion related to the oral care practices of nurses for older acute medical hospitalised patients .
the review revealed that nursing staff know that good nursing includes oral health care , but this knowledge does not always mean that oral health care is administered .
oral health care seems to be separated from other nursing activities and is not discussed when nursing care plans are written , only when oral problems are obvious . |
benign prostatic hyperplasia ( bph ) is a histologic diagnosis that refers to the proliferation of smooth muscle and epithelial cells within the prostatic transition zone , for which dihydrotestosterone ( dht ) is the primary androgen responsible in elderly men . as the initial treatment for symptomatic bph , pharmacotherapy with 1-adrenergic blockers , 5-reductase inhibitors , antimuscarinic agents , or a combination thereof is often used .
type 1 and type 2 isoenzymes of 5-reductase are present throughout the body , and dutasteride , a dual 5-reductase inhibitor , acts competitively and specifically on type 1 and type 2 isoenzymes to inhibit the conversion of testosterone to the more potent dht [ 46 ] . in comparison with finasteride ,
a 5-reductase inhibitor selectively acting on type 2 isoenzyme , dutasteride is a 45-fold greater inhibitor of type 1 5-reductase , and a 2.5-fold greater inhibitor of type 2 5-reductase [ 810 ] .
patients with bph who do not respond suitably to pharmacotherapy often undergo transurethral resection of the prostate ( tur - p ) , the gold standard for the surgical treatment of bph , and microwave thermotherapy in order to improve symptoms of bladder outlet obstruction .
several studies assessing clinical and histopathological perioperative effects of dutasteride have been reported [ 1214 ] , in which staining of cd31 or cd34 in endothelium was mainly used to detect microvessels .
data delineating histopathological effects of long - term dutasteride administration on bph tissue are still lacking .
therefore , we conducted a retrospective comparative study to analyze histopathological changes in bph tissue between patients given dutasteride for a relatively long time and controls without preoperative medication .
blood vessels are composed of aorta , arteries , arterioles , capillaries , venules , veins , and cava . in the prostatic tissue , arteries and arterioles
are identifiable with light microscopy as blood vessels with thick walls of obvious muscular media .
there are no specific histologic features that accurately distinguish small arteries from larger arterioles . for convenience
, arterioles are said to have a diameter of less than 100 m . on the contrary , veins and venules are identified as blood vessels with thin muscular wall and the latter is also supposed to be less than 100 m in diameter .
capillaries have neither a muscular media nor an elastic lamella , while a single but complete layer of endothelial cells lies on a basement membrane .
additionally , it was reported that the vascular endothelial markers cd31 and cd34 are occasionally expressed even in lymphatic endothelial cells [ 16 , 17 ] , which may make it more difficult to definitely distinguish capillaries and lymphatic vessels .
the definition of microvessels does not appear in textbooks of pathology , but seems to be explained as blood vessels of the microcirculatory system , that is , capillaries , arterioles , and venules . in the present study , we assessed parameters concerning arteries and arterioles as well as veins and venules in resected prostate tissue , as they have solid vascular walls and are firmly identified by examining sections stained with hematoxylin and eosin using light microscopy
. additionally , we counted the number of vascular vessels in the tissue by staining endothelial cells with anti - cd31 antibody .
arteries / arterioles and veins / venules are much larger than capillaries , and it is reasonable to hypothesize that they contribute more to the operative blood loss in the perioperative period .
the aim of this study was to assess the histopathological effects of dutasteride administration in the tissue of patients with benign prostatic hyperplasia in a retrospective study .
dutasteride reduced artery / arteriole and vein / venule densities and vein / venule area .
this study was conducted in accordance with the helsinki declaration after approval by the ethics committee of kanto rosai hospital .
the committee approved the use of oral consent documented in the electronic chart for each patient , as the study was retrospective and nonrandomized .
this is a retrospective study to investigate effects of dutasteride administered before transurethral resection of the prostate in japanese bph patients .
twenty - seven bph patients whose prostate volume determined by transabdominal ultrasonography was more than 40 cm were enrolled between february 2010 and april 2011 .
their serum psa levels were less than 4.0 ng / ml , otherwise transrectal prostate biopsy had already shown no cancer in the prostate .
they had not received lhrh agonists , antiandrogens , estrogens , and 5-reductase inhibitors and had not experienced any type of prostatic surgery or radiation therapy to the pelvis before .
patients with severe cardiovascular disease , liver disease , renal failure , or bleeding tendency were excluded , as they were not suitable for tur - p .
patients were administered 0.5 mg of dutasteride once daily ( glaxosmithkline , uk ) for some period ( n = 15 ) , or else they underwent a transurethral resection of the prostate ( tur - p ) without dutasteride treatment ( n = 12 ) . a daily 0.5
age , body height , body weight , body mass index , and duration of administration of dutasteride were recorded preoperatively .
the transurethral resection was carried out by three surgeons with irrigation in 3% d - sorbitol ( uromatic s , baxter , usa ) .
patients were not transfused with autologous or allogeneic blood of any type in the perioperative period .
operation time for tur - p , amount of intravenous crystalloid infusion during tur - p , and weight of resected prostate tissue were recorded as perioperative data .
pre- and postoperative blood hemoglobin ( hb ) , hematocrit ( hct ) , and serum sodium ( na ) levels were also determined . as histopathological data , resected prostate tissue was assessed for artery / arteriole density ( ad ) , vein / venule density ( vd ) , and glandular density ( gd ) .
additionally , artery / arteriole area ( aa ) , vein / venule area ( va ) , and glandular area ( ga ) were also evaluated as a proportion of the whole area .
some sections were subjected to elastica van gieson staining in order to distinguish between elastic fibers , collagen fibers , and muscle fibers .
additionally , all sections were immunohistochemically stained with anti - cd31 monoclonal antibody ( jc70a , dako , denmark ) following the manufacturer 's recommendations .
then , a section of one chip at the center of the tissue slide as well as an additional four sections of chips around the first section were scanned with a microscopy ( bx51 , olympus , japan ) at a magnification of 40 .
the histology of each section was captured into a windows computer with a digital camera ( ds - fi1 , nikon instruments , japan ) using image - analyzing software ( nis - elements d , nikon instruments , japan ) .
a scale bar of 1000 m was placed on each image for calibration in the histological analysis .
thereafter , images of sections were analyzed with image - analyzing software ( image j , http://rsbweb.nih.gov/ij/ ) in order to count numbers of arteries / arterioles , veins / venules , and glands in sections stained with hematoxylin and eosin .
outer lines of whole sections were traced with a freehand - selections tool of image j in order to measure the traced areas .
outlines of the lumens of arteries / arterioles and veins / venules were also traced in a similar manner to measure their intraluminal areas .
intraglandular areas were thresholded and measured also using image j. the sums of the number of arteries / arterioles or veins / venules in five sections from a patient divided by the sum of the whole areas of five sections were regarded as ad and vd , respectively .
the sums of all artery / arteriole areas or of all vein / venule areas in five sections divided by the sum of the whole areas of five sections were regarded as aa and va , respectively .
ga was similarly calculated . supposing that vascular lumens are completely round , the diameter ( d ) of a lumen was calculated by the formula ; d=4a/ , where a is the measured area of the lumen .
density and area of cd31-expressing vessels were similarly calculated with sections stained with anti - cd31 antibody .
more precisely , outlines of the lumens of cd31-expressing vessels were traced to measure their intraluminal areas .
the sums of the number of cd31-expressing vessels in five sections from a patient divided by the sum of the whole areas of five sections were regarded as cd31d .
the sums of all areas of cd31-expressing vessels in five sections divided by the sum of the whole areas of five sections were regarded as cd31a .
the statistical analysis was performed using the mann - whitney u test for intergroup comparisons .
spearman 's rank correlation test and the linear regression analysis were performed to analyze correlations between ad / vd and the duration of administration of dutasteride and to draw least squares regression lines .
there were no significant differences in age , height , body weight , body mass index , and prostate volume at the first visit between the control and dutasteride - treated groups . in the dutasteride - treated group ,
the period of dutasteride administration was 16.3 8.1 weeks and reduction in prostate volume was 28.2% 30.2% .
there were no significant differences in operation time , amount of intravenous crystalloid infusion during tur - p , weight of resected prostate tissue , and perioperative changes in hb , hct , and serum na .
the numbers of vascular vessels detected in all 27 cases either with hematoxylin / eosin staining or with cd31 staining were 724 and 3390 in total , that is , 26.8 and 125.6/case , respectively .
the difference in the number of vessels detected either with hematoxylin / eosin staining or with cd31 staining was more in vessels whose diameters were less than 100 m compared with larger vessels , as shown in figure 1 .
figure 2 shows the typical histology of bph with hematoxylin and eosin staining and with elastica van gieson staining .
arteries / arterioles and veins / venules were able to be distinguished by the thick muscular media .
as the identification of arteries / arterioles and veins / venules was similar between elastica van gieson staining and staining with hematoxylin and eosin , the histological data for arteries / arterioles and veins / venules were basically obtained using sections stained with hematoxylin and eosin . with hematoxylin / eosin staining , the numbers of arteries / arterioles and veins / venules were 182 and 542 in all 27 cases , that is , 6.7 and 20.1/case , respectively , while vessels without media such as capillaries were not counted .
arteries / arterioles , veins / venules , and capillaries were essentially indistinguishable with cd31 staining .
ad , vd , and va were significantly lower in the dutasteride - treated group than the control group .
cd31d and cd31a were also similar between the two groups . as shown in figure 4 ( lower panels ) , vd and va were negatively correlated with the duration of administration of dutasteride ( p = 0.002 and 0.003 , resp . ) .
neither ad nor aa showed a significant correlation with the duration of dutasteride treatment ( p = 0.099 and 0.089 , resp .
cd31d and cd31a were not correlated with the duration of administration either ( data not shown ) .
blood vessels allow haematopoietic cells to eliminate the organism for immune surveillance , supply oxygen / nutrients , and dispose of waste leading to beneficial effects for tissue growth and regeneration .
angioblasts differentiate into endothelial cells and then assemble to form a vascular vessel ( vasculogenesis ) followed by subsequent sprouting expansion of the vascular network ( angiogenesis ) .
human and rat prostate endothelial cells play an important role in androgen responses in the prostate , as they express functional androgen receptor and are androgen sensitive [ 19 , 20 ] .
hahn et al . showed that the perioperative administration of dutasteride had no effects on microvessel density ( mvd ) in prostatic tissue of bph patients given pre - operative dutasteride for 2 or 4 weeks compared with placebo controls in a prospective , randomized , and multicenter study , where mvd was counted by the number of microvessels immunostained with anti - cd34 antibody within an area of 0.754 mm of a resected chip .
accordingly , it follows that there are approximately 70 microvessels per mm in their control specimens , while the sum of the arteries / arterioles and veins / venules in the control specimens in our study was only 1.22 per mm .
. also showed that dutasteride had no effect on prostatic mvd in bph patients given pre - operative dutasteride for 2 to 4 weeks compared with controls given no drug , where mvd was calculated as a mean of counts of cd34-positive microvessels in 10 consecutive high - power fields .
the cd31d , density of cd31-positive vessels , is much lower in the present study compared with the densities of microvessels in other studies .
the distribution of cd31-positive vessels is not even throughout the resected prostatic chips with some portion containing few vessels . in the present study ,
all cd31-positive vessels in five chips were counted , then the cd31d was calculated by dividing the number of the vessels by the whole area of those chips .
thus , the calculated densities were not very high . concerning preoperative finasteride administration and mvd in resected prostatic tissue , donohue et al
. reported that inhibition of expression of vascular endothelial growth factor and reduction of mvd were observed after two weeks administration of 5 mg daily of finasteride , where mvd was assessed by counting cd31-positive microvessels in 10 nonoverlapping , consecutive high - power fields .
furthermore , lekas et al . reported in a prospective , randomized study that finasteride administration for 25.3 weeks on average to bph patients resulted in the suppression of mvd as well as vascular endothelial growth factor and hypoxia - inducible factor-1 expression in a time - dependent manner , where mvd was assessed by counting cd34-positive microvessels in the three most vascularized high - power fields .
the vessels with media identified with hematoxylin / eosin staining in the present study seem mainly to be arterioles and venules of relatively smaller diameter , as the peak of the distribution of calculated diameters of those vessels was 50100 m .
the duration of dutasteride administration of 16.3 weeks in the present study was relatively long compared with that in previous studies , up to 4 weeks .
this may be why dutasteride administration before tur - p in this study caused reductions of ad , vd , and va in the prostatic tissue partly in a time - dependent manner . otherwise , the counting of numerous capillaries in other reports might have concealed more important influences of dutasteride on larger blood vessels . in other words
, although larger blood vessels tend to be influenced by dutasteride more than capillaries , the much more numerousness of the latter contributes to the computed values of the vessel densities and gets changes of the larger vessels indiscernible .
actually , androgens are reported to limit the number of new vessels developed while they contribute to the presence of larger vessels and androgen deprivation therapy induces changes in relatively large vessels .
measurements of luminal areas of blood vessels in the prostate were not made in the preceding reports describing effects of 5-reductase inhibitors , which can be a useful tool for evaluating effects of drugs on blood vessels . as they showed clearer effects of dutasteride including time dependency , veins / venules might be more susceptible to dutasteride than arteries / arterioles .
this may be because the numbers of capillaries in the bph tissues outweigh those of larger vessels with media and capillaries may not be influenced by dutasteride .
then , it may be worth evaluating larger vascular vessels with media by morphologically and morphometrically investigating specimens under direct vision and not only relying on staining with endothelial markers such as cd31 and cd34 . in the rat prostate , levels of angiogenetic factors such as hypoxia - inducible factor-1 and vascular endothelial growth factor ( vegf ) were lower in the dutasteride - treated groups than in the control group . in human bph tissues ,
vegf expression was significantly associated with androgen receptor and type 2 5-reductase expression , but not with type 1 5-reductase expression .
these result could support the present finding of decreased vascularity in bph tissues with dutasteride administration possibly through the suppression of androgen signaling pathways .
andriole et al . reported that prostate cancer patients administered 10 mg of dutasteride daily for 1 week followed by 5 mg daily for more than 45 days in total showed a decrease in mvd by 45% in cancer tissue in addition to a trend toward increased apoptosis of cancer cells when compared with controls without dutasteride administration . in this paper ,
the dose of dutasteride was much higher than that used for bph patients , and so the significant decrease in mvd in prostatic tissue might have been demonstrated after a shorter period of dutasteride administration than in the present paper .
another possibility is that the interstitial microvessels in malignant prostatic tissue differ from those in benign tissue in quality .
andriole et al . also reported in a more recent article that the administration of 0.5 mg of dutasteride daily over a 4-year study period ( reduce study ) reduced the risk of incident prostate cancer and acute urinary retention by 22.8% and 77.3% , respectively , compared with the control .
as the administration of finasteride for as little as two weeks reduced prostatic mvd , the actions of finasteride on prostatic microvessels may be faster and more profound than those of dutasteride .
additional inhibition of the type 1 5-reductase by a dual 5-reductase inhibitor may antagonize the microvessel suppressive activity of type 2-specific 5-reductase inhibitors . in benign prostatic tissue
, we did not detect differences in glandular density and the proportion of intraglandular area regardless of dutasteride administration .
such morphometrics seem rarely reported , probably because glands in benign prostatic tissue have complicated folds .
we could calculate values by thresholding intraglandular spaces with the thresholding function of image j. in the report by andriole et al .
described above , dutasteride caused a decrease of 18% in the width of benign prostatic epithelial cells in patients with prostate cancer .
considering that densities and areas of arteries / arterioles and veins / venules , but not glands in benign prostatic tissue decreased with dutasteride as reported in this study , the main target of dutasteride in reducing bph size could be the interstitium including blood vessels , not glands .
perioperative 46 weeks ' administration of dutasteride had no effect in reducing blood loss during or after tur - p [ 12 , 13 ] , while two patients in the control group in the latter article required a postoperative blood transfusion compared to no patient in the dutasteride - treated group .
similarly in the present study , blood loss was comparable between the two groups as estimated from the perioperative changes in blood hemoglobin and hematocrit .
thus , the reduction in the density and area of blood vessels in bph did not directly lead to decreased blood loss in the perioperative period . in conclusion , dutasteride administration for 16.3 8.1 weeks before tur - p caused reductions of ad , vd , and va in bph tissue partly in a time - dependent manner .
blood loss estimated by the perioperative changes in blood hb and hct was comparable between the control and dutasteride - treated groups . | objectives .
dutasteride , a dual 5-reductase inhibitor , is used to treat benign prostatic hyperplasia .
nevertheless , its histopathological effects on the morphometrics of blood vessels and glands are still controversial .
the aim here was to assess the histopathological effects of dutasteride in cases of benign prostatic hyperplasia in a retrospective study .
methods .
patients with benign prostatic hyperplasia more than 40 cm3 in prostatic volume were administered 0.5 mg of dutasteride daily or left untreated prior to receiving a transurethral resection of the prostate .
images of sections stained with hematoxylin / eosin and with anti - cd31 antibody were analyzed .
results . in the dutasteride - treated group ,
the duration of administration was 16.3 8.1
weeks .
artery / arteriole density and vein / venule density in benign prostatic tissue were both lower in the dutasteride - treated group than in the control group .
the vein / venule area as a percentage of the whole area was also lower in the dutasteride - treated group , while the artery / arteriole area did not show a significant difference .
glandular / cd31-expressing vessel densities as well as glandular / cd31-expressing vessel areas were comparable between the two groups .
conclusions .
dutasteride reduced the artery / arteriole and vein / venule densities and the proportion of vein / venule area in the tissue of patients with benign prostatic hyperplasia . |
a cross - sectional design was employed and written informed consent was obtained from all the subjects taken up for the study .
all asymptomatic hiv patients who were admitted to the hospital at the time of evaluation were included in the study .
staging of hiv seropositive cases was carried out as per centers for disease control ( cdc ) classification .
although interviewers knew of the seropositive status , they were not aware of the stage of hiv disease prior to the psychological assessment . an equal number of normal persons were selected as controls .
the controls were comparable with the cases regarding their age , education , sex , monthly income and occupation
.
confirmed hiv seropositivityphysically asymptomatic in stage ii or iiiage between 15 and 50 years .
confirmed hiv seropositivity physically asymptomatic in stage ii or iii age between 15 and 50 years .
confirmed hiv seronegativityage between 15 and 50 yearsphysically asymptomatic at the time of evaluation .
confirmed hiv seronegativity age between 15 and 50 years physically asymptomatic at the time of evaluation
.
past history or presence of central nervous system disordershistory of head injury with loss of consciousnesspast history of psychiatric illnesspatients who qualify acquired immune deficiency syndrome ( aids ) defining criteria .
past history or presence of central nervous system disorders history of head injury with loss of consciousness past history of psychiatric illness patients who qualify acquired immune deficiency syndrome ( aids ) defining criteria .
after enrolment into the study , a semi - structured proforma was used to record demographic details and assess risk factors for hiv infection .
physical examination , mental status evaluation and relevant laboratory investigations ( hemogram , urine routine examination , liver function test , hepatitis b antigen , venereal disease research laboratory and others if required ) were carried out to exclude cases and controls not eligible for the study .
psychiatric diagnoses were made as per the icd-10 classification of mental and behavioral disorders diagnostic criteria for research .
all subjects underwent the following psychiatric rating scales :
general health questionnaire ( ghq)mini mental state examination ( mmse)hospital anxiety and depression scale ( hads)sensation seeking scale ( indian adaptation ) .
general health questionnaire ( ghq ) mini mental state examination ( mmse ) hospital anxiety and depression scale ( hads ) sensation seeking scale ( indian adaptation ) .
the socio - demographic characteristics of the group were compared by the chi - square test . for comparing the scores on psychiatric rating
all asymptomatic hiv patients who were admitted to the hospital at the time of evaluation were included in the study .
staging of hiv seropositive cases was carried out as per centers for disease control ( cdc ) classification .
although interviewers knew of the seropositive status , they were not aware of the stage of hiv disease prior to the psychological assessment . an equal number of normal persons were selected as controls .
the controls were comparable with the cases regarding their age , education , sex , monthly income and occupation .
confirmed hiv seropositivityphysically asymptomatic in stage ii or iiiage between 15 and 50 years .
confirmed hiv seropositivity physically asymptomatic in stage ii or iii age between 15 and 50 years
confirmed hiv seronegativityage between 15 and 50 yearsphysically asymptomatic at the time of evaluation .
confirmed hiv seronegativity age between 15 and 50 years physically asymptomatic at the time of evaluation
past history or presence of central nervous system disordershistory of head injury with loss of consciousnesspast history of psychiatric illnesspatients who qualify acquired immune deficiency syndrome ( aids ) defining criteria .
past history or presence of
central nervous system disorders history of head injury with loss of consciousness past history of psychiatric illness patients who qualify acquired immune deficiency syndrome ( aids ) defining criteria .
after enrolment into the study , a semi - structured proforma was used to record demographic details and assess risk factors for hiv infection . physical examination , mental status evaluation and relevant laboratory investigations ( hemogram , urine routine examination , liver function test , hepatitis b antigen , venereal disease research laboratory and others if required ) were carried out to exclude cases and controls not eligible for the study .
psychiatric diagnoses were made as per the icd-10 classification of mental and behavioral disorders diagnostic criteria for research .
all subjects underwent the following psychiatric rating scales :
general health questionnaire ( ghq)mini mental state examination ( mmse)hospital anxiety and depression scale ( hads)sensation seeking scale ( indian adaptation ) .
general health questionnaire ( ghq ) mini mental state examination ( mmse ) hospital anxiety and depression scale ( hads ) sensation seeking scale ( indian adaptation ) .
the socio - demographic characteristics of the group were compared by the chi - square test
. for comparing the scores on psychiatric rating scales the mann - whitney u - test was used .
a total of 200 subjects were included in the study of which 100 were cases ( hiv seropositive ) and 100 were controls ( hiv seronegative ) .
this selection bias was due to the fact that the hospital catered to predominantly male population .
of the hiv seropositive patients 75 were married compared with 73 of the seronegative subjects . the difference was not statistically significant ( = 0.03 ; df = 1 ; p = 0.872 ) .
though 75% of the hiv seropositive subjects were married however , only 5 - 6% was staying with their spouses due to the nature of their employment .
duration of hiv seropositivity was 0 - 6 months in 55 patients ; nine patients each were seropostive for 6 - 12 months , 12 - 18 months and 18 - 24 months ; seven patients each were seropositive for 24 - 30 and 30 - 36 months ; while two patients each were positive for 36 - 42 months and > 42 months .
the risk factors for hiv infection included heterosexual promiscuity ( n = 85 ) , multiple injections / tattooing ( n = 3 ) , transfusion of unscreened blood ( n = 2 ) , health worker handling infected blood ( n = 1 ) while in nine patients no risk factor could be identified . neither cases nor controls showed any cognitive dysfunction on mmse ( score < 24 ) .
the observed ghq-12 caseness ( a cut - off score of 2 ) for hiv and control groups was 49 and 9 respectively [ table 2 ] .
the difference was statistically significant ( = 38.85 ; df = 1 ; p < 0.0001 ) .
all the patients who ultimately were given a psychiatric diagnosis had abnormal scores on ghq .
thus in the present study , ghq-12 had a sensitivity of 100% and specificity of 92% for detecting psychiatric morbidity in asymptomatic hiv patients .
demographic characteristics of hiv positive and negative subjects results of ghq and hads results of sensation seeking scale psychiatric disorders in asymptomatic hiv subject
the fundamental premise of modern psychiatry is that emotional life and behaviors have biological underpinnings that develop and are shaped within a broader social context . perhaps that interplay is nowhere more evident than in the psychobiology of hiv infection .
the changing face of the hiv pandemic from one of accelerating deterioration to a more chronic course suggests that the role of clinical psychiatry must expand to encompass new populations and to adapt to changing neuromedical aspects of illness .
the transition model proposes that adverse psychological phenomenon ( e.g. , adjustment disorders , mood disorders and anxiety disorders ) may be anticipated at key points such as discovery of seroconversion , initiation of anti - retroviral treatment , onset of physical symptoms , advance in hiv related illness stage and hiv related bereavement and that they reflect a breakdown in coping capacities in the face of hiv .
various studies have suggested that four factors are important in explaining the increased psychiatric morbidity in hiv patients .
they are pre - existing psychiatric morbidity , neurotoxic property of hiv itself , terminal nature of diagnosis and social stigmatization . in the present study ,
the psychiatric morbidity was assessed in asymptomatic hiv patients ( stage ii and iii of cdc classification ) as psychiatric manifestations in advanced stages , i.e. , aids may be due to opportunistic or central nervous system infections .
these guidelines were not followed by many of the studies , which resulted in erroneous observations .
some studies have kept sexually transmitted diseases patients as controls in view of similar social stigmatization , others high risk hiv seronegative persons ( homosexuals and iv drug users ) and still others rheumatoid arthritis . control subjects in this study were the normal healthy persons who had the same atmosphere and background as of the cases .
normal persons were kept as controls to find out the exact phenomenology of asymptomatic hiv patients and their difference in sensation seeking .
most of the studies have assessed one or at the most two of the variables and that too on symptomatic population .
the age range of the patients in present study table 1 was on par with other studies .
on the other hand , the western studies had higher number of homosexual transmission and intravenous drug abuse .
the observed ghq-12 caseness ( a cut - off score of 2 ) for hiv and control groups [ table 2 ] was within the ranges reported in literature . in the present study , ghq-12 had a sensitivity of 100% and specificity of 92% indicating that it is a good screening test for psychiatric morbidity in asymptomatic hiv patients .
the finding of absence of cognitive dysfunction in patients in the present study is in agreement with an earlier study .
contrary findings of 10.5% cognitive impairment in an earlier indian study was probably due to inclusion of symptomatic ( stage iv cdc ) hiv patients in that study .
we can conclude from the above observations that asymptomatic hiv patients do not result in any gross cognitive dysfunction .
singh et al . studied cognitive function in asymptomatic hiv seropositive individuals by using luria - nebraska neuropsychological battery and reported slower fine motor speed and slower speed of information processing and no difference in any other cognitive domain .
this indicates that the utility of mmse as a good screening test for assessment of cognitive dysfunction in asymptomatic hiv patients needs further evaluation .
the observation of higher prevalence of anxiety on hads [ table 2 ] in hiv seropositive individuals was similar to earlier studies .
the very knowledge of harboring a potentially fatal infection of uncertain prognosis , with bleak treatment prospects and possible suffering and social isolation can be sufficiently stressful to induce anxiety
the finding of significantly higher depression on hads [ table 2 ] in hiv seropositive subjects is similar to earlier studies . among hiv patients ,
clinical depression has been reported frequently , but estimates concerning its prevalence has varied considerably ( 30 - 61% ) , but always greater than in the general population ( 4 - 40% ) .
it is not yet determined whether hiv itself increases the likelihood of major depressive disorder beyond the expected increases found in other chronic diseases .
hiv infection can be associated with anergic - apathetic - fatigue states , presumably mediated by release of somnogenic lymphokines .
various studies have brought out factors , which led to higher rates of depression in hiv patients : decline of immunocompetence , adverse life events , pre - existing mood disorder , noting the onset of constitutional symptoms and initiating retroviral therapy .
it is imperative that depression in hiv positive patients must be identified and treated because depression has been associated with a lower likelihood of receiving anti - retrovirals and poor adherence , which leads to a worse outcome in hiv infected persons and increased mortality .
sensation seeking is a personality factor that could have an important role in hiv risk behavior .
sensation seeking focuses on the need for new and varied experiences through uninhibited behavior , these include dangerous activities , a non - conventional life - style and a rejection of monotony .
sensation seeking trait was assessed under the four different characteristics in the present study table 3 .
thrill and adventure seeking , experience seeking and boredom susceptibility revealed statistically significant higher scores in asymptomatic hiv patients compared with hiv seronegative controls .
high sensation seeking trait may play a vital role in engaging in high - risk behavior resulting in this dreaded illness .
more complementary studies are needed to identify different aspects of sensation seeking and methods of effective intervention for prevention of hiv . in the present study ,
hiv seropositive patients had significantly higher incidence of psychiatric disorders including alcohol dependence , adjustment disorder and sexual dysfunction [ table 4 ] .
five out of the nine hiv seropositive patients with sexual dysfunction had no concurrent axis 1 diagnosis .
impaired sexual functioning contributes to diminished quality of life for seropositive patients , many of whom report this as a significant loss in their lives .
the high prevalence of sexual dysfunction in these patients is an important finding given the low base rates of this disorder in male population under age 44 .
overall , there was moderate agreement between interviewer 's diagnosis of depression and patient 's self - report of depressive symptoms on an assessment scale in the present study , consistent with the findings of an earlier study .
the significantly higher prevalence of alcohol dependence syndrome confirmed the findings by rosenberg et al . who reported alcohol as the strongest predictor of hiv drug risk behaviors , stronger than drug use generally .
most patients with substance abuse disorders had developed these disorders before acquiring hiv which is in agreement with earlier report .
substance abusers are prone to have high risk sexual behaviors , as there is a higher rate of non - inhibition , impaired judgment and impulsivity . for these same reasons ,
mounting evidence suggests that these patients have accelerated and more severe neurocognitive dysfunction compared with non - substance - abusing hiv - infected populations .
psychiatric morbidity is significantly higher in asymptomatic hiv patients as compared to hiv seronegative controls . among the individual disorders , alcohol dependence syndrome , sexual dysfunction and adjustment disorder
high sensation seeking and substance abuse found in hiv seropositive patients may play a vital role in engaging in high - risk behavior resulting in this dreaded illness . | background : psychiatric morbidity in human immunodeficiency virus ( hiv ) patients is being studied all over the world .
there is paucity of indian literature particularly in asymptomatic hiv individuals.aim:the aim of the following study is to establish the prevalence and the determinants of psychiatric morbidity in asymptomatic hiv patients.materials and methods : a cross - sectional study was undertaken to assess psychiatric morbidity as per icd-10 dacryocystorhinostomy criteria in 100 consecutive asymptomatic seropositive hiv patients and an equal number of age , sex , education , economic and marital status matched hiv seronegative control .
all subjects were assessed with the general health questionnaire ( ghq ) , mini mental status examination , hospital anxiety and depression scale ( hads ) and sensation seeking scale ( sss ) and the scores were analyzed statistically.results:asymptomatic hiv positive patients had significantly higher ghq caseness and depression but not anxiety on hads as compared to hiv seronegative controls .
on sss asymptomatic hiv seropositive subjects showed significant higher scores in thrill and adventure seeking , experience seeking and boredom susceptibility as compared to controls .
hiv seropositive patients had significantly higher incidence of total psychiatric morbidity . among the individual disorders , alcohol dependence syndrome , sexual dysfunction and adjustment disorder
were significantly increased compared with hiv seronegative controls.conclusion:psychiatric morbidity is higher in asymptomatic hiv patients when compared to hiv seronegative controls . among the individual disorders , alcohol dependence syndrome , sexual dysfunction and adjustment disorder were significantly increased compared with hiv seronegative controls .
high sensation seeking and substance abuse found in hiv seropositive patients may play a vital role in engaging in high - risk behavior resulting in this dreaded illness . |
high - frequency ultrasound is a noninvasive diagnostic method with several applications in the field of internal medicine and , recently , clinical dermatology as well as dermatooncology and dermatocosmetics.1,2 it can represent a
noninvasive histological tool for the quantification of the cutaneous structure or of the cutaneous pathology by monitoring the evolution of cutaneous lesions and the assessment of topical therapies efficacy in chronic inflammatory disorders ( morphea , scleroderma , psoriasis ) , for the quantification of the intrinsic and extrinsic senescence process , as well as the efficacy of topical or generally applied therapies.3 it can also be used as an important research tool of the cutaneous senescence process and the efficacy of various anti - aging therapies.4,5 the use of high - frequency ultrasound in the assessment of the regenerative effects of various anti - aging therapies is an exciting and important area for research . from all the available imaging methods , high - frequency ultrasound ( dermascan 20 mhz cortex technology ) is a new study tool focused on the assessment of the cutaneous physiological and pathological features , having a penetration depth of up to 2.5 cm .
collagen is an echogenic marker protein , synthesized by dermal fibroblasts , which can be identified by high - frequency ultrasound .
the changes of the extracellular matrix , consisting in variations of the dermal density and echogenicity throughout the physiological senescence process , or during several anti - aging therapies quantify subtle reactions at molecular , cellular , and tissular level that influence the local homeostasis.6 high - frequency ultrasound allows an objective assessment of the cutaneous structure : the thickness of the epidermis and dermis as well as the dermal density , which are useful parameters for the quantification of the cutaneous regeneration process.7,8 several changes of the cutaneous structure after topical prednisone therapy in children , or anti - aging natural supplement therapies in adults were ultrasonographically successfully assessed.9 previous studies have shown that the thickness of the epidermis and dermis , as well as the dermal density are important parameters that assess the cutaneous regeneration process .
the neosynthesis of the protein structures induces an increase of the dermal echogenicity and density , local cell architecture changes , and an increase of the dermis and epidermis thickness .
the epidermis is the morphological expression of the changes in the subjacent dermis.7 it has been proved that certain ultrasonographic markers , such as sleb ( subepidermal low echogenity band ) or the leps / lepi ratio ( number of low echogenic pixels in the upper dermis / number of low echogenic pixels in the lower dermis ) can quantify the cutaneous senescence process , as well as the efficacy of various anti - aging therapies.2,10,11 as far as the anti - aging therapy is concerned , viniferol , a standardized extract from bordeaux vine stalks , is one of the newest and very efficient anti - wrinkle and anti - aging agents .
the active ingredient of viniferol is -viniferine , a generic name for a new class of phytoalexins characterized as resveratrol dimers .
besides the well - known antioxidant effect of this flavonoide - containing extract , it has a direct action on the protein expression genes involved in the proliferation and differentiation of integumentary cells .
these are retinoid - like effects which decrease cellular differentiation and increase cellular growth . due to this action , viniferol profoundly restructures and regenerates the skin and due to its antioxidant properties , it reestablishes the metabolic balance of the cutaneous cells , slowing down the tissular degeneration and disorganization process.12 even though the general anti - aging effects of the flavonoids are well known , until now no scientific studies investigated the action of viniferol at the cutaneous level by using high - frequency ultrasound .
the purpose of the study is the assessment , with the help of high - frequency ultrasound , of the cutaneous changes induced by topical use of products containing viniferol .
eighty female subjects , aged 2275 years , who requested prophylaxis and anti - aging therapy with flavonoids , were prospectively included in the study . half ( 50% ) of the subjects belonged to fitzpatrick phototype class ii and 50% to phototype class iii.13 the study excluded patients with known allergies to topical flavonoids , cutaneous facial lesions , resurfacing or other anti - aging therapies in the last 2 months , or those who used phototherapy or oral contraception .
the subjects taken into the study were divided into two categories : a study group and a control group .
the study group followed the proposed anti - aging therapy for 12 weeks , according to a standard protocol . in the morning , a hydrating emollient cream , based on occlusive hydrating agents was applied at the facial level ( including the zygomatic area ) by lightly massaging the area for 2 minutes.14 in the evening , an anti - aging product containing viniferol , was applied in the same manner .
no other cosmetic products were used by the subjects during the 12 weeks of study .
the control group followed a placebo therapy for 12 weeks , using only moisturizing cream in the morning and evening , applied at the facial level , including the zygomatic area .
for every subject , ultrasonographic images were taken from the zygomatic level at the beginning and 12 weeks after the local application of the emollient , hydrating product , and the anti - aging , viniferol - based cream .
the study was approved by the ethical committee of university of medicine and pharmacy iuliu hatieganu , cluj - napoca , romania .
every subject was informed about the nature and purpose of the study , and signed a written consent before enrolling into the study .
the ultrasonographic evaluation was performed with the dermascan equipment ( cortex technologies , hadsund , denmark ) , containing a 20 mhz transducer , that allows the in vivo acquirement of sectional cutaneous images up to a depth of 2.5 cm .
dermascan consists of three major components : a transducer , an elaboration system , and a database ( figure 1 ) . the ultrasonic wave is partially reflected at the boundary between adjacent structures and generates echoes of different amplitudes .
the intensity of the reflected echoes is evaluated by a microprocessor and visualized as a colored two- dimensional image.1 the color scale of echogenicity is : white yellow red green blue black .
normally , the epidermal echogenicity appears as a white band , the dermis is expressed as a two - color composition : yellow and/or red , and the subcutaneous layer appears either green or black ( figure 2 ) .
the transducer was applied on the integument , on the left zygomatic area , parallel to the zygomatic arcade , after previously having cleaned the cutaneous surface and applied ultrasonographic gel .
the gain curve was adjusted at a value of 20 db , at a speed of ultrasound at tissue level of 1580 m / s .
the obtained images were further processed with image analysis software ( dermavision , cortex technology ) . for every subject , the following parameters were measured on the images obtained before and after therapy : thickness of the epidermis and dermis ( mm ) , the number of lep ( low echogenic pixels ) , mep ( medium echogenic pixels ) , hep ( high echogenic pixels ) , leps / lepi ratio .
the thickness of the epidermis was obtained by calculating the mean of three measurements performed at three different sites of each image ( the two extremities of the analyzed image and the center of the image ) .
the thickness of the dermis was obtained by measuring the distance between the dermo - epidermal and the dermo - hypodermic junction at the same three different sites and by establishing the mean of the three values .
the analysis software used has the pixel amplitude corresponding to a numerical scale set between 0255 . by selecting a certain interval from the 0255 scale
, we obtained values corresponding to a certain pixel type , present in the analyzed image .
thus , the 030 interval corresponds to lep , the 50150 interval to mep , and the 200255 interval to hep .
the number of lep were determined separately in the upper ( lepu ) and lower ( lepl ) dermis .
the boundary between these regions was the line chosen as parallel to the epidermal entrance echo and dividing the dermal image into two parts of equal thickness .
the ratio of lep number in the upper and lower dermis ( leps / lepi ) was calculated .
statistical analysis was performed with spss ( v 15.0 ; spss inc , chicago , il ) .
the data we obtained were analyzed , calculating the mean and standard deviation for the quantitative variables of every group and the proportions for the qualitative variables .
the difference of means before and after treatment was tested using a t - test for paired samples , and the relationship between different parameters was assessed through spearman s correlation coefficients .
eighty female subjects , aged 2275 years , who requested prophylaxis and anti - aging therapy with flavonoids , were prospectively included in the study . half ( 50% ) of the subjects belonged to fitzpatrick phototype class ii and 50% to phototype class iii.13 the study excluded patients with known allergies to topical flavonoids , cutaneous facial lesions , resurfacing or other anti - aging therapies in the last 2 months , or those who used phototherapy or oral contraception .
the subjects taken into the study were divided into two categories : a study group and a control group .
the study group followed the proposed anti - aging therapy for 12 weeks , according to a standard protocol . in the morning , a hydrating emollient cream , based on occlusive hydrating agents was applied at the facial level ( including the zygomatic area ) by lightly massaging the area for 2 minutes.14 in the evening , an anti - aging product containing viniferol , was applied in the same manner .
no other cosmetic products were used by the subjects during the 12 weeks of study .
the control group followed a placebo therapy for 12 weeks , using only moisturizing cream in the morning and evening , applied at the facial level , including the zygomatic area .
for every subject , ultrasonographic images were taken from the zygomatic level at the beginning and 12 weeks after the local application of the emollient , hydrating product , and the anti - aging , viniferol - based cream .
the study was approved by the ethical committee of university of medicine and pharmacy iuliu hatieganu , cluj - napoca , romania .
every subject was informed about the nature and purpose of the study , and signed a written consent before enrolling into the study .
the ultrasonographic evaluation was performed with the dermascan equipment ( cortex technologies , hadsund , denmark ) , containing a 20 mhz transducer , that allows the in vivo acquirement of sectional cutaneous images up to a depth of 2.5 cm .
dermascan consists of three major components : a transducer , an elaboration system , and a database ( figure 1 ) . the ultrasonic wave is partially reflected at the boundary between adjacent structures and generates echoes of different amplitudes .
the intensity of the reflected echoes is evaluated by a microprocessor and visualized as a colored two- dimensional image.1 the color scale of echogenicity is : white yellow red green blue black .
normally , the epidermal echogenicity appears as a white band , the dermis is expressed as a two - color composition : yellow and/or red , and the subcutaneous layer appears either green or black ( figure 2 ) .
the transducer was applied on the integument , on the left zygomatic area , parallel to the zygomatic arcade , after previously having cleaned the cutaneous surface and applied ultrasonographic gel .
the gain curve was adjusted at a value of 20 db , at a speed of ultrasound at tissue level of 1580 m / s .
the obtained images were further processed with image analysis software ( dermavision , cortex technology ) . for every subject , the following parameters were measured on the images obtained before and after therapy : thickness of the epidermis and dermis ( mm ) , the number of lep ( low echogenic pixels ) , mep ( medium echogenic pixels ) , hep ( high echogenic pixels ) , leps / lepi ratio .
the thickness of the epidermis was obtained by calculating the mean of three measurements performed at three different sites of each image ( the two extremities of the analyzed image and the center of the image ) .
the thickness of the dermis was obtained by measuring the distance between the dermo - epidermal and the dermo - hypodermic junction at the same three different sites and by establishing the mean of the three values .
the analysis software used has the pixel amplitude corresponding to a numerical scale set between 0255 . by selecting a certain interval from the 0255 scale
, we obtained values corresponding to a certain pixel type , present in the analyzed image .
thus , the 030 interval corresponds to lep , the 50150 interval to mep , and the 200255 interval to hep .
the number of lep were determined separately in the upper ( lepu ) and lower ( lepl ) dermis .
the boundary between these regions was the line chosen as parallel to the epidermal entrance echo and dividing the dermal image into two parts of equal thickness .
the ratio of lep number in the upper and lower dermis ( leps / lepi ) was calculated .
statistical analysis was performed with spss ( v 15.0 ; spss inc , chicago , il ) .
the data we obtained were analyzed , calculating the mean and standard deviation for the quantitative variables of every group and the proportions for the qualitative variables .
the difference of means before and after treatment was tested using a t - test for paired samples , and the relationship between different parameters was assessed through spearman s correlation coefficients .
forty female subjects with a mean age of 49.60 14.19 years ( range 2275 years ) were included in the study group .
twelve subjects ( 30% ) were under the age of 40 , 16 subjects ( 40% ) between 4060 years and twelve ( 30% ) over the age of 60 .
the control group was composed of subjects aged 2275 years as well , divided into the above mentioned age categories .
all subjects involved in the study tolerated the therapy , without evoking adverse effects ( erythema , pruritus , ocular disturbance ) .
subjectively , post - flavonoid therapy , a significant hydration of the skin throughout the day and an increase of the cutaneous tonicity was noticed .
the thickness of the epidermis in the study group initially was 0.129 0.237 mm , and the thickness of the dermis 1.434 0.241 mm .
after therapy , an increase of the mean thickness of the epidermis ( 0.129 0.237 mm vs 0.150 0.323 mm , p < 0.000 ) , and of the dermis ( 1.434 0.241 mm vs 1.569 0.219 mm , p <
the thickness of the dermis increased mainly in the 4060 years age group ( 1.413 0.280 mm vs 1.569 0.279 mm , p = 0.001 ) , and less , but still significantly in the < 40 years group ( 1.416 0.266 mm vs 1.585 0.150 mm , p = 0.015 ) , while in those > 60 years , the increase was not statistically significant ( 1.480 0.157 mm vs 1.554 0.204 mm , p = 0.097 ) . at the same time , at the dermal level , the number of lep decreased ( 15,153.53 3589.86 vs 12,958.48 3628.35 , p < 0.0001 ) , but this aspect was only noticed in the lower dermis ( 6949.75 1966.93 vs 6257.62 2224.88 , p = 0.016 ) , not in the upper dermis ( 7290.55 1794.60 vs 6940.65 2150.30 , p = 0.168 ) .
overall , the leps / lepi ratio increased significantly after flavonoid therapy ( 1.092 0.330 vs 1.259 0.631 , p = 0.011 ) .
we also noticed an increase of mep ( 3359.72 1457.36 vs 3983.47 1401.24 , p = 0.013 ) and hep ( 460.27 323.93 vs 750.90 493.82 , p <
the general variation pattern of the quantifiable ultrasonographic parameters after flavonoid therapy is illustrated in table 1 and figure 3 .
if we consider the variation of the ultrasonographic parameters after topical flavonoid therapy according to the phototype class of the subjects , it can be noticed that after therapy , there is a significant increase of the leps / lepi ratio in the subjects belonging to phototype class ii , not iii ( table 2 ) . in the placebo group , we noticed no significant thickening of the epidermis and a slight increase of the dermis after therapy ( 1.433 0.34 mm vs 1.486 0.14 mm ) .
the number of lep at dermal level also show a slight increase ( 13,213 1284 vs 15,374 2318 , p = 0.1 ) due to an optimal hydration of the skin and a discrete decrease of high echogenity pixels ( 421.8 121.18 vs 368.3 104.03 , p = 0.07 ) .
the leps / lepi ratio showed no particular display according to the age or phototype of the subjects .
thus , locally applied flavonoids induce the neosynthesis of the fibrillary structures , but also of glycosaminoglycans , which display intense hydrophilic properties , favoring the cutaneous hydration .
it is well known that flavonoids have important anti - aging properties not only at a cutaneous level , but at the level of the entire organism .
viniferol , a molecule with proven anti - aging and antioxidant properties , exhibits a complex action at the cutaneous level : it interacts with fibroblastic receptors , amplifies the interrelation fibrocyte - extracellular matrix , modulates the adhesiveness molecules and interferes with the oxidative stress process and nonenzymatic glycation , with a regenerative effect at cutaneous level.15 after therapy , a significant increase of the mean thickness of the epidermis and dermis was noticed , which once again confirms the presence of a complex , regenerative dermal process , induced by flavonoids.16,17 the dermal thickness increased the most in the 4060 years age group , to a lesser extent , but still significantly under the age of 40 years , and insignificantly over 60 years .
we can affirm that topical flavonoid products have the best efficacy on mature integument , with specific structural and hormonal characteristics .
in young subjects ( < 40 years ) the thickness of the dermis increases discretely as the dermis is a young connective tissue , rich in glycosaminoglycans and thus , properly hydrated.18 after the age of 60 , the extracellular matrix ( fibrocytes , fibers , glycosaminoglycans ) of the skin undergoes characteristic degenerative changes .
therefore , the flavonoid - based anti - aging therapy has less effective regenerative changes in this age interval . these regenerative changes could be amplified though by the use of products able to interfere with the characteristic aging mechanisms for this specific age group . at the same time , concomitantly with the change in dermal thickness , the number of lep decreased in the lower dermis , but not in the upper dermis .
the decrease in the number of lep in the lower dermis is proportional to the significant increase of mep and hep that quantify protein neosynthesis , as well as cytoarchitectural reorganizations of the extracellular matrix .
according to the literature , the cutaneous imaging parameters that can be considered as objective in the evaluation of the cutaneous aging / regeneration process are the following : lep quantifies the degree of cutaneous hydration , inflammatory processes , solar elastosis , collagen degeneration .
the leps / lepi ratio allows an appreciation of the density and integrity of the extracellular matrix , both from the upper and lower dermis , which vary according to age , uv - ray exposure , and therapy.16 the data obtained from the study shows important ultrasonographic changes at the cutaneous level after anti - aging therapy .
flavonoids have a complex action at the dermal level , interfering with several mechanisms involved in the senescence process .
they act at the level of fibrocytes , on specific receptors , turning inactive mature cells into young , metabolically active ones.19 also , studies performed in order to assess the phytooestrogen activity of viniferol ( evaluation of gene expression , coding for typical differentiation proteins ) have shown that it influences gene expression and cell proliferation both at the epidermal and dermal levels .
therefore viniferol decreases gene expression in loricrine , fillagrin , and keratin 11 , and increases gene expression in calgranulin b.20 the synthesis of the protein structures is initiated at the intracellular level .
the trophocollagen molecules , elastin , and the glycosaminoglycans are extracellularly assembled into microfibrils , fibers , or proteoglycans .
depending on the biochemical structure , the level of organization , architectural orientation , and quantity , the proteins show a certain cutaneous echogenity degree .
the low echogenity pixels that quantify the hydration degree of the extracellular matrix especially in the lower dermis , are replaced by medium and high echogenity pixels , quantifying protein synthesis .
once these are assembled into mature fibers , the local echogenicity increases , and are expressed as hyperechogenic areas ( hep ) .
the increase of the leps / lepi ratio quantifies the replacement of the hypoechogenic pixels from the lower dermis with medium and high echogenic pixels as a result of protein neosynthesis .
type i collagen , that is predominant at the dermal level ( punctiform hyperechogenic pixels ) is organized in fibers , visible as hyperechogenic bands , having a parallel display in the lower dermis .
these hyperechogenic bands , visible especially on photoprotected sites , represent an ultrasonographic marker of the intrinsic aging process.21 the collagen
elastin protein structure , in different organization stages , is quantified by pixels of different amplitude .
the post - therapeutic changes , visible on the sonograms as an increase of medium and high echogenic pixels , are suggestive of increased protein synthesis , the assembly of molecules to filaments , fibrils , and eventually , collagen fibers . according to our observations , there is a direct relationship between the genetically determined cutaneous phototype , the structure and repartition of echogenic proteins ( collagen , elastin ) and the different response to anti - aging therapies.22 if we consider the significant changes of the ultrasonographic parameters after anti - aging therapy depending on the phototype of the subject , a significant increase of the leps/ lepi ratio is present in the subjects in phototype ii class , but not class iii .
this observation would justify the correlation of the anti - aging therapy with the cutaneous phototype .
further studies are necessary to confirm the different reactivity of the phototype classes to local therapies .
the synthesis process of fibrillary proteins depends on several factors , involving complex , targeted , and personalized therapies .
according to the wear and tear theory , throughout life , intrinsic and extrinsic mechanisms associated to the senescence process reduce the regenerative capacity of cells , leading to post - translational changes at the level of intracellular and extracellular proteins , such as oxidation and nonenzymatic glycation .
flavonoids , through complex mechanisms , interfere with the reactions involved in the senescence process , and induce the synthesis of the extracellular matrix . according to our data , viniferol - based products are more efficient in the 4060 years age group , characterized by complex biological changes at the cutaneous level .
viniferol shows real and important anti - aging properties , since it interferes concomitantly with the genetic , oxidative , immunologic , and metabolic mechanisms that are involved in the cutaneous aging process.23 prophylaxis of the aging process should start before the age of 40 years , preferably in the critical age group ( 2040 years ) , which is characterized by important changes at the tissular , cellular , and molecular levels.24 the optimization of anti - aging therapy , according to special studies , requires targeted , personalized therapies , adapted to the hormonal , genetic , oxidative , immunologic , and metabolic status of the subject , and is capable of interfering with deficient mechanisms in certain age groups .
high - frequency ultrasound can be used as a modern , noninvasive , histological tool for the assessment of various anti - aging therapies , with applications in the fields of pharmacology and dermato - cosmetology .
high - frequency ultrasound is a modern , noninvasive method that allows the assessment of the efficacy of anti - aging therapies .
after topical flavonoid - based therapy , a significant increase of the epidermis and dermis was noticed , especially in the 4060 years age group .
concomitantly , other cutaneous changes take place : decrease in the number of lep and increase in the mep and hep .
anti - aging prophylaxis should be initiated in the 2040 years critical age group , involving personalized therapies also adapted to the phototype . | ultrasonography allows the quantification of dermal density and echogenicity changes during the physiological senescence process .
some active ingredients are able to slow down the tissular degeneration and disorganization process .
the purpose of this study was to assess the cutaneous changes induced by the topical use of products containing viniferol as active ingredient , using high - frequency ultrasound .
the study was performed over 12 weeks and included 80 healthy caucasian female subjects , aged 2275 years , divided into two groups : the study group and the control group .
the product was applied according to a predetermined protocol .
the measurements performed for each subject were : the thickness of the epidermis and dermis ( mm ) , the number of low , medium , and high echogenic pixels , and the number of low echogenic pixels in the upper dermis / number of low echogenic pixels in the lower dermis .
all the parameters showed a significant improvement .
ultrasound measurements showed an increase of the mean thickness of the epidermis ( p < 0.0001 ) and dermis ( p < 0.0001 ) following the application of the viniferol product as compared to the control group .
the changes in the dermal echogenicity confirm the efficacy and direct action of viniferol upon the cutaneous fibroblasts .
no side effects related to the treatment were recorded .
the study proves the efficacy of this active ingredient in the cutaneous senescence process as well , as the fact that anti - aging prophylaxis should be initiated in the 2040 year critical age group .
this interval involves specific changes in dermal echogenicity that quantify intense molecular , biochemical and structural changes , being thus mostly and highly responsive to the anti - aging therapy . |
patellofemoral pain ( pfp ) accounts for approximately 25% of knee injuries in athletes ( taunton et al . , 2002 ) .
pfp patients are often not able to perform exercises like running , as increased patellofemoral joint compression forces ( pfjcf ) may aggravate pfp pathology .
overloading of the patellofemoral joint in pfp patients could eventually lead to severe chronic injuries , such as osteoarthritis ( buckwalter and brown , 2004 ) .
conservative treatment ( such as rehabilitation ) is important to manage pfp ( dixit et al . , 2007 ) .
although it has been reported that br has lower pfjcf compared to fr , this may be due to methodological issues ; as running speeds were lower for br than fr trials ( flynn and soutaslittle , 1995 ) .
another study found no difference in pfjcf between fr and br at similar , but unnaturally slow speed ( sussman et al . , 2000 ) .
further research is therefore required to establish differences between pfjcf in br compared to fr at the same speed .
pfjcf is influenced by multiple inter - related factors : knee extensor moment , patellar moment arm , quadriceps muscle force and patellar tendon force .
the quadriceps muscle force is related to the knee extensor moment and the patellar tendon moment arm and the patellar tendon force is dependent on the knee angle and the quadriceps force ( gill and o'connor , 1996 ) .
the within subject differences in maximum pfjcf between fr and br will therefore depend on the peak knee extensor moment and the knee angle at this peak moment .
the main factors that influence the knee moment are the magnitude of the ground reaction force ( grf ) , position of the knee relative to the grf vector , and angular accelerations of the lower limb segments .
besides these individual biomechanical factors , propulsive mechanisms of br and fr might also explain differences in pfjcf .
the telescopic inverted pendulum ( tip ) approach can be used to explore the predominant propulsive mechanisms in fr and br ( jacobs and van ingen schenau , 1992 ; papa and cappozzo , 1999 ) .
pendular movement , such as observed in walking , can be simulated by a simple inverted pendulum model where the stance limb is modeled as a rigid segment that rotates around the ankle ( mcgeer , 1990 ; garcia et al . , 1998 ) .
such movement would have relatively low knee extensor and high hip flexor moments . running on
the other hand involves a large compression and passive recoil of the stance limb ( telescopic motion ) and can therefore be better modeled by a spring mass model ( seyfarth et al . , 2002 ) .
the aims of this study were to ( 1 ) investigate if br had a reduced peak pfjcf compared to fr at the same speed , and ( 2 ) if this force was reduced in br , to investigate how changes in relevant biomechanical parameters resulted in this reduced pfjcf .
it was hypothesized that ( 1 ) pfjcf would be lower in br compared to fr , and ( 2 ) that this would coincide with a reduced peak knee moment in br as a result of grf alterations in br .
heel strike running has been associated with increased grf ( lieberman et al . , 2010 ) and foot ground contact has been found to be reversed in br relative to fr ; toe heel contact in br versus heel
twenty moderately active healthy subjects , without any recent knee injury or pain , were recruited for this study .
ethical approval was obtained from the human research ethics committee of the school of healthcare studies at cardiff university , and written consent was obtained from all subjects .
the subjects were asked to run along a 7-m walkway in forward ( fr ) and backward ( br ) directions at a speed between 2.8 and 3.4 m / s .
kinematic data were collected at 200 hz using an eight camera vicon mx motion analysis system ( oxford metrics group ltd . , uk ) .
16 reflective markers were placed using the lower limb plug - in - gait marker set .
ground reaction force data were collected at 1000 hz using two kistler force plates ( kistler instruments ltd . , switzerland ) .
data of three subjects were excluded from analysis , due to missing pelvis markers during part of the data collection .
the data of 17 subjects ( 7 males and 10 females , age : 286 years , height : 1.710.07 m and mass : 70.720.3
inverse dynamics calculations were performed within vicon nexus software ( version 1.6.1 , oxford metrics group ltd . , uk ) .
, usa ) , combining kinematic and kinetic data with values for the patellar tendon moment arm ( dpt ) from literature ( tsaopoulos et al . , 2006 ) .
a polynomial was fitted to these extrapolated data , resulting in an equation for dpt based on knee angle ( ) , and body height .
tsaopoulos et al . ( 2006 ) demonstrated that dpt was expected to be scaled by body height , but not by body mass:(1)dpt = a(1.092 + 0.020.0000012)witha=0.04ifheight1.65ma=0.045if1.65m <
the pfjcf was calculated as follows:(2)pfjcf = rfqfplfqwith(3)fq = mk(max)dptand(4)rfqfpl=0.633 + 0.010.000052where rfq - fpl is the the ratio of the quadriceps to patellar tendon force , fq is the quadriceps tendon force and mk(max ) is the peak knee moment .
( 4 ) ) . to investigate the kinematics and kinetics of br and fr ,
a telescopic inverted pendulum ( tip ) model approach was used , as described in the introduction ( fig .
the magnitude of the grf was calculated at the time of peak knee moment ( mk(max)):(5)|grf|=fy2+fz2with fy as the horizontal and fz as the vertical component of the grf .
the orientation of the grf relative to the ground ( grf ) at the time of mk(max ) was calculated in the sagittal plane , with 0 being perpendicular to the ground , grf>0 pointing in anterior and grf<0 in posterior direction .
the location of the grf relative to the foot ( coploc ) was calculated at the time of mk(max ) by dividing the distance between the projection of the center of pressure ( cop ) on the foot and the metatarsal marker by the length of the foot .
the position of the cop relative to the foot was also calculated at foot strike ( coplocfs ) , with foot
the foot , shank and thigh segment angular accelerations ( foot , shank and thigh ) were calculated using the line between the calcaneus and metatarsal marker , the calcaneus and knee marker , and the asi and knee marker respectively .
statistical differences for the output variables between fr and br were determined in spss ( version 18.0.2 ) with an independent t - test .
stepwise linear regression analysis was used to investigate which variables most influenced pfjcf and subsequently mk(max ) .
the pfjcf and mk(max ) were significantly higher and the knee was slightly more flexed in fr ( table 1 ) .
peak hip flexor moments ( mh(max ) ) were significantly higher in br ( table 1 ) .
2 ) showed that the stance leg is shortened during the deceleration phase and extended during the push - off phase in both fr and br . in fr
the stance leg flexed slightly more at mk(max ) ( table 1 ) and extended more during the push - off phase than in br ( fig .
2 ) . in both fr and br , mk(max ) occurred at similar though significantly different approach angles of the contact leg ( l ; fig .
therefore in both situations the body was upright and leaning forward slightly at mk(max ) ( as l was close to , but smaller than 90 )
. there was no significant difference between fr and br for the magnitude ( |grf| ) and orientation of the grf ( grf ) at mk(max ) ( table 2 ) .
the cop location on the foot ( coploc ) at mk(max ) was further backward and moving slower forward along the foot in fr ( table 2 ) .
the angular acceleration ( foot ) of the foot at mk(max ) was significantly different and in opposite directions between fr and br ( table 3 ) .
the acceleration of the shank segment ( shank ) at mk(max ) was in the same direction , but significantly smaller in fr ( table 3 ) .
there was no significant difference between the angular accelerations of the thigh segment ( thigh ) at mk(max ) ( table 3 ) .
stepwise regression analysis with the pfjcf as dependent variable and mk(max ) , mh(max ) , k ( knee flexion angle ) , l , and |grf| at mk(max ) as predictors confirmed that mk(max ) predicted the majority of variance in pfjcf ( 93.0% , adjusted r=0.930 ) .
another stepwise regression analysis with mk(max ) as the dependent variable and k , l , |grf| , grf , coploc , copdt , foot , shank , thigh at mk(max ) as potential predictors showed that 54.8% ( adjusted r=0.548 ) of the variance in mk(max ) was predicted by k , coploc , and |grf| .
the cop location on the foot at foot strike ( coplocfs ) was closer to the forefoot in br compared to fr ( table 2 ) .
when investigating fr and br separately pfjcf was not correlated to coplocfs ; however when data were pooled there was a significant but weak correlation ( r=0.260 and p=0.008 , as shown in fig .
pfjcf was significantly lower in br than in fr and this difference was not due to running speed , confirming our first hypothesis .
the reduction in pfjcf was lower than that found by flynn and soutaslittle ( 1995 ) ( 27% versus 46% decrease ) .
the higher decrease in pfjcf observed by flynn and soutaslittle ( 1995 ) was most likely due to the difference in running speed between their fr and br trials .
kinematics at the peak knee moment ( mk(max ) ) were similar between br and fr which seems consistent with an earlier study ( van deursen et al . , 1998 ) .
however , kinetics differed with higher mk(max ) in fr and higher mh(max ) in br .
this predominantly agreed with flynn and soutaslittle ( 1995 ) , as knee flexion angles were similar ( fr : 44 and 38 , br : 41 and 38 ) and peak knee moments were similar for br ( 124 and 123 nm ) but lower in fr ( 157 and 246 nm ) .
tip ( telescopic inverted pendulum ) analysis demonstrated that in both br and fr mk(max ) occurred during the loading response , with the body upright and leaning slightly forward ; mk(max ) thereby positively contributed to the support moment .
the forward lean indicated that mk(max ) contributed to push - off in fr only , as in br a backward lean would be required for mk(max ) to contribute to push - off .
this was demonstrated by the reduced mk(max ) and increased mh(max ) in br , which would be expected in pendular movement .
also , in fr the stance leg extended more during the push - off phase ( and flexed slightly more during the deceleration phase ) than in br , consistent with our interpretation that fr involves a more telescopic movement .
regression analysis showed that mk(max ) could predict the majority of the differences in pfjcf , confirming the first part of the second hypothesis that reduced pfjcf in br was caused by a reduced mk(max ) .
although the magnitude ( |grf| ) and orientation ( grf ) of the grf at mk(max ) did not differ between br and fr , the location of the grf was further back on the foot in fr ( larger coploc ) .
this would result in a larger moment arm between the grf vector and the knee joint , as the knee flexion angle ( k ) at mk(max ) was similar between br and fr .
stepwise regression analysis showed that the variance in mk(max ) was best predicted by k , coploc and |grf| .
k and coploc both influence the magnitude of the moment arm of the grf vector relative to the knee joint .
mk(max ) therefore relied most on the position and magnitude of the grf , partly confirming the second part of our second hypothesis .
the main factor influencing the peak knee moment was therefore coploc , indicating that foot strike has a large impact on pfjcf .
although angular accelerations of the lower limb segments and joint angles were different between br and fr trials , these were not significant predictors of mk(max ) , and therefore are considered to have minimal influence on pfjcf .
the differences in pfjcf observed between br and fr were not consistent in all subjects ( fig .
investigation of the cop location at foot strike ( coplocfs ) showed that this was closer to the heel in fr .
there was a weak correlation between coplocfs and pfjcf when fr and br data were pooled .
the relatively low pfjcf observed in some of the subjects during fr may therefore be due to running style ( such as heel versus forefoot strike ) .
we would expect lower knee moments resulting in lower pfjcf in forefoot strike runners , as they have lower loading rates of the foot ( oakley and pratt , 1988 ) and a lower grf ( lieberman et al . , 2010 ) .
this agrees with our findings that pfjcf was reduced if the cop was closer to the forefoot .
however , this conclusion did not apply to all subjects during fr ( see fig .
4 , right lower corner ) . clearly , further research is required to investigate whether it is the br style that resulted in a reduced pfjcf or whether an adapted fr style could also be advised to pfp patients .
this study had several limitations ; as pfjcf can not be measured in vivo it was estimated with simplified models .
this study focused on compressive forces only and did not include the direction and location of the forces acting on the patellofemoral joint .
the use of more complex models of the knee and the additional calculation of patellofemoral joint stresses ( ratio of pfjcf to the contact area ( mcginty et al . , 2000 ) ) would have provided insight into the distribution and direction of the forces acting on the joint surface .
there is however a strong relationship between the patellofemoral contact area and knee flexion angles ( salsich et al . , 2003 ; besier et al . , 2005 ; escamilla et al . ,
2008 ) . as mk(max ) occurred at similar knee flexion angles in br and fr , it can be assumed that the patellofemoral contact area would be comparable , and patellofemoral joint stresses would be directly related to pfjcf .
estimation of joint stresses requires complex and computationally intense methods ( farrokhi et al . , 2011 ) ; as similar trends could be expected in patellofemoral joint stresses and compression forces between br and fr , this study included compression forces only .
future research may involve more detailed analysis of the forces acting on the patellofemoral joint during backward and forward running .
the patellar tendon moment arm was important in the calculations of the pfjcf , as it defined the magnitude of the pfjcf relative to the knee moment .
there is controversy in literature on how this moment arm should be estimated ( tsaopoulos et al . , 2006 ) .
we assumed the patellar tendon moment arm depended on knee angle , as the majority of studies demonstrated that the patellar tendon moment arm changes significantly during the first 45 of knee flexion ( smidt , 1973 ; herzog and read , 1993 ; baltzopoulos , 1995 ; kellis and baltzopoulos , 1999 ; tsaopoulos et al .
, 2006 , 2007 ) , and only limited studies found the moment arm to change little with knee angle ( gill and o'connor , 1996 ) .
this study demonstrated that pfjcf was reduced in br compared to fr , and that this was not due to a difference in running speed .
it can be concluded that br can be used as part of rehabilitation of pfp patients , to continue to exercise without increased pfjcf . although br can be suggested for rehabilitation , only a limited number of studies investigated br as part of rehabilitation of knee injured patients .
a case study showed that br allowed exercising with decreased pfp ; however if implemented incorrectly it can lead to overuse injury ( satterfield et al . ,
obviously , rehabilitation programs need to include other components , such as muscle strengthening ( dixit et al . , 2007 ;
dixit et al . , 2007 ) and/or taping ( dixit et al . , 2007 ) .
the reduced pfjcf in br compared to fr may also prevent overloading and thereby the development of chronic conditions such as osteoarthritis .
however pfjcf was not decreased in br compared to fr in all subjects , and pfjcf was lower when the cop was closer to the forefoot .
the cop location , that was closer to the heel at peak knee moment in fr than in br , was the main predictor of the increased knee extensor moments .
certain fr styles may therefore also be able to reduce pfjcf , and could be useful in injury prevention or rehabilitation .
| patellofemoral pain ( pfp ) is a common injury and increased patellofemoral joint compression forces ( pfjcf ) may aggravate symptoms .
backward running ( br ) has been suggested for exercise with reduced pfjcf.the aims of this study were to ( 1 ) investigate if br had reduced peak pfjcf compared to forward running ( fr ) at the same speed , and ( 2 ) if pfjcf was reduced in br , to investigate which biomechanical parameters explained this . it was hypothesized that ( 1 ) pfjcf would be lower in br , and ( 2 ) that this would coincide with a reduced peak knee moment caused by altered ground reaction forces ( grfs).twenty healthy subjects ran in forward and backward directions at consistent speed .
kinematic and ground reaction force data were collected ; inverse dynamic and pfjcf analyses were performed.pfjcf were higher in fr than br ( 4.51.5 ; 3.41.4bw ; p<0.01 ) .
the majority of this difference ( 93.1% ) was predicted by increased knee moments in fr compared to br ( 15754 ; 12451 nm ; p<0.01 ) .
54.8% of differences in knee moments could be predicted by the magnitude of the grf ( 2.30.3 ; 2.40.2bw ) , knee flexion angle ( 446 ; 417 ) and center of pressure location on the foot ( 2511 ; 126% ) at time of peak knee moment .
results were not consistent in all subjects.it was concluded that br had reduced pfjcf compared to fr .
this was caused by an increased knee moment , due to differences in magnitude and location of the grf vector relative to the knee .
br can therefore be used to exercise with decreased pfjcf . |
developmental dysplasia of the hip ( ddh ) denotes a wide spectrum of pathologic conditions , ranging from subtle acetabular dysplasia to irreducible hip dislocation.1 it is a very common to suspect and diagnose hip dysplasia when the children start walking in india due to lack of screening programme .
the principles of treatment of dislocated hip joint for an older child are quite different from those of a new born.2 in a child of walking age and beyond , achieving a concentrically reduced hip while minimizing complications is more challenging . in the older child
the reduction of hip is difficult because of adaptive shortening of the extraarticular soft tissues , acetabular dysplasia , capsular constriction , increased femoral anteversion , fibrofatty tissue in the acetabulum , hypertrophied ligamentum teres , and fixed inversion of limbus .
if left untreated , these dysplastic changes lead to osteoarthritis in early adulthood.34 bracing , traction , closed reduction , open reduction , and femoral or pelvic osteotomies are the frequently used treatment modalities for older children.57 the incidence of osteonecrosis of the femoral head has been reduced by avoiding immobilization of the hip in extreme abduction and by using femur - shortening osteotomies when appropriate.8 pelvic osteotomy continues to gain favor for the treatment of selected patients over 18 months of age.910 traction has been used before reduction of a dislocated hip to decrease the risk of ischemia of the femoral head , but data supporting use of this treatment are inconsistent.1113 closed or open reduction without a bony realignment procedure is associated with residual dysplasia.1415 femoral shortening has been shown to facilitate reduction and does not appear to increase the prevalence of osteonecrosis of the femoral head.1617 many authors have reported success with a single - stage procedure consisting of open reduction , capsulorrhaphy , femoral shortening , and pelvic osteotomy.1823 the one - stage surgical treatment for ddh , consisting of open reduction , femoral shortening , and pelvic osteotomy , is a demanding procedure , technically , than a staged procedure .
we report retrospective analysis to evaluate the radiographic and functional results of one - stage triple operative procedure of open reduction , femoral shortening , and salter 's osteotomy in older children .
between january 2005 and june 2010 , 25 patients ( 30 hips ) underwent one - stage triple procedure of open reduction , femoral shortening derotation , and salter 's osteotomy for the treatment of ddh .
the average age was 3.9 years ( range 18 months 8 yrs ) at the time of open reduction . only the patients with idiopathic congenital dislocation of the hip were included in the study .
clinical data regarding pain symptoms , gait pattern , range of hip joint motion and presence of trendelenburg sign were recorded for each patient preoperatively and on the latest followup using the modified mckay 's criteria.24 tnnis classification system was used to assess the degree of dislocation of the femoral head.25 radiographic evaluations included examination of preoperative and followup plain radiographs to classify patients according to the severin 's grading system.26 avascular necrosis was assessed using bucholz and ogden 's classification.2728 open reduction followed the recommendations of salter29 for the innominate osteotomy surgical technique and the femoral osteotomy was similar to that described by predrag klisic.30 the operative procedure started with the modified smith - petersen anterior approach ( bikini incision ) [ figure 1a ] .
the muscles were reflected from the inner and outer aspects of the iliac crest and the hip joint was exposed [ figure 1b ] .
this was followed by the release of iliopsoas , excision of pulvinar , release of ligamentum teres , and incision of transverse acetabular ligament .
gentle traction was applied to the extremity to assess the degree of contracture of the muscles that crossed the hip joint .
due to the soft tissue contractures , the femoral head still was not reducible without applying excessive pressure .
peroperative photographs showing ( a ) bikini incision ; ( b ) exposure of the hip joint ; ( c ) femoral osteotomy fixed with 4-hole small fragment plate ; ( d ) pelvic osteotomy performed by using a gigli saw ; ( e ) bone graft taken from the proximal fragment of the ilium ; ( f ) pelvic osteotomy fixed with a triangular bone graft and two k - wires ; ( g ) reduction of the hip joint through a separate lateral approach , the proximal end of femur was exposed subperiosteally and a transverse osteotomy was made at the level of lesser trochanter .
the amount of overlap in the femur was evaluated to determine the amount of femoral shaft to be resected .
the osteotomy site was internally fixed with a four - hole small fragment plate [ figure 1c ] .
shortening of 12 cm was found to be necessary to allow easy reduction of the femoral head while maintaining normal soft tissue tension across the hip . at the time of open reduction
femoral anteversion in excess of 60 as determined by forward angulation of the head and neck of femur with relation to the coronal plane of the femur is considered an indication for derotational osteotomy .
derotation was achieved by externally rotating the distal femoral fragment in relation to the proximal fragment until the patella pointed directly forward before applying the plate .
it is important to avoid excessive derotation of femur as posterior instability may result if it is combined with salter 's innominate osteotomy . at the time of initial reduction , if coverage of the femoral head was poor ( which was assessed by the evidence of inadequate coverage of femoral head anteriorly with hip in extension and laterally with hip in adduction ) or if the acetabulum was clearly maldirected , then we proceed for salter 's osteotomy . in this study
all 25 patients ( 30 hips ) required salter 's osteotomy for stabilization of the hip joint .
a pelvic osteotomy was performed as described by salter using a gigli saw [ figure 1d ] .
a full - thickness bone graft was removed from the anterior part of the iliac crest and trimmed to the shape of a wedge ; the base should correspond approximately to the distance between the anterior superior and inferior spines .
after osteotomy , the distal segment of the innominate bone containing the entire acetabulum was shifted forward , downward , and outward so that the osteotomy site was opened anterolaterally .
the bone graft was inserted on its edge into the osteotomy site and held with two k - wires [ figure 1e , f ] .
the hip was reduced concentrically , and the stability was assessed with the limb in the weight - bearing position , i.e. in slight abduction and extension [ figure 1 g ] .
redundant capsule was excised when necessary and capsulorrhaphy performed with interrupted absorbable sutures . throughout the operation ,
the mean blood loss which is measured by weighing sponges and recording suction volumes is 220 ml ( range 200- 250 ml ) .
the average time for the combined operation of open reduction and osteotomy was 2.5 hours ( range 2.25 - 2.75 hours ) .
postoperatively , the hips were immobilized in a 1 hip spica in position of slight flexion , abduction and internal rotation for 6 weeks .
this was changed to an abduction splint for a further 4 weeks which allows non weight bearing flexion and extension mobilization of the hip joints .
k - wires were removed after consolidation at the osteotomy site usually at 68 weeks .
partial weight bearing started after 10 weeks with crutches for 2 weeks , followed by full weight bearing .
patients with bilateral dysplasia ( n=5 ) had the procedures on both hips at an interval of 36 months ( mean 4.5 months ) as per patient 's convenience .
the patients were followed up in the outpatient clinic at 3 month intervals during the first 2 years after surgery and then at yearly intervals .
they were evaluated clinically during each visit for the range of motion of the affected hip , the quality of gait , and the presence of any pain .
each patient 's preoperative and postoperative radiographs were evaluated to determine the degree of displacement of the femoral head ( the tnnis grade ) , the acetabular index , c e angle , the continuity of the shenton 's line , and the presence or absence of avascular necrosis .
all the patients with a minimum follow up of two years were included in this study .
open reduction followed the recommendations of salter29 for the innominate osteotomy surgical technique and the femoral osteotomy was similar to that described by predrag klisic.30 the operative procedure started with the modified smith - petersen anterior approach ( bikini incision ) [ figure 1a ] .
the muscles were reflected from the inner and outer aspects of the iliac crest and the hip joint was exposed [ figure 1b ] .
this was followed by the release of iliopsoas , excision of pulvinar , release of ligamentum teres , and incision of transverse acetabular ligament .
gentle traction was applied to the extremity to assess the degree of contracture of the muscles that crossed the hip joint .
due to the soft tissue contractures , the femoral head still was not reducible without applying excessive pressure .
peroperative photographs showing ( a ) bikini incision ; ( b ) exposure of the hip joint ; ( c ) femoral osteotomy fixed with 4-hole small fragment plate ; ( d ) pelvic osteotomy performed by using a gigli saw ; ( e ) bone graft taken from the proximal fragment of the ilium ; ( f ) pelvic osteotomy fixed with a triangular bone graft and two k - wires ; ( g ) reduction of the hip joint through a separate lateral approach , the proximal end of femur was exposed subperiosteally and a transverse osteotomy was made at the level of lesser trochanter .
the amount of overlap in the femur was evaluated to determine the amount of femoral shaft to be resected .
the osteotomy site was internally fixed with a four - hole small fragment plate [ figure 1c ] .
shortening of 12 cm was found to be necessary to allow easy reduction of the femoral head while maintaining normal soft tissue tension across the hip . at the time of open reduction femoral anteversion in excess of 60 as determined by forward angulation of the head and neck of femur with relation to the coronal plane of the femur is considered an indication for derotational osteotomy .
derotation was achieved by externally rotating the distal femoral fragment in relation to the proximal fragment until the patella pointed directly forward before applying the plate .
it is important to avoid excessive derotation of femur as posterior instability may result if it is combined with salter 's innominate osteotomy . at the time of initial reduction , if coverage of the femoral head was poor ( which was assessed by the evidence of inadequate coverage of femoral head anteriorly with hip in extension and laterally with hip in adduction ) or if the acetabulum was clearly maldirected
all 25 patients ( 30 hips ) required salter 's osteotomy for stabilization of the hip joint .
a pelvic osteotomy was performed as described by salter using a gigli saw [ figure 1d ] .
a full - thickness bone graft was removed from the anterior part of the iliac crest and trimmed to the shape of a wedge ; the base should correspond approximately to the distance between the anterior superior and inferior spines .
after osteotomy , the distal segment of the innominate bone containing the entire acetabulum was shifted forward , downward , and outward so that the osteotomy site was opened anterolaterally .
the bone graft was inserted on its edge into the osteotomy site and held with two k - wires [ figure 1e , f ] .
the hip was reduced concentrically , and the stability was assessed with the limb in the weight - bearing position , i.e. in slight abduction and extension [ figure 1 g ] .
redundant capsule was excised when necessary and capsulorrhaphy performed with interrupted absorbable sutures . throughout the operation ,
the mean blood loss which is measured by weighing sponges and recording suction volumes is 220 ml ( range 200- 250 ml ) .
the average time for the combined operation of open reduction and osteotomy was 2.5 hours ( range 2.25 - 2.75 hours ) .
postoperatively , the hips were immobilized in a 1 hip spica in position of slight flexion , abduction and internal rotation for 6 weeks .
this was changed to an abduction splint for a further 4 weeks which allows non weight bearing flexion and extension mobilization of the hip joints .
k - wires were removed after consolidation at the osteotomy site usually at 68 weeks .
partial weight bearing started after 10 weeks with crutches for 2 weeks , followed by full weight bearing .
patients with bilateral dysplasia ( n=5 ) had the procedures on both hips at an interval of 36 months ( mean 4.5 months ) as per patient 's convenience .
the patients were followed up in the outpatient clinic at 3 month intervals during the first 2 years after surgery and then at yearly intervals .
they were evaluated clinically during each visit for the range of motion of the affected hip , the quality of gait , and the presence of any pain .
each patient 's preoperative and postoperative radiographs were evaluated to determine the degree of displacement of the femoral head ( the tnnis grade ) , the acetabular index , c e angle , the continuity of the shenton 's line , and the presence or absence of avascular necrosis .
all the patients with a minimum follow up of two years were included in this study .
there were 20 unilateral dislocations ( 9 right unilateral dislocations , 11 left unilateral dislocations ) and 5 bilateral dislocations .
the average age at the time of operation was 3.9 years ( range 1.68 years ) .
immediate postoperative shortening of lower limb was 12 cm ( average 1.5 cm ) which improved to 0.21 cm ( average 0.3 cm ) during the latest followup examination .
femur derotated to an average of 23.2 ( range 2030 ) to correct the anteversion that was noted during the operation .
surgical and followup data on the final clinical evaluation , the clinical results according to mckay 's scoring system [ table 2 ] were also favorable
. at preoperative evaluation , 23 hips ( 76.6% ) were in fair condition and 7 hips ( 23.3% ) were in poor condition . at the end of the study
, there were 13 hips ( 43.3% ) in excellent condition , 14 ( 46.6% ) in good condition , 2 ( 6.6% ) in fair condition , and 1(3.3% ) in poor condition .
mckay 's criteria for clinical evaluation according to severin 's classification [ table 3 ] fourteen ( 46.6% ) hips were severin 's class v and 16 ( 53.3% ) were severin 's class iv prior to surgery .
postoperatively , 25 hips ( 83.3% ) were rated class i ( 19 hips ) and class ii ( 6 hips ) .
five hips ( 16.6% ) were rated class iii ( 4 hips ) and class vi ( one hip ) at the latest followup .
cases 17 and 20 are illustrative of excellent clinical and radiographic ( class i ) results [ figures 2a c and 3a , b ] . cases 7 , 13 , and 16 showed good clinical and radiographic ( class ii ) results [ figures 4a c , 5a c , and 6a c ] .
p radiograph of right hip joint ( case 17 ) showing ( a ) dislocation ; ( b ) adequate reduction at 8 months after surgery ; ( c ) well - reduced hip joint within the remodeled acetabulum at 5.7 years followup ( a ) x - ray pelvis including both hip shows ddh of left hip ( case 20 ) in a 6.9 year old girl ( b ) x - ray of same patient at 2.7 year followup shows concentric reduction with radiographic result severin 's class i a 2.5-year - old boy with untreated ddh of the left hip joint ; postoperative x - ray left hip anteroposterior view ( case 7 ) showing ( a ) ddh of left hip ( b ) postoperative well reduced hip joint after single - stage triple procedure ; ( c ) reduction maintained at 3.9 years after surgery preoperative x - ray pelvis anteroposterior view of a 3.9 year old girl ( case 13 ) showing ( a ) bilateral ddh ; ( b ) followup x - ray 2.2 years after surgery shows well - reduced hip joints ; ( c ) well functional result with patient sitting cross legged anteroposterior radiograph of a 5.8-year - old girl ( case 16 ) showing ( a ) unilateral dislocation of the hip right side ; ( b ) followup radiograph 7 months after surgery with adequate reduction ; ( c ) followup radiograph after 7.6 years of surgery , with well - developed congruous hip joint according to tnnis classification [ table 4 ] of the degree of dislocation severity ( preoperatively ) , there were 4 patients ( 13.3% ) in grade i , 14 ( 46.6% ) in grade ii , and 12 ( 40% ) in grade iii , as compared to only 1 ( 3.3% ) of grade ii postoperatively .
tnnis classification for severity of dislocation the acetabular index was measured at each followup radiographs to assess acetabular development at the final followup .
the average acetabular index was 234.1 ; it decreased from 425 preoperatively to 212 postoperatively .
one patient ( case 5 ) developed lateral physeal damage ( type ii classification of bucholz and ogden ) . at the latest followup ,
the clinical results were good , although the radiographic results were severin 's class iii .
the hip was considered to be adequately reduced when the radiographs showed an intact shenton 's line , a center - edge angle of more then 15 , and an acetabular index of less then 28. the adequacy of acetabular remodeling was evaluated by comparing the immediate postoperative radiograph with the latest followup radiograph .
improvement of the acetabular index by at least 8 and to 20 or less in the most recent radiographs was considered to be adequate correction .
evaluation of gait revealed that three hips ( cases 9 , 18 , and 21 ) had a mild gluteus medius lurch and two ( cases 5 and 15 ) had a short legged gait .
one patient had infrequent discomfort , and three noted weakness ( a mild trendelenburg limp ) .
in general , the subjective evaluation did not correlate with the objective findings ; it was therefore of little value in assessing the end results .
one patient ( case 21 ) had an open reduction and innominate osteotomy performed in a false acetabulum .
most recent followup shows severin 's radiographic rating class vi [ figure 7a c ] .
repeat arthrotomy at the time of plate removal disclosed extensive scarring but no other intraarticular obstruction or pathology .
anteroposterior ratiograph of right hip joint of a 5.7-year - old male child ( case 21 ) showing ( a ) untreated ddh ( b ) immediate postoperative x - ray with residual dislocation ; ( c ) severin 's class vi at the latest followup , 3.9 years after surgery
failure to treat at an early age may lead to gait abnormalities , limitation of motion of the hip , joint pain , and osteoarthritis .
however , the age beyond which surgical treatment is contraindicated has been the subject of debate due to the risk of serious complications . concentric reduction in older children may be a challenge , more so when the femoral head is flattened or when the acetabulum is dysplastic
. reduction may be hindered by soft tissue contracture with undue pressure on the femoral head .
this may result in poor development of hip joint and avascular necrosis of the femoral head .
femoral osteotomy , including shortening , derotation osteotomy , combined with open reduction , capsulorrhaphy , and pelvic osteotomy , is an extensive procedure that can safely be undertaken in one stage .
this one stage procedure which has been used in older children is the method of choice in the treatment of late diagnosed congenital dislocation of the hip . with femoral shortening as the fundamental step
, this method can be applied in high dislocations to prevent undue pressure on the femoral head , which is the main cause of avascular necrosis , joint stiffness , and failures .
there have been numerous reports discussing the merits of a more aggressive surgical approach that includes a single procedure combining an open reduction with a femoral or pelvic osteotomy .
salter and dubos showed 93.6% good to excellent results in children of younger age group.31 klisic and jankovic ( n=60 ) reported 63% excellent to good results in the age group of 515 years.30 karaka et al .
operated on 47 patients ( 55 hips ) , who were 4 years and more , with 67% good or excellent clinical results and 65% good or excellent radiological results.32 ryan et al .
operated on 15 patients ( 18 hips ) , who were 4 years and more , with 100% good or excellent clinical results ( mckay 's criteria ) and 100% severin 's classification class i ( 10 ) and ii ( 8).34 umer et al .
have reported 86.2% good to excellent clinical results and 51.7% severin 's class i at final evaluation.35 the results of the salter 's osteotomy for treatment of ddh in 30 hips treated beyond 18 months of age were reviewed .
the results of the study showed a significant improvement in the radiological features of the operated hip joints according to severin 's classification . at the followup none of the patient had class v severity compared to 14 hips ( 46.6% ) prior to surgery .
there was only one hip in class iv post operatively as compared to 16 hips ( 53.3% ) preoperatively .
notably 83.3% of hips achieved an improvement of class i ( 19 hips ) and class ii ( 6 hips ) at followup compared to none before surgery .
similarly , favorable results were also observed when clinical parameters were compared before and after surgery . at final followup , excellent to good results
only 1 ( 3.3% ) hip joint demonstrated poor condition at followup compared to 7 ( 23.3% ) prior to surgery
. principle of preserving the shape and capacity of the acetabulum by salter 's innominate osteotomy is important for the future function and development of the joint as compared to pemberton osteotomy which alters the shape of acetabulum and decreases its capacity .
late - diagnosed congenital dislocation of the hip with one - stage open reduction , femoral shortening , capsulorrhaphy , and pelvic osteotomy corrects associated femoral and acetabular deformities .
this operation improves the cover of the femoral head and provide stability in the weight - bearing position .
this procedure can be done safely , with reliable results and without an increase in the risk of avascular necrosis .
the limitation of this study is that all the patients were not followed to skeletal maturity .
the presence of residual hip dysplasia and problems relating to subluxation , trochanteric overgrowth and avascular necrosis if any should be recognized early and treated appropriately to ensure optimum results . | background : the principles of treatment of congenital dislocation of hip in old children are different than those of infants and neonates .
the purpose of this study is to evaluate the radiographic and functional results of one - stage treatment ( open reduction , femoral shortening derotation , and salter 's osteotomy ) of ddh in older children.materials and methods : between january 2005 and june 2010 , 25 patients ( 30 hips ) underwent one - stage triple procedure of open reduction , femoral shortening derotation , and salter 's osteotomy for the treatment of ddh .
preoperatively , they were classified according to the tnnis class .
clinical outcomes were assessed using the modified mckay 's criteria to measure pain symptoms , gait pattern , trendelenburg sign status , and the range of hip joint movement .
radiographic assessment was made using severin 's scoring method to measure the centre - edge angle and dysplasia.results:the mean age at the time of operation was 3.9 years ( range 1.68 years ) , and the average duration of followup was 4.1 years ( range 27.6 years ) .
the mckay 's score was excellent in 13 hips , good in 14 hips , fair in 2 , and poor in 1 hip .
the severin 's class i and ii was found in 25 ( 83.3% ) hips at the time of final evaluation as compared to none at the time of presentation.conclusions:young children having ddh can safely be treated with an extensive one - stage triple procedure of open reduction , femoral shortening derotation , and salter 's osteotomy , without increasing the risk of avn . early diagnosis and intervention is the successful treatment of patients suffering from ddh . |
the use of electronic health records ( ehrs ) and other electronic clinical systems that capture data about clinical encounters and patient s care continue to grow rapidly.1,2 linking clinically derived data from these different systems can greatly facilitate clinical research and operational and quality initiatives.1 there are several possible approaches to make these data available for such uses , including ( 1 ) maintaining separate repositories for different data sources and linking data after extraction , and ( 2 ) incorporating data from different sources into a new data warehouse .
each approach presents unique benefits and challenges , with the institutional choice of approach depending on a variety of conceptual and pragmatic factors . with any approach , evaluation of data quality
is necessary to ensure the validity and generalizability of findings from clinical research and operational and quality initiatives .
however , data quality evaluation of data linked from different sources faces a number of unique challenges given the complexities of the underlying information systems .
the paper presents a data quality evaluation of a new joint data warehouse formed using the second approach , the knowledge program ( kp ) data warehouse at the cleveland clinic . after designing and implementing the approach to form the kp
, we focused on evaluating and adjusting our strategy by collecting and assessing data quality using two commonly used metrics of quality : completeness and concordance .
completeness refers to the proportion of cases where data are recorded in the system,3 and concordance is a measure of value agreement;4 neither relies on the presence of a gold standard .
the primary objective of our analysis was to determine the completeness and concordance of common clinical data variables available in the joint warehouse when compared to a patient s medical record , in preparation for its use in quality and research activities .
a novel aspect of this project was that a gold standard was not used . selecting a gold standard
may have introduced errors , due to the fact that the ehr was populated with data from multiple sources at variable intervals . to disseminate our findings to others , we discuss the challenges associated with evaluating the data quality of the joint warehouse in this comparative fashion and relate the lessons learned .
most commonly , validation studies of electronic data employ conventional statistical tools such as sensitivity , specificity , and positive and negative predictive values , typically comparing the electronic data with a manual medical record review or direct patient report.5 additional measures that have been recommended when validating data include the following : the ratio of true positive cases to false negative cases ( tpfn ratio)5 and the ratio of false negatives cases to the total number of patients in a database multiplied by 10,000 ( dbfind).5 these methods require the existence of a reference- or gold standard .
other methods , such as the kappa value , are prevalence dependent with limited generalizability to other settings where prevalence differs.6 although a manual review of data stored in the ehr seems an obvious choice for such a reference standard when validating data housed in a joint database , several issues complicate this decision . the complexity of data feeds received by a joint database like the kp results in data being obtained from several different sources , such that there is no single complete reference .
data tied to a patient , rather than an encounter , varies over time depending on the date at which it is assessed . in a large , multisite institution ,
health - level 7 ( hl7 ) feeds from different locations rarely turn on at a single time point , making it difficult to assess a firm start date for evaluation .
the complexity of available locations for entering data into the ehr presents perhaps the greatest challenge , sometimes resulting in less information being available upon review of a single encounter in the ehr , as compared to the joint warehouse .
these factors motivate the evaluation of data quality using criteria that do not assume either source to be complete ; namely , descriptive statistics in terms of ( 1 ) the presence or absence of a given value in each source ( completeness ) , and ( 2 ) concordance between the two values in cases where they are both present . in this study , completeness and concordance were assessed for demographic information , common laboratory results , vital signs , diagnoses , and medications .
these data variables represent the major categories of data in the kp data warehouse that are used for secondary analyses .
we anticipated that the assessment would identify three major characteristics of data quality in the kp , including the following : ( 1 ) completeness and concordance would be highest with data elements that do not require manual entry into the ehr ( e.g. , laboratory results ) ; ( 2 ) when demographics and vital signs were present , there would be a high level of agreement between the two sources ; and , ( 3 ) the highest levels of disagreement would be with medication and diagnoses data , due to the variety of locations and ways in which they can be stored , in addition to the wide range of options available for data entry into the ehr .
most commonly , validation studies of electronic data employ conventional statistical tools such as sensitivity , specificity , and positive and negative predictive values , typically comparing the electronic data with a manual medical record review or direct patient report.5 additional measures that have been recommended when validating data include the following : the ratio of true positive cases to false negative cases ( tpfn ratio)5 and the ratio of false negatives cases to the total number of patients in a database multiplied by 10,000 ( dbfind).5 these methods require the existence of a reference- or gold standard .
other methods , such as the kappa value , are prevalence dependent with limited generalizability to other settings where prevalence differs.6 although a manual review of data stored in the ehr seems an obvious choice for such a reference standard when validating data housed in a joint database , several issues complicate this decision . the complexity of data feeds received by a joint database like the kp results in data being obtained from several different sources , such that there is no single complete reference .
data tied to a patient , rather than an encounter , varies over time depending on the date at which it is assessed . in a large , multisite institution ,
health - level 7 ( hl7 ) feeds from different locations rarely turn on at a single time point , making it difficult to assess a firm start date for evaluation .
the complexity of available locations for entering data into the ehr presents perhaps the greatest challenge , sometimes resulting in less information being available upon review of a single encounter in the ehr , as compared to the joint warehouse .
these factors motivate the evaluation of data quality using criteria that do not assume either source to be complete ; namely , descriptive statistics in terms of ( 1 ) the presence or absence of a given value in each source ( completeness ) , and ( 2 ) concordance between the two values in cases where they are both present . in this study , completeness and concordance were assessed for demographic information , common laboratory results , vital signs , diagnoses , and medications .
these data variables represent the major categories of data in the kp data warehouse that are used for secondary analyses .
we anticipated that the assessment would identify three major characteristics of data quality in the kp , including the following : ( 1 ) completeness and concordance would be highest with data elements that do not require manual entry into the ehr ( e.g. , laboratory results ) ; ( 2 ) when demographics and vital signs were present , there would be a high level of agreement between the two sources ; and , ( 3 ) the highest levels of disagreement would be with medication and diagnoses data , due to the variety of locations and ways in which they can be stored , in addition to the wide range of options available for data entry into the ehr .
the cleveland clinic has developed the kp data warehouse to more effectively use clinical data to inform clinical operations , quality , and research .
the kp data warehouse was first used in the neurological institute ( ni ) , which consists of four departments : neurology , neurosurgery , psychiatry , and physical medicine and rehabilitation .
this warehouse includes direct hl7 feeds from the ehr ( epic , verona , wis . ) , and laboratory , radiology , and scheduling systems ( figure 1 ) .
the kp data warehouse also serves as the main repository for patient - reported outcome measures ( proms ) that are collected at the point of care through a web - based system.7 the availability of proms was one of the catalysts to develop this warehouse and include feeds from additional data sources .
the focus of this investigation was on the quality of warehouse data compared to the ehr , since that can be also be used as a data source for aggregate analyses .
a manual review of electronic charts was performed by first reviewing an electronic chart based on one specific encounter in a commercially available ehr , followed by a comparison with the same data available in the kp database .
completeness and concordance were determined for the five categories shown in table 1 . for this study
the ni was chosen as a focus for this review as it represented the largest user of the data warehouse .
the sample size was chosen to limit the time - consuming process of manual review , but still provide adequate power to estimate the sensitivity of a chosen diagnosis , with a reasonable margin of error ( e.g. , coronary artery disease with an expected prevalence of 18 percent ) . in order to represent adequately all 15 centers in the ni , a random sample of charts were proportionally stratified by center , based on the patient volumes of each center .
manual chart abstractions of the ehr were performed by a physician who reviewed the most recent ni - based encounter during the study period .
clinical information in the reviewed encounter note was abstracted , including vital signs , diagnoses , and medications , regardless of whether it was entered as free - text or through autopopulation of clinical data from other areas in the ehr .
scanned documents were reviewed only if they were recorded at or before the date of the encounter .
information available in the other areas of ehr at the time of the review was abstracted for demographic information , laboratory results , vital signs , active medications , and diagnoses .
level of agreement of data elements between the ehr and kp was determined for the following diagnoses : cancer , coronary artery disease , diabetes , stroke , chronic renal insufficiency , depression , hypertension , atrial fibrillation , hypothyroidism , obesity , and sleep apnea .
three areas of the electronic chart were considered when evaluating diagnoses : past medical history , problem list , and the encounter diagnosis section . notes from nursing , home care , physical and occupational therapy , speech therapy , pastoral care , social work , telephone encounters , and financial counselors were excluded from the review , as was additional documentation unrelated to a specific encounter .
data from the kp data warehouse on the 250 subjects were electronically extracted and placed into a study data set , which included all laboratory values , vital signs , diagnoses , and medications in the kp data warehouse up until the time of extraction . a data registry registered nurse reformatted the extracted data set and merged this with the manually abstracted ehr data on these subjects .
the kp data extraction into the study data set occurred at a single time point .
the data sets for this study ( table 2 ) were housed in the research electronic data capture ( redcap ) tool hosted at cleveland clinic.8 redcap is a secure , web - based application designed to support data capture for research studies , providing the following : ( 1 ) an intuitive interface for validated data entry ; ( 2 ) audit trails for tracking data manipulation and export procedures ; ( 3 ) automated export procedures for seamless data downloads to common statistical packages ; and ( 4 ) procedures for importing data from external sources .
descriptive statistics were computed for demographics ( mean and standard deviation for age , frequency counts , and percentages for gender , race , etc . ) .
completeness of demographic , lab , diagnoses , medical use , and vital signs data was determined by calculating the percent of cases in which each was present in the ehr and kp , respectively , and computing the discrepancy ( number and percent ) between the two sources .
concordance was determined for categorical variables by calculating the number and percent of cases in which the data values were identical ( including missing ) in both data sets .
comparisons between the ehr and kp were made both excluding and including free text ehr fields ( scanned documents and progress notes ) .
completeness and concordance of common clinical data values in the joint warehouse was performed without identifying a gold standard . having a gold standard is a potential source of errors , since the ehr contains data from a variety of sources that are entered at different intervals .
the cleveland clinic has developed the kp data warehouse to more effectively use clinical data to inform clinical operations , quality , and research .
the kp data warehouse was first used in the neurological institute ( ni ) , which consists of four departments : neurology , neurosurgery , psychiatry , and physical medicine and rehabilitation .
this warehouse includes direct hl7 feeds from the ehr ( epic , verona , wis . ) , and laboratory , radiology , and scheduling systems ( figure 1 ) .
the kp data warehouse also serves as the main repository for patient - reported outcome measures ( proms ) that are collected at the point of care through a web - based system.7 the availability of proms was one of the catalysts to develop this warehouse and include feeds from additional data sources .
the focus of this investigation was on the quality of warehouse data compared to the ehr , since that can be also be used as a data source for aggregate analyses .
a manual review of electronic charts was performed by first reviewing an electronic chart based on one specific encounter in a commercially available ehr , followed by a comparison with the same data available in the kp database .
the ni was chosen as a focus for this review as it represented the largest user of the data warehouse .
the sample size was chosen to limit the time - consuming process of manual review , but still provide adequate power to estimate the sensitivity of a chosen diagnosis , with a reasonable margin of error ( e.g. , coronary artery disease with an expected prevalence of 18 percent ) . in order to represent adequately all 15 centers in the ni , a random sample of charts were proportionally stratified by center , based on the patient volumes of each center .
manual chart abstractions of the ehr were performed by a physician who reviewed the most recent ni - based encounter during the study period .
clinical information in the reviewed encounter note was abstracted , including vital signs , diagnoses , and medications , regardless of whether it was entered as free - text or through autopopulation of clinical data from other areas in the ehr .
scanned documents were reviewed only if they were recorded at or before the date of the encounter .
information available in the other areas of ehr at the time of the review was abstracted for demographic information , laboratory results , vital signs , active medications , and diagnoses .
level of agreement of data elements between the ehr and kp was determined for the following diagnoses : cancer , coronary artery disease , diabetes , stroke , chronic renal insufficiency , depression , hypertension , atrial fibrillation , hypothyroidism , obesity , and sleep apnea .
three areas of the electronic chart were considered when evaluating diagnoses : past medical history , problem list , and the encounter diagnosis section . notes from nursing , home care , physical and occupational therapy , speech therapy , pastoral care , social work , telephone encounters , and financial counselors were excluded from the review , as was additional documentation unrelated to a specific encounter .
data from the kp data warehouse on the 250 subjects were electronically extracted and placed into a study data set , which included all laboratory values , vital signs , diagnoses , and medications in the kp data warehouse up until the time of extraction . a data registry registered nurse
reformatted the extracted data set and merged this with the manually abstracted ehr data on these subjects .
the kp data extraction into the study data set occurred at a single time point .
the data sets for this study ( table 2 ) were housed in the research electronic data capture ( redcap ) tool hosted at cleveland clinic.8 redcap is a secure , web - based application designed to support data capture for research studies , providing the following : ( 1 ) an intuitive interface for validated data entry ; ( 2 ) audit trails for tracking data manipulation and export procedures ; ( 3 ) automated export procedures for seamless data downloads to common statistical packages ; and ( 4 ) procedures for importing data from external sources .
descriptive statistics were computed for demographics ( mean and standard deviation for age , frequency counts , and percentages for gender , race , etc . ) .
completeness of demographic , lab , diagnoses , medical use , and vital signs data was determined by calculating the percent of cases in which each was present in the ehr and kp , respectively , and computing the discrepancy ( number and percent ) between the two sources .
concordance was determined for categorical variables by calculating the number and percent of cases in which the data values were identical ( including missing ) in both data sets . comparisons between the ehr and kp were made both excluding and including free text ehr fields ( scanned documents and progress notes ) .
completeness and concordance of common clinical data values in the joint warehouse was performed without identifying a gold standard . having a gold standard is a potential source of errors , since the ehr contains data from a variety of sources that are entered at different intervals .
the mean age of the patients in this study was 51.4 ( sd = 16.1 ) , consisting of more women than men ( 59.6 percent vs. 40.4 percent ) .
the group was primarily white ( 79.2 percent ) , and a minority was black ( 14.0 percent ) .
the majority of patients were married ( 62.0 percent ) , followed by single ( 19.6 percent ) , divorced ( 9.2 percent ) , and widowed ( 5.2 percent ) .
the results comparing completeness and concordance between the two sources are displayed in tables 35 .
data elements for demographic data ( table 3 ) were present in more cases in the ehr ( 100 percent ) compared to the kp data warehouse ( 98.499.6 percent ) in all instances except for date of death , for which there were more data elements in the kp data warehouse than the ehr ( 3.2 percent vs. 1.2 percent ) . in cases where the demographic data elements were present in both the ehr and the kp data warehouse , values between the two sources were concordant over 98 percent of the time .
select labs ( table 4 ) were recorded in the kp data warehouse in 1.6 percent ( fasting blood sugar ) to 52.4 percent ( creatinine , blood urea nitrogen , and glucose ) of cases .
the ehr review was more complete relative to the kp data warehouse with a 0.86.4 percent difference in the presence of specific lab values between the two .
vital signs were recorded in 25.6 percent ( height ) to 69.6 percent ( blood pressure ) of cases . for all vital signs ( table 5 ) ,
there was complete agreement in values for laboratory results and vitals when they were present in both the ehr and kp data set .
the prevalence for the selected diagnoses varied widely in both the ehr and kp data warehouse ( 3.229.6 percent vs. 2.825.2 percent ; table 5 ) .
hypertension and depression were the most frequently documented diagnoses , with rates of 18.4 and 25.2 percent , respectively , in the kp data warehouse .
for all diagnoses except stroke , a higher prevalence was identified using ehr data than kp data , with the discrepancies ranging from 0.4 to 11.2 percent .
the documented use of medication also varied widely . according to the kp data warehouse ,
14 percent were on diabetic medications , 47.2 percent were on antihypertensives , and 30.8 percent were on aspirin .
in contrast to the other data variables assessed in this study , there was higher prevalence of medication use in the kp records than in the ehr review ranging from 3.6 to 6.8 percent higher rates than the ehr .
analysis of completeness and concordance of the data was repeated , comparing the kp to the ehr review limited to variables available in structured fields ( appendices 1 and 2 ) , excluding ehr data present in scanned documents or free - text fields .
this provides a comparison of the discretely available electronic data in the two data sets .
as expected , there were more cases of matching data between the structured fields of the ehr and the kp data warehouse .
still , vital signs , laboratory values , and diagnoses were more complete in the ehr than in the kp data warehouse ; there were 2.86.8 percent more cases with vitals , 14.4 percent more cases with laboratory values , and 09.6 percent more cases with diagnoses in the ehr than in the kp . in our comparison ( n = 250 ) , completeness of demographic data (
excluding date of death ) was 100 percent in the ehr , and 98.499.6 percent in the kp data warehouse .
data from ehr were available only in a nonstructured format for up to 2.4 percent of laboratory values , 2.4 percent of vitals , and 1.6 percent of diagnoses .
in this evaluation comparing completeness and level of agreement of the kp data warehouse and the ehr , there was a mismatch in available data in most of the categories assessed up to 6.4 percent of laboratory values , 9.2 percent of vital signs , and 11.2 percent of diagnoses , which could potentially have an impact on analyses using these data .
the ehr generally had more complete data than the kp data warehouse except for information on deaths and medications , for which the kp data warehouse had more .
when data were available in both sources , there was a high , but imperfect , concordance in values .
the reasons for these discrepancies were explored in depth , and our findings highlight many factors that need to be considered when constructing , evaluating , and utilizing an external data set derived from an ehr and other clinical data sources . the biggest factor that has an impact on the completeness of data was the start date of data feeds into the kp data warehouse .
data were deposited into the kp data warehouse from the ehr only after specific data feeds were turned on , and any data recorded in the ehr prior to this were not available in the kp data warehouse .
this lack of historical data resulted in the absence of up to 9.6 percent of evaluated diagnoses in the kp data warehouse compared to the ehr ( appendix 2 ) .
similarly , there were separate kp data feeds for different laboratory test categories and locations within the health system , which were turned on in a staggered fashion .
laboratory test results were not available in the kp data warehouse if the tests were performed and reported before the data feeds to the warehouse were turned on .
this staggered implementation of laboratory feeds was responsible for the majority of the mismatches in laboratory values in this current study .
because of the uneven nature of missing values , with laboratory values available for some but not all patients , this issue may not be apparent to an end user who might falsely conclude that certain laboratory tests were not performed .
the systematic missingness of data has the potential to confound analyses if it is not identified and adequately addressed in the study design or analysis .
thus , when constructing and utilizing an external data source , it is necessary to have a clear understanding of when data variables for different collection locations are accessible .
an unexpected finding of this analysis was that , in contrast to other data variables , there was a higher prevalence of medications in the kp data warehouse compared to the data from the ehr review .
after investigation , this was found to be due to a systematic issue with medication data feeds
finding this significant unexpected error is an example of why it is critical to systematically assess the data quality of all data variables .
another relevant factor contributing to the completeness of data in the kp and other data warehouses that obtain data from ehrs was the inability to incorporate provider - entered nonstructured clinical data , such as free - text or scanned documents .
we found that outside results ( e.g. , laboratory tests ) were sometimes entered into the ehr in the form of a scanned document .
other data including vital signs , medications , or medical history were occasionally entered only as free - text in an encounter note , even though discrete fields for data entry were available and recommended as part of standard documentation practices .
data that are in nonstructured parts of the ehr were not sent to the kp data warehouse , which resulted in less complete data in the kp in these areas compared to the manual ehr review .
our analysis indicates that up to 2.4 percent of external laboratory information and vitals were not recorded in the kp due to this issue ( table 4 and appendix 1 ) .
although these percentages are fairly low , they reflect a limitation of performing automated surveys of electronic data sources .
these findings may differ for databases from other institutions due to differences in documentation practices across specialties and institutions .
the imperfect concordance between the kp and ehr in this study when variables were present in both data sets was due to the dynamic nature of some of the clinical data elements in the ehr .
patient - level data , such as medications , diagnoses , or demographics ( except for death ) can change over time , sometimes outside of a clinic visit .
encounter - based data , such as laboratory values or an encounter note , are typically static and do not change once entered unless a correction or amendment is made . the kp data warehouse is designed to only receive updates to a patient s data when a clinical visit is completed , and only if the visit is in a clinical area utilizing the kp . these factors ,
along with an occasional difference in timing of data reviews of the ehr and kp data , resulted in imperfect agreement for some demographic data between the two databases .
given the dynamic nature of some data variables in an ehr and other clinically derived data sources , it is critical to be aware of the frequency and extent of data updates to the external warehouse .
ideally , there would be real - time feeds that immediately upload any changes in the original data set to the joint database .
scheduled updates are an alternative , however , these types of processes may be resource intensive to set up .
lack of documentation in the ehr led to missing data in both the kp data warehouse and original ehr .
for example , blood pressure measurements were available for only 78.8 percent ( 197 of 250 ) , height measurements for 28.8 percent ( 72 of 250 ) , and weight measurements for 51.2 percent ( 128 of 250 ) of encounters in the original ehr ( table 4 ) .
missing documentation is a significant limitation of data sets derived from ehrs and other clinically derived data sources.9,10 phenotyping with ehr data is an area of active research to combat the problem of missing or variably recorded data .
with ehr - driven phenotyping , raw ehr data is transformed into clinically relevant features using heuristic rules or modeling to increase the sensitivity and specificity of identifying specific diagnoses or patient characteristics.11 a better understanding of data quality and the implications of missingness can provide information on uncertainty in ehr - derived phenotypes .
this current analysis could be part of an initial step toward the development of algorithms to better identify these select conditions . in this kp data quality evaluation , completeness and concordance between the kp data warehouse and data from a manual review of the ehr
the kp receives direct feeds from the laboratory , radiology , and scheduling systems , in addition to feeds from the ehr , and receives regular updates from the social security death index on deaths .
given the different direct data sources that feed into the kp , a comparison with the manual ehr review as the gold standard would have misguided our analysis .
indeed , although the kp data warehouse did have less complete information in most data variables compared to the ehr , the kp had more data on deaths .
this supports the practice of avoiding the designation of any single data set as the gold standard when evaluating data quality of a joint data warehouse , especially one that has multiple data sources .
the biggest factor that has an impact on the completeness of data was the start date of data feeds into the kp data warehouse .
data were deposited into the kp data warehouse from the ehr only after specific data feeds were turned on , and any data recorded in the ehr prior to this were not available in the kp data warehouse .
this lack of historical data resulted in the absence of up to 9.6 percent of evaluated diagnoses in the kp data warehouse compared to the ehr ( appendix 2 ) .
similarly , there were separate kp data feeds for different laboratory test categories and locations within the health system , which were turned on in a staggered fashion .
laboratory test results were not available in the kp data warehouse if the tests were performed and reported before the data feeds to the warehouse were turned on .
this staggered implementation of laboratory feeds was responsible for the majority of the mismatches in laboratory values in this current study . because of the uneven nature of missing values , with laboratory values available for some but not all patients
, this issue may not be apparent to an end user who might falsely conclude that certain laboratory tests were not performed .
the systematic missingness of data has the potential to confound analyses if it is not identified and adequately addressed in the study design or analysis .
thus , when constructing and utilizing an external data source , it is necessary to have a clear understanding of when data variables for different collection locations are accessible .
an unexpected finding of this analysis was that , in contrast to other data variables , there was a higher prevalence of medications in the kp data warehouse compared to the data from the ehr review .
after investigation , this was found to be due to a systematic issue with medication data feeds
this resulted in an incorrect inflation of medications designated as active . finding this significant unexpected error is an example of why it is critical to systematically assess the data quality of all data variables .
another relevant factor contributing to the completeness of data in the kp and other data warehouses that obtain data from ehrs was the inability to incorporate provider - entered nonstructured clinical data , such as free - text or scanned documents .
we found that outside results ( e.g. , laboratory tests ) were sometimes entered into the ehr in the form of a scanned document .
other data including vital signs , medications , or medical history were occasionally entered only as free - text in an encounter note , even though discrete fields for data entry were available and recommended as part of standard documentation practices .
data that are in nonstructured parts of the ehr were not sent to the kp data warehouse , which resulted in less complete data in the kp in these areas compared to the manual ehr review .
our analysis indicates that up to 2.4 percent of external laboratory information and vitals were not recorded in the kp due to this issue ( table 4 and appendix 1 ) .
although these percentages are fairly low , they reflect a limitation of performing automated surveys of electronic data sources .
these findings may differ for databases from other institutions due to differences in documentation practices across specialties and institutions .
the imperfect concordance between the kp and ehr in this study when variables were present in both data sets was due to the dynamic nature of some of the clinical data elements in the ehr .
patient - level data , such as medications , diagnoses , or demographics ( except for death ) can change over time , sometimes outside of a clinic visit .
encounter - based data , such as laboratory values or an encounter note , are typically static and do not change once entered unless a correction or amendment is made . the kp data warehouse is designed to only receive updates to a patient s data when a clinical visit is completed , and only if the visit is in a clinical area utilizing the kp .
these factors , along with an occasional difference in timing of data reviews of the ehr and kp data , resulted in imperfect agreement for some demographic data between the two databases .
given the dynamic nature of some data variables in an ehr and other clinically derived data sources , it is critical to be aware of the frequency and extent of data updates to the external warehouse .
ideally , there would be real - time feeds that immediately upload any changes in the original data set to the joint database .
scheduled updates are an alternative , however , these types of processes may be resource intensive to set up .
lack of documentation in the ehr led to missing data in both the kp data warehouse and original ehr .
for example , blood pressure measurements were available for only 78.8 percent ( 197 of 250 ) , height measurements for 28.8 percent ( 72 of 250 ) , and weight measurements for 51.2 percent ( 128 of 250 ) of encounters in the original ehr ( table 4 ) .
missing documentation is a significant limitation of data sets derived from ehrs and other clinically derived data sources.9,10 phenotyping with ehr data is an area of active research to combat the problem of missing or variably recorded data .
with ehr - driven phenotyping , raw ehr data is transformed into clinically relevant features using heuristic rules or modeling to increase the sensitivity and specificity of identifying specific diagnoses or patient characteristics.11 a better understanding of data quality and the implications of missingness can provide information on uncertainty in ehr - derived phenotypes
. this current analysis could be part of an initial step toward the development of algorithms to better identify these select conditions .
in this kp data quality evaluation , completeness and concordance between the kp data warehouse and data from a manual review of the ehr were compared , rather than designating the ehr as the gold standard .
the kp receives direct feeds from the laboratory , radiology , and scheduling systems , in addition to feeds from the ehr , and receives regular updates from the social security death index on deaths .
given the different direct data sources that feed into the kp , a comparison with the manual ehr review as the gold standard would have misguided our analysis .
indeed , although the kp data warehouse did have less complete information in most data variables compared to the ehr , the kp had more data on deaths .
this supports the practice of avoiding the designation of any single data set as the gold standard when evaluating data quality of a joint data warehouse , especially one that has multiple data sources .
this study identifies several factors that should be evaluated when developing a joint data warehouse and subsequently assessing the quality of its data .
these include the frequency and method for updating the data warehouse , start date , and maintenance of data feeds , and the dynamic nature of data in the source ehr . the additional limitations of using clinically derived data sources should also be considered , such as an inability to capture information recorded in nonstructured formats and incomplete documentation with data feeds . in addition , when evaluating the quality of an external data set with multiple and complex data sources , assessing completeness and concordance between data set and source data may be more appropriate than designating one to be a gold standard .
this study provides an innovative example of how the quality of a data set with multiple sources can be evaluated without designating a gold standard for comparison . | introduction : the use of clinically derived data from electronic health records ( ehrs ) and other electronic clinical systems can greatly facilitate clinical research as well as operational and quality initiatives .
one approach for making these data available is to incorporate data from different sources into a joint data warehouse .
when using such a data warehouse , it is important to understand the quality of the data .
the primary objective of this study was to determine the completeness and concordance of common types of clinical data available in the knowledge program ( kp ) joint data warehouse , which contains feeds from several electronic systems including the ehr.methods:a manual review was performed of specific data elements for 250 patients from an ehr , and these were compared with corresponding elements in the kp data warehouse .
completeness and concordance were calculated for five categories of data including demographics , vital signs , laboratory results , diagnoses , and medications.results:in general , data elements for demographics , vital signs , diagnoses , and laboratory results were present in more cases in the source ehr compared to the kp .
when data elements were available in both sources , there was a high concordance .
in contrast , the kp data warehouse documented a higher prevalence of deaths and medications compared to the ehr.discussion:several factors contributed to the discrepancies between data in the kp and the ehr including the start date and frequency of data feeds updates into the kp , inability to transfer data located in nonstructured formats ( e.g. , free text or scanned documents ) , as well as incomplete and missing data variables in the source ehr.conclusion:when evaluating the quality of a data warehouse with multiple data sources , assessing completeness and concordance between data set and source data may be better than designating one to be a gold standard .
this will allow the user to optimize the method and timing of data transfer in order to capture data with better accuracy . |
conventional glass ionomer cements ( gics ) that undergo setting through an acid - base reaction between an ion - leachable glass and aqueous polyacid.1 they have been widely used in restorative dentistry because of their advantageous properties including biocompatibility,2 adhesion to tooth structure,3 and fluoride release.4 however , these materials have some clinical limitations such as prolonged setting reaction time , moisture sensitivity during initial setting , dehydration , and rough surface texture , which can hamper mechanical resistance.5 to overcome these shortcomings , resin - modified glass ionomer cements ( rmgics ) were developed .
in addition to the conventional gic formulation , these materials contain monomers and photo initiators.6 cement setting starts when two components are mixed together due to an acid - base reaction .
light exposure causes the creation of cross bonds between polymeric chains and polymerization of methacrylate.7 thus , the reaction may be finished immediately after light exposure , so that operators can have a longer working time.8 metal - reinforced glass ionomer cements were introduced to the dental market in 1977 .
the addition of silver - amalgam alloy powder to conventional materials increase the physical strength of the cement and provide radiopacity.9 they can be used to restore class ii cavities by tunnel preparation , deciduous teeth ( especially class i ) , core build - ups and retrograde root filling . recently , several faster - setting , high - viscosity conventional gics , originally developed for use with the atraumatic restorative treatment11 have become available .
gics by some authors.10 these materials set faster and having a higher viscosity because of presence of finer glass particles , anhydrous polyacrylic acids of high molecular weight , and a high powder - to - liquid mixing ratio.10,11 the setting reaction is the same as the acid - base reaction of a typical conventional gic .
one of the latest developments in gics is a nanofiller gic which contains nano - sised powder particles and fluorapatite .
the benefit gained from rmgics and bonded nanofiller technology is that they can provide the glass ionomer with improved polish and esthetic properties . although some studies5,6,12 exist concerning the mechanical properties of conventional gics and resin - modified gics , there is no sufficient information about novel nanofiller gics .
gics have been used for different purposes in dentistry in restoring incipient carious lesions especially in primary teeth . in the dental market , there are various gics possessing distinctive properties ; and recent ones are added every day ; it is crucial to have knowledge about the physical and mechanical properties of different brands and new products when choosing gics as restorative material .
therefore , the aim of this study was to evaluate surface roughness and hardness of six different gics and determine whether there is any correlation between gics surface roughness and hardness .
a nanofiller gic ( ketac n100 ) , a resin - modified gic ( fuji ii lc ) , 3 conventional gics ( fuji ix , ionofil molar , aqua ionofil plus ) , and a silver - reinforced gic ( argion molar ) were used in this study .
the chemical compositions of these materials are listed in table 1 . a sectional teflon mold ( 8 mm diameter 2 mm deep ) was used to prepare the sample .
the mold was first mounted on top of a glass plate and a mylar strip .
the materials were mixed or handled according to their manufacturers instructions and inserted into the molds slightly excessively .
the mylar strip was positioned on the mold , and a second glass plate was then placed on top of the filled mold . a slight pressure was applied , and the bulk of the extruded excess cement was removed .
for the light - curing gics ( ketac n100 and fuji ii lc ) , specimens were cured with 20 s exposures by a halogen light - curing unit ( hilux , benliolu , ankara , turkey ) .
the light intensity was measured with a radiometer ( hilux , benliolu , ankara , turkey ) before starting the experiment .
the prepared samples were then stored in a distilled water at 37 c for 24 h. eleven samples were prepared from each material . for the surface roughness test ,
5 samples from each material were used and kept in the mold throughout the experiment .
the surface roughness of each specimen was measured in 5 different positions initially before polishing using a surface profilometer ( surftest 211 , mitutoyo , tokyo , japan ) .
the cut - off value for surface roughness was 0.8 mm , and the traversing distance of the stylus was 4.0 mm .
the radius of the tracing diamond tip was 5 m , the measuring force and speed were 4 mn ( 0.4 gf ) and 0.5 m s , respectively .
the average roughness value ( ra , m ) of an individual disc was taken as the mean of the ra ( average of peaks and valleys of a surface ) values measured in 5 different positions .
each sample was then polished sequentially with coarse , medium , fine , and superfine aluminum oxide abrasive discs ( sof - lex , 3 m , st paul , mn , usa ) .
the discs were mounted on a slow - speed hand piece ( ca111 , bien air dental , bienne , switzerland ) rotating at approximately 20 000 rpm .
the process was carried out by a single operator as the manufacturer s directions with light hand pressure in a circular pattern for 20 s , per each sof - lex disc .
after the polishing procedure was completed , surface roughness was measured in 5 different positions parallel to the first measurements using the surface profilometer . after polishing ,
1 sample of the tested materials was prepared for scanning electron microscopy ( ism-840a , jeol , tokyo , japan ) .
samples were sputter coated with gold to a thickness of approximate - samples were used . after being polished with aluminum oxide abrasive discs , the hardness of the upper surfaces
was measured using the vickers microhardness measuring instrument ( hmv microhardness tester , shimadzu , japan ) .
a 200-gf load was applied through the indenter with a dwell time of 15 s. five readings were taken for each specimen , and the mean vickers hardness value was recorded .
one - way analysis of variance ( anova ) with the post - hoc tukey - kramer hsd multiple - range test was used to determine significant differences among the materials in each test a significance level of =.05 . paired samples t - test
a nanofiller gic ( ketac n100 ) , a resin - modified gic ( fuji ii lc ) , 3 conventional gics ( fuji ix , ionofil molar , aqua ionofil plus ) , and a silver - reinforced gic ( argion molar ) were used in this study .
the chemical compositions of these materials are listed in table 1 . a sectional teflon mold ( 8 mm diameter 2 mm deep ) was used to prepare the sample .
the mold was first mounted on top of a glass plate and a mylar strip .
the materials were mixed or handled according to their manufacturers instructions and inserted into the molds slightly excessively .
the mylar strip was positioned on the mold , and a second glass plate was then placed on top of the filled mold . a slight pressure was applied , and the bulk of the extruded excess cement was removed .
for the light - curing gics ( ketac n100 and fuji ii lc ) , specimens were cured with 20 s exposures by a halogen light - curing unit ( hilux , benliolu , ankara , turkey ) .
the light intensity was measured with a radiometer ( hilux , benliolu , ankara , turkey ) before starting the experiment .
the prepared samples were then stored in a distilled water at 37 c for 24 h. eleven samples were prepared from each material .
for the surface roughness test , 5 samples from each material were used and kept in the mold throughout the experiment .
the surface roughness of each specimen was measured in 5 different positions initially before polishing using a surface profilometer ( surftest 211 , mitutoyo , tokyo , japan ) .
the cut - off value for surface roughness was 0.8 mm , and the traversing distance of the stylus was 4.0 mm .
the radius of the tracing diamond tip was 5 m , the measuring force and speed were 4 mn ( 0.4 gf ) and 0.5 m s , respectively .
the average roughness value ( ra , m ) of an individual disc was taken as the mean of the ra ( average of peaks and valleys of a surface ) values measured in 5 different positions .
each sample was then polished sequentially with coarse , medium , fine , and superfine aluminum oxide abrasive discs ( sof - lex , 3 m , st paul , mn , usa ) .
the discs were mounted on a slow - speed hand piece ( ca111 , bien air dental , bienne , switzerland ) rotating at approximately 20 000 rpm .
the process was carried out by a single operator as the manufacturer s directions with light hand pressure in a circular pattern for 20 s , per each sof - lex disc .
after the polishing procedure was completed , surface roughness was measured in 5 different positions parallel to the first measurements using the surface profilometer . after polishing ,
1 sample of the tested materials was prepared for scanning electron microscopy ( ism-840a , jeol , tokyo , japan ) .
samples were sputter coated with gold to a thickness of approximate - samples were used . after being polished with aluminum oxide abrasive discs , the hardness of the upper surfaces
was measured using the vickers microhardness measuring instrument ( hmv microhardness tester , shimadzu , japan ) .
a 200-gf load was applied through the indenter with a dwell time of 15 s. five readings were taken for each specimen , and the mean vickers hardness value was recorded .
one - way analysis of variance ( anova ) with the post - hoc tukey - kramer hsd multiple - range test was used to determine significant differences among the materials in each test a significance level of =.05 . paired samples t - test
mean and standard deviations of surface roughness ( ra ) are shown in table 2 .
when the materials were compared before and after polishing surface roughness values , resin - modified gic ( fuji ii lc ) , conventional gic ( fuji ix ) , and silver - reinforced gic ( argion molar ) showed statistically significant differences ( p<.05 ) .
there were no statistically significant difference between before and after polishing surface roughness values in nano - filler gic ( ketac n100 ) and conventional gics ( ionofil molar , aqua ionofil plus ) ( p>.05 ) .
the smoothest surface before the polishing procedure was observed in nanofiller gic ( ketac n100 ) .
this was followed by resin - modified gic , fuji ii lc ; then silver - reinforced gic , argion molar , conventional gics , aqua ionofil plus , fuji ix , and ionofil molar , respectively . before the polishing procedure
, there were no significant differences among the surface roughness values of all materials ( p<.05 ) except between the nanofiller gic ketac n100 ( p>.05 ) .
after the finishing procedure , it was observed that the surface roughness was the lowest in nano - filler gic ketac n100 and highest in conventional gic and aqua ionofil plus .
the sem photomicrographs obtained in this study showed that , regardless of the commercial brand , all the conventional gics presented voids and cracks on their surfaces ( figure 2 ) . mean and
the result of the microhardness test indicated that the microhardness value of silver - reinforced gic argion molar was greater than that of the other gics . among the all conventional gics examined , fuji ix showed the lowest hardness value .
when the hardness values of all tested gics were compared , the differences between materials ( except aqua ionofil plus with ionofil molar and ketac n100 with fuji ii lc ( p>.05 ) ) were found statistically significant ( p<.05 ) .
linear regression showed that there is no correlation between surface roughness and hardness of tested gics ( t = 1.141 , p = .264 )
mean and standard deviations of surface roughness ( ra ) are shown in table 2 .
when the materials were compared before and after polishing surface roughness values , resin - modified gic ( fuji ii lc ) , conventional gic ( fuji ix ) , and silver - reinforced gic ( argion molar ) showed statistically significant differences ( p<.05 ) .
there were no statistically significant difference between before and after polishing surface roughness values in nano - filler gic ( ketac n100 ) and conventional gics ( ionofil molar , aqua ionofil plus ) ( p>.05 ) .
the smoothest surface before the polishing procedure was observed in nanofiller gic ( ketac n100 ) .
this was followed by resin - modified gic , fuji ii lc ; then silver - reinforced gic , argion molar , conventional gics , aqua ionofil plus , fuji ix , and ionofil molar , respectively . before the polishing procedure
, there were no significant differences among the surface roughness values of all materials ( p<.05 ) except between the nanofiller gic ketac n100 ( p>.05 ) .
after the finishing procedure , it was observed that the surface roughness was the lowest in nano - filler gic ketac n100 and highest in conventional gic and aqua ionofil plus .
the sem photomicrographs obtained in this study showed that , regardless of the commercial brand , all the conventional gics presented voids and cracks on their surfaces ( figure 2 ) .
the result of the microhardness test indicated that the microhardness value of silver - reinforced gic argion molar was greater than that of the other gics . among the all conventional gics examined , fuji ix showed the lowest hardness value .
when the hardness values of all tested gics were compared , the differences between materials ( except aqua ionofil plus with ionofil molar and ketac n100 with fuji ii lc ( p>.05 ) ) were found statistically significant ( p<.05 ) .
linear regression showed that there is no correlation between surface roughness and hardness of tested gics ( t = 1.141 , p = .264 )
in the present study , the values of average ra for all tested gics before and after polishing were within range of 0.440.78 m and 0.480.98 m range , respectively .
bollen et al13 examined the relation between the ra values of titanium implants and bacterial adhesion and reported that the critical surface roughness ( ra ) for bacterial colonization is 0.2 m .
surface roughness higher than 0.2 m is likely to increase significantly bacterial adhesion , dental plaque maturation , and acidity , which act on material surfaces , thus increasing caries risk . in this study , all gics presented surface roughness below this value both before and after polishing .
however , other studies reported no appreciable difference in plaque accumulation between surfaces polished by different methods that resulted in ra values within 0.71.4 m range.14,15 similar to the results of the current study several authors have shown the smoothest surfaces of gics are obtained with the mylar strip.16,17 however , the correct anatomic contour of the restoration is rarely achieved by using only a mylar strip.18 for finishing and polishing procedures , several systems can be used such as fluted carbide burs , diamond burs , white abrasive stones , and aluminum - oxide discs . however ,
the literature showed that aluminum - oxide discs allow better surface characteristics for gics.16,19 therefore , in the present study , aluminum oxide discs were chosen for the polishing procedure of gics .
finishing and polishing of esthetic restorative materials always pose a difficulty because particles and matrix differ in hardness and thus can not be abraded uniformly.20,21 for a finishing system to be effective , the cutting particles must be harder than the filler materials .
otherwise , the polishing agent will only remove the matrix and leave the particles protruding from the surface.22 in the present study , nanofiller gic ketac n100 showed smoother surfaces than the other tested gics both before and after polishing ( p<.05 ) .
some in vitro studies have also demonstrated that the addition of nanofillers provides enhanced surface wear and polish relative to some other commercially available dental materials.23 the silver - reinforced gic and argion molar had average roughness values ( ra ) of 0.64 m and 0.75 m , respectively .
these values were near to the average roughness value ( ra ) of resin - modified gic , fuji ii lc , nanofiller gic , and ketac n100 .
the silver reinforcing can improve the wear resistance as well as the brittleness of gics .
however , the reinforcing effects of metal additives to glass ionomers have been the subject of many controversies .
although some investigators found significant differences between the strengths of conventional and reinforced materials,27,28 , others found no difference.2426 the absence of interfacial bonding , which is critical for efficient transfer of stress from the matrix to the reinforcement , may explain why metal - reinforced materials have not proved to be stronger or more durable than their metal - free counterparts.29 metal - reinforced gics have been superseded by highly viscous conventional gics like fuji ix .
highly viscous conventional gics has enhanced mechanical properties by their improved chemistry and also they are tooth colored .
preliminary studies have shown that highly viscous gics have either comparable or superior mechanical properties and wear resistance to metal - reinforced cements.30 particle size has been shown to play an important role in how well a material polishes .
some studies have been recorded the highest values of surface roughness for the materials with larger particle sizes.12,22 on the other hand , for the gics , gladys et al12 noted that a material with small particle sizes ( photac fil , mean particle size 5.56 m ) was 10 times rougher after being polished than ionosit fil ( mean particle size 9.3 m ) , which has larger particles .
others have shown that smaller glass particles are abraded rather than larger particles.31 in the present study , after polishing , the ionofil molar material with small particle sizes ( mean particle size 5.0 m ) was rougher than the others ( fuji ii lc , 5.9 m ) and argion molar ( 8 m ) with the larger particle sizes ( table 1 ) .
adversely , ionofil molar with a small particle size showed smoother surfaces than the fuji ix with an average particle size of 10 m and aqua ionofil plus with an average particle size of 8.0 m .
hence , other parameters such as differences in shape , distribution , and number of particles ; interfacial bonding between particles ; and interfacial bonding between particles and matrix may contribute to a material s surface finish.12 also , the storage media of gic specimens can affect their surface roughness . in the present study ,
the prepared gic specimens were stored in distilled water at 37 c for 24 h to mimic clinic conditions .
the chemical dissolution process can produce an increase in surface roughness.32 however ; one study evaluated the effects of storage media upon the surface micromorphology of resin - based materials and revealed no statistically significant difference in surface roughness between specimens exposed to distilled deionized water or artificial saliva.33 gics liquid component might influence their surface roughness as well .
the conventional gic is usually supplied as a powder and polyacid liquid . when the polyacid is present in solution , an increase in the viscosity of the liquid occurs , making the cement paste progressively more difficult to manipulate .
hence , nowadays manufacturers are using the polyacrylic acid in solid form for blending with glass ionomer powder rather than using poly - acid liquid .
water - mixed gics have a low viscosity in the early mixing stages plus improved shelf life because there is no possibility of gelation occurring in its liquid.34 gics surface hardness may be affected by the polishing process because the later involves polishing of the glass particles and the abrasion of the matrix or depleted glass layer . in the present study
, the highest hardness values were observed in conventional gics , except in the highly viscous conventional gic , fuji ix .
there are some studies supporting that surface hardness of conventional gics is higher than the surface hardness of resin - modified gics.31,35 it has been claimed that the inclusion of resins in the resin - modified gic does not improve the surface microhardness of these materials.36 the findings of the present study suggest that insertion of resin particles as in fuji ii lc or nanoparticles as in nanofiller gic ketac n100 did not enhance the surface hardness of these materials . also , in the present study , there was no linear regression found between surface roughness and hardness , so it can be concluded that the surface roughness of gics might be affected by other factors like the differences in the composition of tested gics rather than their surface hardnesses .
the sem photomicrographs obtained in this study showed that all conventional gics presented voids and cracks on their surface . despite the differences in mean values of the roughness , sem examination revealed a similar morphological surface pattern for each material .
the cracks could be produced during specimen processing for sem analysis , while the voids were probably included during mixing of gic powder and liquid .
within the limits defined in the present study , the following conclusions can be drawn :
different surface roughness and hardness values were obtained from gics , and this finding may be due to their different compositions.the smoothest surfaces for all gics were obtained when cured in contact with a mylar strip.while the smoothest surfaces were obtained for nanofiller gic ketac n100 , the roughest surfaces were obtained especially from conventional gics.the silver reinforced gic had the highest microhardness value.there was no correlation between surface roughness and hardness of gics .
different surface roughness and hardness values were obtained from gics , and this finding may be due to their different compositions .
the smoothest surfaces for all gics were obtained when cured in contact with a mylar strip .
while the smoothest surfaces were obtained for nanofiller gic ketac n100 , the roughest surfaces were obtained especially from conventional gics . | objectives : the aim of this study was to evaluate surface roughness and hardness of a nanofiller gic , a resin - modified gic , three conventional gics , and a silver - reinforced gic.methods:for each material , 11 spcecimens were prepared and then stored in distilled water at 37 c for 24 h. the surface roughness of 5 specimens was measured using a surface profilometer before polishing and after polishing with coarse , medium , fine , superfine aluminum oxide abrasive sof - lex discs respectively . the hardness of the upper surfaces of the remaining 6 specimens was measured with a vickers microhardness measuring instrument.results:all tested gics showed lower surface roughness values after the polishing procedure .
surface finish of nanofiller gic was smoother than the other tested gics after polishing .
this was followed by resin - modified gic , fuji ii lc ; then silver - reinforced gic , argion molar , conventional gics , aqua ionofil plus , fuji ix , and ionofil molar , respectively .
the result of the hardness test indicated that the microhardness value of silver - reinforced gic was greater than that of the other gics .
when the hardness values of all tested gics were compared , the differences between materials ( except aqua ionofil plus with ionofil molar and ketac n100 with fuji ii lc ( p>.05 ) ) were found statistically significant ( p<.05).conclusions : according to the results of this study , it can be concluded that the differences in the composition of gics may affect their surface roughness and hardness . |
skin tumors present in a variety of clinical forms as a result of which they enter into the differential diagnoses of a number of conditions presenting as swellings in the skin and subcutaneous tissue .
the cutaneous adnexal neoplasms , in themselves , constitute a wide spectrum because they can differentiate along one or more of the adnexal lineages . among the adnexal tumors ,
the mixed tumor of the skin , also known by the name chondroid syringoma , is an important adnexal neoplasm that shows evidence of both epithelial and mesenchymal differentiation .
although around 400 cases of chondroid syringomas have been reported worldwide , only eight cases of hyaline cell rich chondroid syringoma have been reported till 2002 .
we report here a case of hyaline cell - rich chondroid syringoma which was also unusual in its location .
a 45-year - old female presented with a slow - growing , painless swelling in the tip of her left index finger since 1 year .
this swelling was a single , firm , well - circumscribed mass measuring around 2.5 cm in diameter .
a clinical diagnosis of implantation dermoid was made and an excision biopsy was performed and the specimen sent for histopathological evaluation . grossly , the tumor was a globular , well - circumscribed , firm , gray - white mass of 2.5 cm diameter , with a cut surface that was glistening white and punctuated by tiny cystic spaces .
histopathological examination revealed a well - circumscribed proliferation of epithelial cells interspersed with foci of fibrous , myxoid , and chondroid stroma [ figures 1a and 1b ] .
most of these cells , which were arranged in broad sheets and nests , had a plasmacytoid appearance with an ovoid eccentric nucleus having fine granular chromatin and a deeply eosinophilic hyaline - like cytoplasm [ figure 2 ] .
interconnecting tubuloalveolar structures lined by two layers of cuboidal epithelial cells and ductal structures lined by one- to two - layered epithelial cells were also evident [ figure 3 ] .
the stroma showed intense positivity with periodic acid - schiff ( pas ) and alcian blue stain and metachromatic staining with toluidine blue .
this constellation of findings led to the diagnosis of hyaline cell rich chondroid syringoma .
( a)well - circumscribed proliferation of epithelial cells interspersed with foci of fibrous and myxoid stroma ( h and e ; 100 ) b : proliferation of epithelial cells interspersed with foci of fibrous and myxoid stroma ( h and e ; 400 ) broad sheets of plasmacytoid
hyaline cells ( h and e ; 400 ) tubuloalveolar structures lined by bi layered epithelium ( h and e ; 400 )
chondroid syringoma or cutaneous mixed tumor is a rare adnexal neoplasm with a very low incidence of 0.01%0.098% .
morphologically it is considered to be the cutaneous counterpart of the pleomorphic adenoma of salivary glands , but differs from it in that it rarely recurs .
typically , the tumor is seen in the head and neck region as a slow - growing , painless , subcutaneous nodule . in the present case ,
the lesion was found at the tip of the index finger an unusual location .
this lesion always occurs as a solitary nodule though , rarely , multiple tumors have been documented .
clinically , this tumor can be mistaken for sebaceous cyst , dermoid , neurofibroma , calcifying epithelioma , solitary trichoepithelioma , etc .
microscopy of a classical chondroid syringoma shows a well - circumscribed tumor that is located in the dermis and subcutaneous tissue with no connection to the overlying epidermis .
there is epithelial cell proliferation admixed with a stroma that is fibrous , myxoid , and chondroid .
the epithelial cells are arranged in nests , islands , or ductal and tubular structures .
hirsch and helwig proposed a set of criteria to be fulfilled for the diagnosis of chondroid syringoma ; these are : 1 ) nests of cuboidal or polygonal cells ; 2 ) intercommunicating tubuloalveolar structures lined by two or more rows of cuboidal cells ; 3 ) ductal structures composed of one or two rows of cuboidal cells ;
a classical chondroid syringoma can satisfy most of these criteria although in some only the epithelial and stromal components predominate . in the present case
the apocrine type or type i chondroid syringoma is the most common type and in this form a tubulocystic pattern of the epithelium is noted .
the eccrine type or the type ii chondroid syringoma shows uniform small round tubules lined by a single layer of cuboidal epithelial cells .
a malignant variant of chondroid syringoma has also been described . in some cases the epithelial cells are predominantly plasmacytoid in nature and such lesions
are termed as hyaline cell rich chondroid syringoma or hyaline cell rich apocrine mixed tumor .
it is important to recognize this entity as it can be morphologically confused for a malignant melanoma or an extra - skeletal chondrosarcoma . in the present case although the plasmacytoid cells predominated , the additional findings of a tubulocystic pattern of the epithelium , along with the varied composition of the stroma , helped in arriving at the diagnosis of hyaline cell rich chondroid syringoma .
this diagnostic entity is very rare , with only nine cases having been reported all over the world to the best of our knowledge . although recurrences are uncommon , malignancy arising in a setting of chondroid syringoma is well known .
hence , histopathological examination is advisable in all such cases . although the lesion is rare , it has to be kept in mind when formulating the differential diagnoses of such nodules .
the histopathologist must also be aware of this entity , including the hyaline cell - rich variant , to avoid potential misdiagnosis . | a mixed tumor is a neoplasm that has microscopic features of both epithelial and mesenchymal differentiation .
such mixed tumors are known as pleomorphic adenomas in the salivary glands , and their cutaneous counterparts are called chondroid syringomas .
these tumors commonly occur in the head and neck region of middle - aged men .
hyaline cell rich chondroid syringoma is a rare benign variant of chondroid syringoma composed of cells with eosinophilic hyaline cytoplasm and plasmacytoid features , the origin of which remains elusive .
although very few cases have been reported in literature , it is important to be aware of this entity so as to avoid misdiagnosis on histopathological examination . in this report
we present a case of hyaline cell - rich chondroid syringoma occurring in the finger . |
globally the total incidence of envenoming is as high as 1,841,000 resulting in 94,000 deaths .
india has a high share of this estimated burden of annual envenoming of 81,000 and deaths 11,000 .
an indian study suggested a gross underestimation of snake bite - related deaths in india , which could be three times higher than that of who estimation . to address this important neglected tropical disease who had formulated guidelines for south - east asia . in this lieu
first aid by a mnemonic do it right ( r - reassurance , i - immobilization , gh - get to the hospital ,
we observed the presence of various limb adorning ornaments ( laos ) on victims who are under treatment and found that the possibility of complications increased with these bands like ornaments over the bitten extremity .
we studied 14 hospitalized cases of snake bite , 13 of which were married hindu women and one hindu male .
lao were found to be present on the bitten limbs of all the under treatment victims . on 13 out of 14 occasions
the snake was identified as common cobra ( naja naja ) and on single occasion [ figure 1f ] .
the snakes were positively identified by the victims and their relatives with minimum chance of error as these snakes are very common in this geographic region . on six occasions
in all these cases the affected limb had shown a variable degree of complications ranging from swelling , early necrotic changes to frank gangrene formation . in three cases ,
the lao were tightened due to swelling [ figure 1a , b , j and m ] .
these patients had invariably complained about pain at the point of constriction due to lao . in four cases patients were though initially reluctant but removed the lao after counselling [ figures 1c , e , h and i ] . in three cases ,
metal cutter was used to remove the lao [ figure 1d , g and j ] . in rest of the cases where the lao are loose , we persuaded and removed them without disturbing the tight tourniquet where present [ figure 1j l , n , o and p ] .
the tourniquets were removed after administration of an initial dose of asv and with resuscitative backup .
limb adorning ornaments on the bitten limb ( photograph by subhendu mallik ) thirteen of the fourteen patients were female , who were initially very reluctant to remove the lao , as they were all married hindus . in the hindu culture ,
red bangles and toe rings are symbols of the married status of women and are never removed completely .
these lao are worn for a lifetime and are retained during funerary rites ( including cremation ) , or until the wearer is widowed . the lone male victim [ figure 1l ] readily removed the lao without any reluctance .
use of various lao like bangles and other articles of cultural and religious significance like scared threads is common in women and men in india
. uses of lao are also observed in many populations and places across the globe .
the nonremoval of various lao in patients of snake bites could be due to :
strong reluctance of married female patients to remove these ornamentslack of knowledge to foresee the complication associated with thesethe absence of any mention for the removal of lao in the recommended first aid methods of who searo guidelines and national protocolslack of published evidence about deleterious effects of lao in peer - reviewed journals .
strong reluctance of married female patients to remove these ornaments lack of knowledge to foresee the complication associated with these the absence of any mention for the removal of lao in the recommended first aid methods of who searo guidelines and national protocols lack of published evidence about deleterious effects of lao in peer - reviewed journals .
the compliance of patients for removal of these lao will surely be better if counseled by a known family physician , who is often the first person of contact .
local swelling is a known early feature of most venomous bites and is severe in viperine envenoming with the exception of kraits ( bungarus sps . ) .
we observed that all such lao if not removed in time can be tightened due to swelling and the patient suffers from distressing pain at the site of tightened lao .
moreover , when swelling occurs it becomes difficult to remove the lao and at times may necessitate a metal cutter to cut the metallic ones .
importantly , if a patient with lao is brought with profuse swelling toward the distal part , then the ornaments might be acting as tourniquets . in such situations it should be handled like that of a tight arterial tourniquet and should not be disturbed until the patient is in the hospital , resuscitation facility is available , and antivenom treatment is started .
application of tight arterial tourniquet is highly condemned because of its harmful effects such as ischemia , necrosis , embolism , and increased risk of the sudden neurotoxic blockade on release .
these complications are also expected in the case of a tightened lao acting as a virtual tourniquet .
if early intervention is instituted , even as simple as removing lao , it may go a long way in preventing secondary and tertiary complications .
however , application of a tight tourniquet is still rampant in practice which needs a spread of awareness at community level regarding the deleterious effects of a tourniquet .
the recommended first aid methods of who searo guidelines ( 1999 , 2010 ) and indian protocol ( 2007 , 2009 ) are silent about the associated complication of lao and actions required thereof .
though in indian protocol ( 2009 ) there is a mention to remove the jewelry , it is under the heading of creating awareness among the community about do 's and don'ts .
we feel that it would have been befitting if it was mentioned in the first step of the recommended first aid methods of the protocol .
our observation on lao , its complications , and benefits of early removal could be a potential resource for those revising the who do it right approach any time in the future .
the removal of jewelries from the affected limbs is appropriately mentioned in some of the recommended first aid methods like guidelines for africa prepared by who .
however the real hazard and complications arising due to these ornaments in venomous snake bite is not emphasized with evidence .
from our observation of cases and review of literature , we suggest that the concept of removal of the lao should be included in the first step of recommended first aid methods of all protocols for a better patient outcome . the do it right
mnemonic for the first aid methods , adopted by india should be reframed with the addition of the concept of remove .
reassure only , should also mean for remove ; r = reassure and remove ( reassure the patient .
| limb adorning ornaments ( lao ) can exacerbate the local effects of envenoming in case of venomous snake bite .
cultural presuppositions do inhibit victims from removing symbolically / ritually important jewelry even under circumstances that might increase the dangers of envenoming - induced gangrene formation .
the recommendation to remove the lao is usually skipped in guidelines and if at all included the very real hazard is uncommonly documented .
we observed 14 cases of snake bite with lao on the bitten limb with minimal to severe local complications .
the possible reasons were discussed with recommendations of proper and timely methods of removal of these ornaments .
we suggest inclusion of this concept of ' remove in the recommended first aid methods of the guidelines / protocols . |
the majority of gabaergic inhibitory neurons are parvalbumin - positive basket cells , which form perisomatic synapses on excitatory neurons to exert powerful regulation of neuronal firing [ 2 , 3 ] . in visual cortex ,
the maturation of inhibition , especially perisomatic inhibition , is regulated by visual experience [ 4 , 5 ] . throughout development ,
basket interneurons extend their axonal branches and form synapses all around the soma of the postsynaptic pyramidal cell , which can be visualized as gad65-positive puncta rings [ 6 , 7 ] .
previous studies have shown that visual deprivation from birth retards the developmental maturation of inhibitory circuitry [ 4 , 810 ] .
for example , dark - rearing from birth prevents the normal developmental increase in the maximal inhibitory postsynaptic current ( max ipsc ) onto layer 2/3 neurons of rat visual cortex [ 4 , 8 ] , and decreases the responsiveness to uncaged gaba at perisomatic locations .
changes in inhibition are thought to underlie the increase in spontaneous action potential frequency and the degradation of receptive field properties of visual cortical neurons following dark - rearing .
interestingly , the effects of visual deprivation can be readily reversed by exposure to light [ 4 , 12 ] ( but see ) , and to some extent by overexpression of brain - derived neurotrophic factor ( bdnf ) .
for example , visual deprivation by intraocular tetrodotoxin ( ttx ) injection decreased the number of terminal boutons of parvalbumin - positive basket cells synapsing onto pyramidal neurons in layer 5/6 of mouse visual cortex .
while this result is consistent with a decrease in total inhibitory function by visual deprivation , there has been no direct demonstration of experience - dependent regulation of the total number of somatic inhibitory synapses , especially in layer 2/3 where the effect of dark - rearing is known to regulate inhibitory function [ 4 , 5 , 8 ] .
furthermore , previous studies examining gad65 positive terminal density in layer 2/3 have reported no real change with dark - rearing , as well as with monocular deprivation , in cats .
these results are inconsistent not only with the physiological findings , but also with reports of a reduction in gabaergic neurons following visual deprivation paradigms in rodents as well as primates [ 16 , 17 ] .
this may be due to differences in species or due to limitations in the previous 2 dimensional analysis of gad65 immunohistochemical staining .
to determine anatomical changes in somatic inhibition of layer 2/3 neurons by visual deprivation , we performed a 3 dimensional analysis of somatic inhibitory synapses visualized by gad65 immunohistochemistry . in order to quantify somatic inhibition specifically onto layer 2/3 pyramidal neurons , we used a line of transgenic mice expressing yellow fluorescence protein ( yfp ) in a subset of pyramidal neurons in the cortex . here
we report that the total number of gad65-positive puncta contacting a yfp expressing soma is reduced in dark - reared mice , and is increased back to normal levels by re - exposure to 3 days of light .
experiments were carried out using transgenic mice expressing yellow fluorescence protein ( yfp ) in a subset of layer 2/3 pyramidal neurons in visual cortex ( yfp-16j line , jackson laboratory ) .
normal - reared ( nr ) animals were exposed to 12-hour light/12-hour dark cycles beginning at birth , and sacrificed via transcardial perfusion at postnatal day 35 ( p35 ) . in order to ensure minimal light exposure ,
pregnant females were placed into the dark room a few days before giving birth to the dark - reared ( dr ) and dark - reared followed by light exposure ( le ) litters .
mice were anesthetized by halothane vapors supplied within a lightproof container and sacrificed via transcardial perfusion at p35 .
le animals were kept in the dark room until p35 , and then light exposed on a 12-hour dark/12-hour light schedule for 3 days before being perfused at p38 .
all animal procedures were carried out in accordance with the national institute of health guide for the care and use of laboratory animals , and approved by the university of maryland institutional animal care and use committee ( iacuc ) .
mice were deeply anesthetized with halothane and underwent transcardial perfusion with 4% paraformaldehyde solution in sodium phosphate buffer ( composition in mm : 30 mm nah2po4 , 120 mm na2hpo4 , ph 7.4 ) .
a total of four mice were sacrificed for each experimental group ( nr , dr , and le ) .
brains were kept shielded from light at 4c first in 4% paraformaldehyde solution ( ph 7.4 ) for at least overnight up to 2 months until used for experiment . the day before sectioning , the brains were placed in 30% sucrose ( in phosphate buffered saline [ pbs ] , composition in mm : 137 nacl , 2.7 kcl , 8 na2hpo4 , 2 kh2po4 , ph 7.4 ) at 4c to sink overnight .
visual cortex was sectioned into 20 m coronal slices using a freezing sliding microtome ( leica ) , and stored in cryoprotectant ( 20% sucrose , 30% ethylene glycol , 0.02% sodium azide , in sodium phosphate buffer , ph 7.4 ) at 20c away from light ( less than 2 months ) until antibody incubation .
at least 4 visual cortex sections from each mouse were used per incubation , and a set of experimental conditions ( nr , dr and le ) was run each time . in addition , a second set of sections from the same animals were run again on a different day to control for possible variability in staining across different incubations .
all incubations were done shielded from light to prevent possible bleaching of yfp . on the day of the experiment ,
4 sections from each animal were removed from cryoprotectant , washed 4 times ( 5 minutes each ) in pbs ( ph 7.4 ) , and frozen overnight at 80c in 30% sucrose ( in pbs , ph 7.4 ) . the following day
, brain sections were defrosted at room temperature and rinsed 4 times ( 5 minutes each ) in pbs ( ph 7.4 ) .
the sections were then incubated in 20c methanol for 10 minutes , and rinsed 4 more times ( 5 min each ) in pbs ( ph 7.4 ) .
the brain sections were then incubated in blocking solution ( 10% normal donkey serum [ nds ] , 4% bovine serum albumin [ bsa ] , and 1% triton in pbs , ph 7.4 ) for 1 hour at room temperature . after blocking
, the sections were incubated in gad65 antibody ( rabbit antiglutamate decarboxylase gad65 polyclonal antibody from chemicon ) diluted 1 : 500 in blocking solution for 7 days at 4c on a rotator .
the primary antibody specificity was confirmed by a detection of a single band corresponding to gad65 on an immunoblot ( data not shown ) .
after the 7-day primary antibody incubation , sections were rinsed in pbs ( ph 7.4 ) 4 times ( 5 min each ) .
brain sections were then incubated for 2 hours in secondary antibody ( alexa fluor 633 goat antirabbit igg from molecular probes - invitrogen was diluted 1 : 200 in pbs containing 1.5% nds ) .
the secondary antibody specificity was assessed and confirmed by no alexa 633 signal when incubating brain sections in only secondary antibody without prior primary antibody incubation ( data not shown ) .
sections were then mounted on superfrost vwr microslides , dried over night in the dark , and coverslipped using prolong gold antifade kit mounting solution ( molecular probes - invitrogen ) .
yfp expressing layer 2/3 pyramidal neurons within the visual cortex were identified and imaged using a zeiss lsm510 confocal microscope with a 100x oil emersion objective lens .
area of imaging was selected solely on the basis of the yfp channel , hence the experimenter was blind to the gad65 staining .
images were taken using a dual scan mode , one for yfp ( emission filter : bp505550 ) and the other for alexa633 ( emission filter : lp650 ) .
the confocal microscope settings for the alexa633 channel were held constant across all the sections used in this study , and was based on a pilot experiment run to optimize the imaging conditions for gad65 staining .
the confocal microscope settings for the yfp channel were adjusted for each cell to produce saturating signals within the soma with clear cut - off boundaries .
these adjustments were necessary to account for differences in yfp expression level across different cells and different animals . to obtain 3 dimensional ( 3d ) information ,
20 images were captured through the z - axis at 1 m steps to encompass the whole section thickness ( 20 m ) .
the acquired images were analyzed using the volocity image analysis software ( improvision ) , which generates a 3d image of the acquired stacks .
image stacks were acquired from at least 4 brain sections per animal per experimental group .
one to three intact yfp labeled neuronal soma were selected and cropped from each image stack for a 3d analysis .
both yfp and alexa633 signals were processed using the volocity software by setting a threshold to eliminate background .
only the gad65 puncta with at least 1 voxel overlap with the yfp channel were taken as synapses contacting the yfp neuron , and the others were removed using the volocity software .
number , volume , and fluorescence intensity of gad65 puncta contacting each yfp labeled pyramidal cell body were then quantified .
a total of 96 neuronal soma and associated gad65 staining were analyzed from the 3 experimental groups ( 32 cells per group ) each from 4 mice .
for 2 dimensional ( 2d ) analysis of images , a single section in the middle of the stack ( the 10th section from the top ) was used .
images with prominent blood vessels were excluded as they showed high fluorescence . using the volocity software , the whole field intensity of the gad65 signal and the number of gad65 puncta were measured .
gad65 puncta density was calculated by dividing the number of puncta measured with the area of the image field .
statistical comparison of each measurement was made using a one - way anova , and p < .05 were taken as indicating statistically significant difference across groups .
fisher 's plsd posthoc test was run to determine further which group(s ) differed from each other .
to determine the effect of visual experience on inhibitory synaptic connections onto pyramidal neurons in layer 2/3 of primary visual cortex , we used a line of transgenic mice that express yfp in a few of these neurons ( yfp16j line ) and immunohistochemically labeled inhibitory terminals using an antibody against gad65 . by isolating gad65 synaptic puncta contacting the soma of yfp expressing neurons
, we were able to measure gad65 puncta number per neuronal soma , the volume of each synaptic puncta , and the intensity of gad65 puncta across three different conditions : normal reared ( nr ) , dark reared from birth to 5 weeks of age ( dr ) , and dark reared from birth followed by 3 days of light exposure ( le ) .
one subject from each of the three groups ( nr , dr , and le ) was run in parallel , and immunohistochemistry was performed in duplicate using sections from each mouse to account for possible variability across antibody incubations .
two sections from each animal per run were imaged with confocal microscopy , and on average two intact yfp labeled neurons in layer 2/3 of each section were imaged .
therefore , we imaged a total of 96 yfp expressing neurons from a total of 12 mice .
representative examples of gad65 staining and yfp signal from a visual cortex layer 2/3 from each group is shown in figure 1 .
quantification of a total of 32 cells ( from 4 mice ) per group showed a significant decrease in gad65 puncta number in dark reared mice compared to normal reared and light exposed animals ( nr = 22.5 2.2 gad65 puncta / soma , dr = 13.6 1.5 gad65 puncta / soma , le = 19.5 1.5 gad65 puncta / soma , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 6.724 , p < .002 ; figure 2(a ) ) . during the course of our analysis
, we unexpectedly found changes in the average volume of yfp expressing layer 2/3 neuron soma by visual experience .
specifically , we found that the average soma volume in dark reared mice was significantly smaller than that of normal reared and dark reared exposed to light ( nr = 758.1 38.3 m , dr = 584.2 29.4 m , le = 824.5 47.8 m , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 9.880 , p < .002 ; figure 2(b ) ) .
differences in yfp expression levels could affect cell size measurements , because of the use of fluorescence intensity threshold for delineating the cell 's boundary .
however , we did not find any significant difference in average yfp intensity across the three groups ( in arbitrary units of yfp fluorescence intensity : nr = 3342 80 , dr = 3159 98 , le = 3394 66 ; n = 32 cells each ; anova : f(2 , 93 ) = 2.321 , p > .1 ) .
while there was a trend , visual manipulation did not significantly alter the cell surface area ( nr = 6774 2177 m , dr = 5255 2007 m , le = 7318 2831 m , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 0.205 , p > .8 ) .
the difference in gad65 puncta number between normal reared and dark reared was not solely due to alterations in cell size , because it was still evident when puncta number was normalized to cell volume .
however , the difference between dark reared and light exposure was no longer significant ( nr = 0.032 0.004 gad65 puncta/m , dr = 0.023 0.002 gad65 puncta/m , le = 0.024 0.002 gad65 puncta/m , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 3.519 , p < .03 ) .
next , we determined whether the different rearing conditions affect gad65 puncta size or intensity . to do this , gad65 puncta volume and intensity were compared across normal - reared , dark - reared , and light exposure conditions .
there was no significant difference across the three conditions ( average gad65 puncta volume : nr = 2.32 0.19 m , dr = 2.56 0.19 m , le = 2.92 0.2 m , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 2.122 , p > .1 ; average gad65 puncta intensity [ measured in arbitrary units of fluorescence intensity ] : nr = 2746 16 , dr = 2759 13 , le : 2776 7 , n = 32 cells from 4 mice each group ; one - way anova : f(2 , 93 ) = 1.424 , p > .2 ; figures 2(c ) , and 2(d ) .
these results collectively suggest that visual experience bidirectionally alters gad65 positive terminal number , but not the size or gad65 content of individual inhibitory terminals .
we found that depriving mice of vision for 5 weeks from birth by dark rearing decreases the number of gad65 positive terminals contacting the soma of visual cortex layer 2/3 pyramidal neurons .
the number of gad65 puncta returned to normal levels when dark - reared mice were exposed to light for 3 days .
unexpectedly , we also found that the size of neuronal soma of layer 2/3 neurons in dark - reared mice was significantly smaller than that of normal - reared counterparts and the light exposed group .
the decrease in gad65 puncta by dark rearing is not likely a consequence of smaller soma size in the dark - reared mice , because the difference remained even when gad65 puncta number was normalized to soma size .
interestingly , changes in gad65 puncta number occurred without changes in the size or the average intensity of gad65 puncta .
these results suggest that dark rearing from birth decreases inhibitory input onto layer 2/3 pyramidal neurons primarily by decreasing the number of inhibitory synapses , which could be readily reversed by only 3 days of light .
previous results strongly suggest that dark rearing from birth attenuates the development of inhibitory networks [ 4 , 911 ] , and that exposure to light can initiate the maturation process [ 4 , 12 ] .
most studies that employed gad65 staining methods so far have relied on 2 dimensional analyses of either the gad65 puncta number per given area or average gad65 staining intensity , both of which failed to detect changes with visual deprivation , or number of gad65 puncta rings [ 6 , 10 ] .
we believe this is likely due to differences in either the methodology or species , because when we performed 2 dimensional analysis of our images , we saw significant decreases in both the total gad65 intensity and the gad65 puncta density with dark - rearing ( figure 3 ) . to our knowledge ,
our study is the first to quantify the total number of inhibitory synapses that impinge onto a single pyramidal neuron soma in an intact mouse visual cortex .
our result showing on average about 22 gad65 positive puncta per soma of individual layer 2/3 pyramidal neuron in normal - reared mice , and about 13 gad65 positive puncta per soma in dark - reared mice are in agreement with previous measurements .
mower and guo ( 2001 ) reported approximately 30 gad65 positive puncta per 400 m area in layers 2/3 of normal - reared cat visual cortex .
when this information is used to calculate a theoretical number in 3d by assuming a spherical cell with a diameter of about 10 m , the result is a prediction of approximately 20 inhibitory synapses .
this is in close agreement with a recent 3d analysis of somatic gad65 puncta in organotypic slice cultures derived from mouse cortex , which measured on average about 21 .
( 2002 ) quantified the maximal inhibitory postsynaptic current ( max ipsc ) on individual pyramidal cells within layer 2/3 of the visual cortex of rats , and also the average miniature ipsc ( mipsc ) , which is produced by a single inhibitory terminal bouton onto these pyramidal cells . by dividing the max ipsc value by the value of average mipsc , the data from morales et al .
( 2002 ) suggest approximately 21 inhibitory connections onto pyramidal cells in nr animals , and about 10 inhibitory connections onto neurons in dr animals .
when these numbers are taken in context with a study exploring the number of inhibitory connections a single interneuron makes onto one pyramidal cell within layer 5/6 cells of mouse visual cortex , an interesting scenario is revealed .
this study showed that a single parvalbumin - positive interneuron makes on average 11 inhibitory connections onto the soma of a single pyramidal cell .
a similar number is reported for a basket cell making synapses onto a layer 2/3 neuron in area 18 of cat visual cortex . if each interneuron creates approximately 10 synaptic contacts , and there are a total of about 20 gad65 positive synapses on the soma of a pyramidal cell in normal - reared animals as our data indicate , this suggests that only 2 interneurons influence somatic integration in pyramidal cells .
whether layer 2/3 neurons also receive about 10 synapses from a single interneuron is unclear , but in any case this suggests that a single inhibitory neuron can have a profound effect on controlling the function of a pyramidal neuron .
interestingly , we show that the decrease in gad65 puncta number by dark rearing is rather rapidly reversed by only 3 days of light exposure .
this corresponds well with electrophysiological measures showing that the decrease in max ipsc by dark rearing is reversed by 2 days of light exposure .
despite changes in the number of somatic gad65 puncta , we did not find a significant change in gad65 puncta size and intensity with visual manipulations .
these results suggest that the major determinant of inhibitory strength is by regulation of the number of synaptic contacts .
unexpectedly , we also found a reduction in the soma size of layer 2/3 pyramidal cells in the dark - reared group , which was reversed by exposure to light for 3 days .
however , we did not find a significant change in the cell surface area by visual manipulation .
this suggests that the observed change in soma volume is likely due to cell shrinkage .
however , we can not make firm conclusions as the soma surface area measurements showed large variability within each group . in any case , to date there is only one other report of a reduction in cell size in visual cortex following visual deprivation .
( 1990 ) reported a reduction in cell size in cat visual cortex ( area 17 ) by dark - rearing using a monoclonal antibody that labels putative cell surface - associated proteoglycan on a subset of neurons .
however , proteoglycan comprising extracellular matrix is a known target for regulation by visual experience , hence whether this truly reflects cell size change is unclear .
the smaller soma size of layer 2/3 neurons in mice dark - reared from birth may be due to reduced availability of neurotrophins .
it is known that neurotrophins can regulate cell size , which seems to be specific for distinct neuronal types .
for example , nt-4 application is known to accelerate the developmental increase in soma size of lateral geniculate nucleus ( lgn ) neurons , and reverses monocular deprivation - induced cell shrinkage in the lgn . on the other hand ,
bdnf application increases the soma size of neurons in the visual cortex , including layer 2/3 pyramidal neurons .
dark - rearing is known to down - regulate bdnf - induced activation of trkb receptors , despite increased bdnf protein expression .
therefore , the smaller soma size in layer 2/3 visual cortex of dark - reared mice may reflect the reduction in bdnf signaling .
the reduction in trkb activation by dark - rearing is reported to reverse by 2 hours of light , hence could explain the recovery of soma size changes observed in our study . considering that bdnf
is implicated in the maturation of inhibitory synapses [ 6 , 8 , 26 ] , it may also be responsible for the changes in gad65 puncta number .
whether the soma size and/or the gad65 puncta number changes observed in our study are indeed due to bdnf regulation awaits further examination .
we demonstrate that visual deprivation alters the number of somatic inhibitory synapses on pyramidal neurons in the superficial layers of visual cortex , which can be reversed by a brief exposure to light .
therefore , our results underscore the importance of experience in sculpting inhibitory connectivity in the cortex . | proper functioning of the visual system depends on maturation of both excitatory and inhibitory synapses within the visual cortex .
considering that perisomatic inhibition is one of the key factors that control the critical period in visual cortex , it is pertinent to understand its regulation by visual experience . to do this , we developed an immunohistochemical method that allows three - dimensional ( 3d ) analysis of the glutamic acid decarboxylase ( gad ) 65-positive inhibitory terminals in the visual cortex . using this method on transgenic mice expressing yellow fluorescence protein ( yfp ) in a subset of neurons
, we found that the number of somatic gad65-puncta on individual layer 2/3 pyramidal neurons is reduced when mice are dark - reared from birth and reverted to normal levels by re - exposure to light .
there was no change in gad65-puncta volume or intensity .
these results support the reorganization of inhibitory circuitry within layer 2/3 of visual cortex in response to changes in visual experience . |
eighteen male new zealand white rabbits weighing 3.4 0.3 kg ( mean standard deviation [ sd ] ) were housed in individual cages in a temperature - controlled room ( 20c22c ) and on a 12-hour light
the animals were fed a diet consisting of regular feed with added 1% cholesterol ( w / w ) for 60 days . on day 30 , animals were separated into two groups .
nine animals were treated with four weekly intravenous injections of lde - etoposide ( 6 mg / kg and 30 mg of total lipids ) , and nine animals received four weekly intravenous saline solution in equivalent volume .
one week after the last dose of the treatment , the euthanasia was performed by a lethal intravenous dose of 80 mg / kg of sodium pentobarbital .
the amount of feed was restricted to 150 g. the food intake was assessed daily , and bodyweight was assessed weekly .
this protocol conforms to the guide for the care and use of laboratory animals ( us national institutes of health ) and was approved by the ethics committee of university of so paulo .
after overnight fasting , blood samples were taken for determination of total and high - density lipoprotein ( hdl ) cholesterol and triglycerides by commercial kits ( labtest , so paulo ) and for blood cell count .
lde was prepared according to the method described by maranho et al.19 a lipid mixture of 40 mg cholesteryl oleate , 20 mg egg phosphatidylcholine , 1 mg triolein , and 0.5 mg cholesterol was emulsified in aqueous medium by prolonged ultrasonic irradiation for 3 hours
. a two - step ultracentrifugation ( 195,000 g for 30 minutes followed by 195,000 g for 120 minutes in a th 641 [ thermo scientific , waltham , ma ] rotor at 4c ) of the crude emulsion with density adjustment by addition of solid kbr was done to obtain the lde nanoemulsion .
lde was dialyzed overnight against 2 l of 10 mm tris - hcl buffer ph 8.0 for kbr extraction , and sterilized by passing through a 0.22 m filter . etoposide ( 6 mg ) was associated to nanoemulsion ( 1 ml , 30 mg of total lipids ) by solubilization of etoposide in ethanol ( 10% v : v).20 etoposide was added to the emulsion .
the solution was sonicated for 1 hour at 70c using a sonifier 450 ( branson ultrasonics , danbury , ct ) , equipped with a 1 cm flat titanium probe .
the resultant mixture was centrifuged at 3,500 rpm for 15 minutes to separate free etoposide .
etoposide loaded nanoemulsion was then passed through 0.22 m pore filter and kept at 4c .
the amount of drug associated to the nanoemulsion was always measured by high - performance liquid chromatography before injection .
the yield of association was 85% and the particle diameter , measured by laser light scattering , was about 60 nm .
aorta was excised from the aortic arch to the abdominal artery , opened longitudinally , washed with saline , and placed in 10% buffered formalin .
after fixation , the aorta was stained in scarlet r , and pictures were taken to measure the lesions .
sections taken for each segment were stained in hematoxylin - eosin for total area measurement .
additional sections were stained with anti - lrp-1 ( calbiochem , darmstadt , germany ) , anti - ldl receptor ( lifespan , seattle , wa ) , anti - rabbit macrophage ( ram-11 clone ) ( dako , carpinteria , ca ) , anti - matrix metallopeptidase 9 ( mmp9 ) ( abcam , cambridge , ma ) , anti - tumor necrosis factor ( tnf)- ( r&d systems , minneapolis , mn ) , anti - interleukin ( il)-1 ( r&d systems ) , anti - tubulin ( abcam ) , anti - scavenger receptor class b member 1 ( sr - b1 ) ( millipore , billerica , ma ) , anti - proliferating cell nuclear antigen ( pcna ) ( abcam ) , anti - cluster of differentiation 36 ( cd36 ) ( genetex , irvine , ca ) , anti- actin ( dako ) , and anti - topoisomerase ii ( abcam ) .
all measurements were performed using leica qwin image analysis software ( leica microsystems , wetzlar , germany ) .
comparison between groups of lipid and hematological profiles and bodyweight was assessed by one - way analysis of variance with tukey post test .
all other analysis was assessed using the student s t - test . in all analysis , difference of two - tailed p below 0.05
eighteen male new zealand white rabbits weighing 3.4 0.3 kg ( mean standard deviation [ sd ] ) were housed in individual cages in a temperature - controlled room ( 20c22c ) and on a 12-hour light
the animals were fed a diet consisting of regular feed with added 1% cholesterol ( w / w ) for 60 days . on day 30 , animals were separated into two groups .
nine animals were treated with four weekly intravenous injections of lde - etoposide ( 6 mg / kg and 30 mg of total lipids ) , and nine animals received four weekly intravenous saline solution in equivalent volume .
one week after the last dose of the treatment , the euthanasia was performed by a lethal intravenous dose of 80 mg / kg of sodium pentobarbital .
the amount of feed was restricted to 150 g. the food intake was assessed daily , and bodyweight was assessed weekly .
this protocol conforms to the guide for the care and use of laboratory animals ( us national institutes of health ) and was approved by the ethics committee of university of so paulo .
after overnight fasting , blood samples were taken for determination of total and high - density lipoprotein ( hdl ) cholesterol and triglycerides by commercial kits ( labtest , so paulo ) and for blood cell count .
lde was prepared according to the method described by maranho et al.19 a lipid mixture of 40 mg cholesteryl oleate , 20 mg egg phosphatidylcholine , 1 mg triolein , and 0.5 mg cholesterol was emulsified in aqueous medium by prolonged ultrasonic irradiation for 3 hours . a two - step ultracentrifugation
( 195,000 g for 30 minutes followed by 195,000 g for 120 minutes in a th 641 [ thermo scientific , waltham , ma ] rotor at 4c ) of the crude emulsion with density adjustment by addition of solid kbr was done to obtain the lde nanoemulsion .
lde was dialyzed overnight against 2 l of 10 mm tris - hcl buffer ph 8.0 for kbr extraction , and sterilized by passing through a 0.22 m filter . etoposide ( 6 mg )
was associated to nanoemulsion ( 1 ml , 30 mg of total lipids ) by solubilization of etoposide in ethanol ( 10% v : v).20 etoposide was added to the emulsion .
the solution was sonicated for 1 hour at 70c using a sonifier 450 ( branson ultrasonics , danbury , ct ) , equipped with a 1 cm flat titanium probe .
the resultant mixture was centrifuged at 3,500 rpm for 15 minutes to separate free etoposide .
etoposide loaded nanoemulsion was then passed through 0.22 m pore filter and kept at 4c .
the amount of drug associated to the nanoemulsion was always measured by high - performance liquid chromatography before injection .
the yield of association was 85% and the particle diameter , measured by laser light scattering , was about 60 nm .
aorta was excised from the aortic arch to the abdominal artery , opened longitudinally , washed with saline , and placed in 10% buffered formalin . after fixation
, the aorta was stained in scarlet r , and pictures were taken to measure the lesions .
sections taken for each segment were stained in hematoxylin - eosin for total area measurement .
additional sections were stained with anti - lrp-1 ( calbiochem , darmstadt , germany ) , anti - ldl receptor ( lifespan , seattle , wa ) , anti - rabbit macrophage ( ram-11 clone ) ( dako , carpinteria , ca ) , anti - matrix metallopeptidase 9 ( mmp9 ) ( abcam , cambridge , ma ) , anti - tumor necrosis factor ( tnf)- ( r&d systems , minneapolis , mn ) , anti - interleukin ( il)-1 ( r&d systems ) , anti - tubulin ( abcam ) , anti - scavenger receptor class b member 1 ( sr - b1 ) ( millipore , billerica , ma ) , anti - proliferating cell nuclear antigen ( pcna ) ( abcam ) , anti - cluster of differentiation 36 ( cd36 ) ( genetex , irvine , ca ) , anti- actin ( dako ) , and anti - topoisomerase ii ( abcam ) .
all measurements were performed using leica qwin image analysis software ( leica microsystems , wetzlar , germany ) .
comparison between groups of lipid and hematological profiles and bodyweight was assessed by one - way analysis of variance with tukey post test .
all other analysis was assessed using the student s t - test . in all analysis , difference of two - tailed p below 0.05
as shown in table 1 , total cholesterol concentration in the cholesterol fed rabbits increased 20-fold in the group treated with lde - etoposide and tenfold in the control group from baseline to the end of the 8th week of the cholesterol feeding period .
after the 8-week period , triglyceride values were increased by the diet about tenfold in both groups .
it is worthwhile to point out that there were no differences in the plasma lipids when controls and lde - etoposide treated rabbits were compared at baseline and at 8 weeks .
compared with baseline , after the 8-week experimental period the food intake increased by 15% in both lde - etoposide and control groups ( figure 1 ) , but the bodyweight was unchanged .
the red blood cell count of the animals treated with lde - etoposide fell by 50% and was not significantly altered in the control group . on the other hand , leukocyte count did not diminish in both lde - etoposide and control groups as shown in table 2 .
figure 2 shows the aorta of a rabbit treated with lde - etoposide and that of a control rabbit . by comparing against the controls , lde - etoposide treatment reduced the lesions by 85% as analyzed by macroscopic morphometry , as shown in table 3 .
table 4 presents the results of the microscopic analysis of segments of the aortic arch of rabbits treated with lde - etoposide and those of controls .
the computer - assisted microscopic morphometric analysis showed that the lde - etoposide reduced the intima layer area by 75% .
figures 3 and 4 show the photomicrographs of stained intimal areas of aortic arch tissues stained for different antibodies . in table 5 , there are the percentages of stained areas of each one of the antibody labels calculated by a computer assisted morphometry technique .
lde - etoposide treatment reduced macrophage presence by 50% ( 67.73% 13.03% of stained area in controls and 26.96% 28.46% in lde - etoposide treated rabbits , p < 0.01 ) .
the percentage of stained area by vascular smooth muscle cells ( vsmcs ) was reduced by 60% ( 27.52% 10.63% in the control group and 7.89% 9.06% in the lde - etoposide group , p
lde - etoposide also reduced the expression of the ldl receptors by 60% ( 35.13% 20.13% in the control group and 11.28% 16.80% in the lde - etoposide group , p < 0.05 ) and of the lrp-1 receptor by 90% ( 41.94% 21.35% in the control group and 5.11% 9.92% in the lde - etoposide group , p < 0.001 ) . in respect to the expression of scavenger receptors , lde - etoposide reduced cd36 by 80% ( 40.13% 15.26% in the control group and 8.60% 13.54% in the lde - etoposide group , p < 0.001 ) and sr - b1 by 80% ( 19.86% 8.26% in the controls and 4.87% 8.65% in the lde - etoposide treated rabbits , p < 0.01 ) . regarding the expression of inflammatory cytokines ,
il-1 protein was reduced by 50% ( 55.41% 17.82% in the control group and 24.82% 22.69% in the lde - etoposide group , p
< 0.01 ) and tnf- by 75% ( 20.04% 15.77% in the control group and 5.57% 6.42% in the lde - etoposide group , p < 0.001 ) .
mmp9 protein expression was reduced by 50% ( 14.98% 8.59% in the control group and 6.44% 8.41% in the lde - etoposide group , p < 0.05 ) . as to the expression of proteins related with cell proliferation ,
tubulin was reduced by 80% ( 47.40% 15.38% in the control group and 16.01% 19.82% in the lde - etoposide group , p
< 0.01 ) and topoisomerase ii by 60% ( 42.46% 15.77% in the control group and 8.51% 10.35% of stained area in the lde - etoposide group , p < 0.001 ) ; pcna was equal in lde - etoposide treated and control animals .
as shown in table 1 , total cholesterol concentration in the cholesterol fed rabbits increased 20-fold in the group treated with lde - etoposide and tenfold in the control group from baseline to the end of the 8th week of the cholesterol feeding period .
after the 8-week period , triglyceride values were increased by the diet about tenfold in both groups .
it is worthwhile to point out that there were no differences in the plasma lipids when controls and lde - etoposide treated rabbits were compared at baseline and at 8 weeks .
compared with baseline , after the 8-week experimental period the food intake increased by 15% in both lde - etoposide and control groups ( figure 1 ) , but the bodyweight was unchanged .
the red blood cell count of the animals treated with lde - etoposide fell by 50% and was not significantly altered in the control group . on the other hand , leukocyte count did not diminish in both lde - etoposide and control groups as shown in table 2 .
figure 2 shows the aorta of a rabbit treated with lde - etoposide and that of a control rabbit . by comparing against the controls , lde - etoposide treatment reduced the lesions by 85% as analyzed by macroscopic morphometry , as shown in table 3 .
table 4 presents the results of the microscopic analysis of segments of the aortic arch of rabbits treated with lde - etoposide and those of controls .
the computer - assisted microscopic morphometric analysis showed that the lde - etoposide reduced the intima layer area by 75% .
figures 3 and 4 show the photomicrographs of stained intimal areas of aortic arch tissues stained for different antibodies . in table 5 , there are the percentages of stained areas of each one of the antibody labels calculated by a computer assisted morphometry technique .
lde - etoposide treatment reduced macrophage presence by 50% ( 67.73% 13.03% of stained area in controls and 26.96% 28.46% in lde - etoposide treated rabbits , p < 0.01 ) .
the percentage of stained area by vascular smooth muscle cells ( vsmcs ) was reduced by 60% ( 27.52% 10.63% in the control group and 7.89% 9.06% in the lde - etoposide group , p
lde - etoposide also reduced the expression of the ldl receptors by 60% ( 35.13% 20.13% in the control group and 11.28% 16.80% in the lde - etoposide group , p
< 0.05 ) and of the lrp-1 receptor by 90% ( 41.94% 21.35% in the control group and 5.11% 9.92% in the lde - etoposide group , p < 0.001 ) . in respect to the expression of scavenger receptors , lde - etoposide reduced cd36 by 80% ( 40.13% 15.26% in the control group and 8.60% 13.54% in the lde - etoposide group , p < 0.001 ) and
sr - b1 by 80% ( 19.86% 8.26% in the controls and 4.87% 8.65% in the lde - etoposide treated rabbits , p < 0.01 )
. regarding the expression of inflammatory cytokines , il-1 protein was reduced by 50% ( 55.41% 17.82% in the control group and 24.82% 22.69% in the lde - etoposide group , p
< 0.01 ) and tnf- by 75% ( 20.04% 15.77% in the control group and 5.57% 6.42% in the lde - etoposide group , p < 0.001 ) .
mmp9 protein expression was reduced by 50% ( 14.98% 8.59% in the control group and 6.44% 8.41% in the lde - etoposide group , p < 0.05 ) . as to the expression of proteins related with cell proliferation ,
tubulin was reduced by 80% ( 47.40% 15.38% in the control group and 16.01% 19.82% in the lde - etoposide group , p
< 0.01 ) and topoisomerase ii by 60% ( 42.46% 15.77% in the control group and 8.51% 10.35% of stained area in the lde - etoposide group , p < 0.001 ) ; pcna was equal in lde - etoposide treated and control animals .
in this study , it was shown that in aortas of cholesterol - fed rabbits , lde - etoposide at the 6 mg / kg bw weekly dose for 4 weeks was able to reduce by 85% the total lesion area as measured by macroscopic morphometry .
de la llera - moya et al21 reported that the treatment of cholesterol - fed rabbits with a conventional commercial etoposide preparation at a much larger dose ( 40 mg / kg bw / week dose administered for 8 weeks ) than that used in this present study resulted in a 50% lesion area reduction , which is a very marked difference in effectiveness of the treatments . differently from the protocol used in this present study ,
the etoposide treatment was introduced 1 week before the beginning of the cholesterol feeding , which leads to a preventive rather than curative treatment approach .
anyhow , the largely known toxicity spectrum of etoposide , which encompasses myelosuppression , gastrointestinal symptoms , hypotension , and allergic reactions prohibits the introduction of this drug in the vehicle used in the commercial formulation in the cardiology field.22 in this study , anemia was the only observed toxicity documented in the animals after the lde - etoposide treatment .
other toxicities under scrutiny here , such as weight loss , diminished food intake , leukopenia , and clinically observable alterations , were absent .
in fact , in the classical toxicological tests conducted in mice , the toxicity of etoposide was pronouncedly reduced by association with lde .
the novel formulation showed maximum tolerance dose and 50% lethal dose roughly five times that of the commercial etoposide formulation.12,20 moreover , the toxicity of lde - etoposide was also tested in a small safety study in two patients with refractory lymphoma . in the etoposide , 300 mg
/ m dose/3 weeks , administered over 6 cycles.17 this dose scheme corresponds to those routinely used in the clinical oncology practice . during the treatment , no clinical and laboratorial toxicities , including anemia or other myelotoxicities , were observed in both patients .
interestingly , anemia not accompanied by leucopenia was the only prominent toxicity found in atherosclerotic rabbits treated with commercial etoposide reported by de la llera - moya et al.21 apparently , this may be a species - related toxicity trait that , as we have shown in our previous studies , did not appear in our lde - etoposide treated patients.17 in patients with ovarian carcinoma , the plasma decay curve of etoposide associated with lde was markedly slower than that of the commercial formulation , as shown by azevedo et al.23 in that study , it was shown that etoposide oleate did not dissociate from lde while in the blood stream .
lde - etoposide pharmacokinetic parameters favor the novel formulation , since extension of half - life of etoposide achieved by the association to lde may be an interesting feature in favoring the pharmacological action in chronic diseases .
the antiatherosclerotic effects of lde - etoposide , at the order of 85% lesion area reduction , were apparently greater than those of lde - paclitaxel described in a previous study .
lde - paclitaxel achieved a 60% reduction of the lesion area.14 by performing statistical analysis with the data obtained in the two studies , this difference was found significant at p = 0.02 . similarly to the lde - paclitaxel experiment in a previous study by the same authors,14 lde - etoposide could not only inhibit the vsmc proliferation and invasion of the intima but also the massive macrophage presence in the intima .
this results in the reduction of the intima width that was statistically equal to that observed in the lde - paclitaxel treatments . in respect to the media , thickening of this arterial layer
is not involved in atherogenesis , so the finding that lde - etoposide treatment did not change media width was indeed expected .
the finding of overall reduction of the protein expression of the receptors tested here , namely ldl receptor , lrp-1 , cd36 , and sr - b1 , by lde - etoposide may simply mirror the decrease in the cellularity of the intima , rather than diminution of receptor number / cell .
apparently , the effect of treatment was greater on the expression of lrp-1 than on the expression of the other receptors , a finding that could suggest a selective effect on the receptor rather than diminution of cell number .
ldl receptors are typically found in vsmcs , whereas cd36 is characteristically a macrophage receptor .
24 the fact that the protein expression of both receptors diminished equally reflects the post - treatment diminution of both vsmc and macrophages that was also equal .
the evaluation of pro - inflammatory cytokines il-1 and tnf- showed that lde - etoposide treatment was effective in impairing the protein expression of both .
il-1 is primarily a macrophage - secreted cytokine , whereas tnf- is produced by diverse cell lines present in the inflammation site.25 impairment of cytokine production can be also related with the diminution of their secreting cells by treatment .
the secretion of inflammatory cytokines impairs the production of collagen and other structural proteins by vmsc and increases the expression of mmp9 and other metalloproteinases.2 therefore , the treatment with the novel etoposide formulation promotes an overall inhibition of pro - inflammatory proteins .
the protein expression of two of the three cell proliferation markers analyzed here was inhibited by lde - etoposide treatment , namely the structural protein tubulin that is overexpressed during mitosis26 and topoisomerase ii , that is overexpressed during dna synthesis.27 those results convey the strong effect of the treatment that consisted in the inhibition of the massive invasion of the intima by macrophages and vsmcs .
unexpectedly , the treatment did not inhibit pcna which is a classical marker of dna synthesis.28 it was worthwhile to point out , however , that the expression of this protein was low in the control group .
etoposide stabilizes the complex formed between dna and topoisomerase ii , which leads to accumulation of strand breaks in dna
. etoposide treatment prevents cells from entering mitosis , thus slowing down the progression of the cell cycle , with arrest in g2 phase.29 however , the effects of the drug in nonneoplastic proliferating tissues and inflammatory sites are largely unexplored .
the antiproliferative effect of etoposide may account for the decrease in atheroma formation in the treated rabbits by proliferation inhibition of smooth muscle cells , macrophages , and macrophage - derived foam cells.21 in conclusion , lde - etoposide was effective in reducing the rabbit atherosclerotic lesions by strongly diminishing the proliferation in the intima of vsmc and macrophages , decreasing the production of pro - inflammatory factors such as il-1 , tnf- , and mmp9 .
lipoprotein receptors and proliferation markers were also inhibited by the treatment . because the safety of lde - etoposide treatment has already been shown not only in animal experiments but also in a pilot clinical study , with excellent tolerability , this novel formulation of etoposide can offer a promising strategy for the treatment of atherosclerotic cardiovascular disease . | objectivescholesterol - rich nanoemulsions ( lde ) bind to low - density lipoprotein ( ldl ) receptors and after injection into the bloodstream concentrate in aortas of atherosclerotic rabbits .
association of paclitaxel with lde markedly reduces the lesions . in previous studies , treatment of refractory cancer patients with
etoposide associated with lde had been shown devoid of toxicity . in this study ,
the ability of etoposide to reduce lesions and inflammatory factors in atherosclerotic rabbits was investigated.methodseighteen new zealand rabbits were fed a 1% cholesterol diet for 60 days . starting from day 30 ,
nine animals were treated with four weekly intravenous injections of etoposide oleate ( 6 mg / kg ) associated with lde , and nine control animals were treated with saline solution injections.resultslde-etoposide reduced the lesion areas of cholesterol - fed animals by 85% and intima width by 50% and impaired macrophage and smooth muscle cell invasion of the intima .
treatment also markedly reduced the protein expression of lipoprotein receptors ( ldl receptor , ldl - related protein-1 , cluster of differentiation 36 , and scavenger receptor class b member 1 ) , inflammatory cytokines ( interleukin-1 and tumor necrosis factor- ) , matrix metallopeptidase-9 , and cell proliferation markers ( topoisomerase ii and tubulin).conclusionthe ability of lde - etoposide to strongly reduce the lesion area and the inflammatory process warrants the great therapeutic potential of this novel preparation to target the inflammatory - proliferative basic mechanisms of the disease . |
ovarian cancer ( oc ) is characterized by malignant transformation of ovarian epithelial , stromal , or germ line cells .
it affects approximately 1 in 70 women and has a poor prognosis ( < 50% , 5-year survival rate ) .
early - stage oc presents no obvious symptoms in the majority of patients , and no effective screening method is available at the current time . as a result ,
oc is most commonly diagnosed at stage iii or stage iv . in later stages ,
metastasis often occurs , which can involve the peritoneal surface and adjacent organs , lymph nodes , lungs , and liver , among other sites . for these patients ,
the outcome is poor , with an on average 20% long - term survival rate .
oc can develop as a result of genetic risk factors , such as mutations in brca1 and brca2 genes .
other nongenetic causes , such as chronic inflammation can result in oc development as well .
it has been shown that a higher number of pregnancies and oral contraceptive use correlates to decreased oc risk , suggesting that limited ovulation creates an environment that is less conducive to neoplasm development .
the most common tools used today in diagnostics include measurement of ca-125 levels in the serum , pelvic ultrasonography , and biopstic and histochemical analysis .
the downfall of the screening methodology is that not all ocs exhibit elevated ca-125 levels , and this method has proven to be rather unreliable in oc detection . by the time clear symptoms are present , and a sonogram , biopsy , and immunohistochemistry are performed , the oc is often in its later stages .
primary treatments of stage iii and iv oc include surgery , followed by several rounds of chemotherapy with radiation rarely used . typically , a full hysterectomy and bilateral salpingooophorectomy are performed .
chemotherapy for oc usually includes platinum- and taxane - based agents , with either three or six rounds of intravenous treatment administered postoperatively .
platinum - containing drugs , such as cisplatin act by binding to and cross linking dna and taxane - containing drugs , such as paclitaxel affect microtubular formation .
side effects often include pain , nausea , vomiting , alopecia , and neuropathy , among others .
these side effects typically arise as a result of healthy cells being affected by the treatment , resulting in various organismal imbalances .
in addition , resistance to the drugs can develop , most often as a result of the cells rapidly eliminating the agent . in order to improve oc survival rates ,
it is necessary to investigate new targets and therapies , which can be administered independently or as adjuvants to the traditional methods of surgery and chemotherapy .
innate immunity is the first response to an immunological challenge , and the onset of an innate immune response against pathogens and danger signals is very rapid .
macrophages , granulocytes , dendritic cells ( dcs ) , and natural killer ( nk ) cells are key immune cells that participate in innate immune responses . after contacting pathogens , these cells are able to eliminate them through several mechanisms , such as phagocytosis or generation of reactive oxygen or nitrogen species .
dcs and macrophages are usually called phagocytes due to their capability to engulf foreign material .
pathogens are detected by phagocytes through the expression of conserved pathogen - associated molecular patterns ( pamps ) present on the cell surface of the pathogen .
these molecules are detected by pattern recognition receptors ( prrs ) expressed on immune cells . through prr recognition ,
the phagocytes of the innate immune response are able to distinguish between self and foreign non - self cells .
some of the main prrs involved in the innate immune response are toll - like receptors ( tlrs ) and nod - like receptors ( nlrs ) [ 35 ] .
a microorganism displaying pamps which has become resident within body tissues can be recognized by macrophage prrs .
when this occurs , the phagocyte will internalize the microorganism by phagocytosis , become activated , and eliminate the microorganism . however , in addition to eliminating the microorganism , the activated macrophage will also begin to secrete proteins known as cytokines and chemokines .
cytokine and chemokine release can lead to increased vascular permeability and expression of cellular adhesion molecules , which can in turn increase neutrophil and monocyte recruitment and infiltration to the site of infection , thereby leading to an overall amplification of the inflammatory response [ 6 , 7 ] .
some examples of proinflammatory cytokines include interleukin ( il)-1 , il-6 , and and tumor necrosis factor - alpha ( tnf- ) [ 8 , 9 ] . through cytokine and chemokine release , additional immune cells can be recruited to the area of infection and cause the classical symptoms of inflammation : swelling , redness , heat , and pain .
infection , chronic irritation and inflammation are among the main causes for the initiation of different types of cancer .
indeed , inflammatory cells can contribute to the proliferation , survival , and migration of tumor cells and also play an important role in shaping the tumor microenvironment .
smoking for instance is associated with chronic inflammation of the lungs and with lung cancer , and alcohol abuse has been linked to inflammation and cancer of the liver and the pancreas .
the relationship between inflammation and cancer is complex , and inflammation can have either tumor - promoting or tumor - suppressive effects , depending on the type of inflammation .
thus , the field of tumor immunology is an important part of the ongoing efforts of improving cancer treatments .
tumors are more than cancer cells , being also composed of non - tumor cells ( leukocytes , endothelial cells , fibroblasts , and smooth muscle cells ) and the extracellular matrix . together with the tumor cells , they constitute the tumor microenvironment .
the cytokine profile of the tumor microenvironment is largely a result of factors produced by the tumor cells themselves , nearby cells , and infiltrating white blood cells and can have profound effects on tumor progression .
some cytokines can influence the tumor microenvironment in such a way that will suppress tumor development , while others can contribute to its growth and metastasis .
upregulation of il-12 , for example , will activate nk cells and cytotoxic t lymphocytes , resulting in cancer cell death .
in addition , induction of il-23 leads to the production of interferons ( ifns ) and other tumor - suppressive factors .
such molecules are activated as part of the antitumor immunity response and promote apoptosis of tumor cells . in the case of chronic inflammation ,
the cytokine profile at the tumor microenvironment is dramatically different and is characterized by an increase in immunosuppressive cytokines , such as tnf- and il-6 .
these cytokines can activate pathways that result in production of other inflammatory molecules , capable of recruiting leukocytes , such as macrophages and dcs to the tumor site .
these infiltrating leukocytes in turn can produce factors which aid in the promotion of angiogenesis and vascularization , subsequently contributing to tumor growth and metastasis .
angiogenesis is critical to cancer growth , as it allows for growing vasculature that will provide sufficient nutrients to the tumor to promote its growth .
chronic inflammation can create an environment in the tumor milieu that is conducive to the formation of new blood vessels and thereby facilitates tumor progression .
in addition to proangiogenic factors that arise from leukocyte infiltration , the tumor cells themselves produce soluble factors that potentiate angiogenesis and secrete proinflammatory cytokines , which can indirectly stimulate tumor growth .
as angiogenesis is a relevant process in cancer progression , pathways that trigger it are being investigated for potential drug targets for development of antiangiogenic therapeutics .
a major proinflammatory switch that can subsequently result in proangiogenic stimuli is nuclear factor - kappa b ( nf-b ) .
nf-b comprises a family of transcription factors that regulates the production of various cytokines , chemokines , and antiapoptotic and stress - response factors .
different nf-b proteins can bind to specific sites on the dna to influence transcription of a multitude of inflammatory response genes .
the transcription factors reside in the cytoplasm , in complex with inhibitors of nf-b ( ibs ) and are released , upon phosphorylation of the ibs by inhibitor of nf-b kinase ( ikk ) . at this point ,
the ibs become degraded and the transcription factors are able to translocate into the nucleus to regulate inflammatory responses .
the nf-b proteins regulate inflammation by binding to specific dna sites and upregulating or downregulating the amounts of histone remodeling proteins at the site .
some inflammatory genes which are upregulated by nf-b include a multitude of proinflammatory cytokines and chemokines , matrix metalloproteases , adhesion factors , cyclooxygenase 2 , and inducible nitric oxide synthase .
upregulation of these molecules results in the recruitment of immune cells and the increased production of proinflammatory molecules .
specifically , it can lead to promotion of angiogenesis and increased metastasis by producing chemokines , such as il-8 that promote leukocyte infiltration and inflammation and increased levels of mmps that promote tumor invasion of nearby tissue .
also , nf-b can upregulate factors like tnf- , which can result in inhibition of apoptosis and also stimulates cell proliferation by increasing transcription of molecules such as il-2 and granulocyte - macrophage colony - stimulating factor ( gm - csf ) .
often treatment results were poor , as nf-b proteins participate in many critical cell cycle functions , the broad - scale disruption of which can lead to a multitude of undesirable side effects .
it is thus necessary to investigate potential regulators of nf-b , which might be drug - target candidates for the development of tumor - targeted , antiangiogenic , and proapoptotic treatments .
tlrs are a family of integral membrane proteins which act as sensors of invading pathogens and primarily reside in immune cells , such as dcs and macrophages .
present either at the cell surface membrane or at the endosomal membrane , tlrs recognize specific pamps and initiate a signaling cascade to elicit an immune response .
as depicted in figure 1 , all tlrs have two domains : a leucine - rich domain that senses the pathogen and a toll - interleukin 1 receptor ( tir ) domain which interacts with an adapter molecule to initiate a signaling cascade to promote an immune response . over ten types of tlrs
different tlrs recognize different pamps , such as protein , nucleic acid , or lipid components of bacteria or viruses .
tlr4 for example , recognizes primarily bacterial components , such as lipopolysaccharide , while others , such as tlr3 recognizes dsrna . upon recognition of the foreign pathogens , tlrs signal through adapter molecules in a signaling cascade , which ultimately results in a change in the cytokine expression patterns of those cells ( figure 2 ) .
several adapter molecules have been characterized in the tlr family , and different tlrs signal through one of four adapters : myd-88 , myd-88 adaptor - like ( mal ) , tir - related adaptor protein inducing interferon ( trif ) , or trif - related adapter protein molecule ( tram ) .
it is becoming evident that tumor cells are capable of exploiting tlr signaling pathways to their advantage .
it has been shown that tlr ligands can promote tumor development by stimulating inflammation , particularly through nf-b activation [ 18 , 19 ] . between tumor cells and infiltrating immune cells that express tlrs , these prrs may be partially responsible for the constitutive activation of nf-b , which is often seen in cancers .
if ligands are present to activate tlrs in the tumor milieu , this can lead to an array of cytokines capable of promoting angiogenesis .
interestingly , tlr ligands have also been shown to possess tumor - suppressive effects via activation of pathways that result in promotion of innate and adaptive antitumor responses .
therefore , tlrs are significant to tumor progression and their effects on tumor development are being evaluated .
while tlrs are predominantly expressed in immune cells , in the last years it has been shown that they are also expressed in a variety of tumor cells where they are functional and can help shape the inflammatory profile of the tumor milieu .
thus , the therapeutic use of tlr agonists has been investigated in several cancer models in order to either target tumor cells or immune cells present in the tumor microenvironment .
the rationale being to either induce apoptosis of tumor cells or to activate resident immune cells that can help mount a robust antitumor response .
positive results have been obtained by using tlr agonists as adjuvants for dc vaccination in murine models of sarcoma ( tlr3/9 agonists ) , lung cancer ( tlr9 ) , and melanoma and brain cancer ( tlr7/8 agonist ) [ 22 , 23 ] .
it has also been shown that tlr agonists can enhance the efficacy of t - cell adoptive therapy by promoting a better interaction between t cells and resident activated dcs .
in addition , direct targeting of tlr9 in cancer cells triggered tumor cell death and an increase in survival in a xenograft model of neuroblastoma .
in particular , different tlr agonists were able to induce migration of human tumor colorectal , breast , lung , and glioblastoma cells , an indication of metastatic potential .
further , tlr signaling has been associated with increased human myeloma and lung cells proliferation and viability [ 27 , 28 ] .
these contradictory observations might be a result of the complex nature of the tumor microenvironment . as we have previously reviewed in detail
for example , high levels of il-4 , il-10 , transforming growth factor beta ( tgf- ) , and vascular endothelial growth factor ( vegf ) can be found in ovarian cancer ascites [ 30 , 31 ] .
il-4 , il-10 , and tgf- can affect phagocyte function , suppressing macrophage and dc activity .
indeed , dcs showing low levels of costimulatory molecules have been detected in tumors expressing high levels of vegf .
these dcs are able to render t cells anergic or tolerised , thus abrogating antitumor immune responses .
interestingly , besides an immune paralysis , we and others have shown that tumor - associated dcs , or leukocytes expressing dc markers , are able to produce angiogenic factors and can promote neovascularization in the tumor microenvironment [ 3436 ] .
our previous work unveiled a mechanism whereby immature dcs contribute to ovarian cancer progression by acquiring a proangiogenic phenotype in response to vegf [ 36 , 37 ] .
further , we have shown that specific depletion of tumor - associated dcs reduces ovarian cancer angiogenesis and growth .
interestingly , high amounts of cd4cd25 regulatory t cells have been described in ovarian carcinoma and ascites , with the capability of suppressing antitumor immune responses [ 39 , 40 ] .
in addition , as we have previously shown , tumor endothelium can contribute to the immune suppressive status of ovarian cancer microenvironment , impairing cytotoxic t - cell infiltration .
on the other hand , it has been shown that ovarian cancer is capable of inducing antitumor immune responses , and that cytotoxic t - cell infiltration in ovarian cancer correlates with a better prognosis .
the presence of several cellular leukocyte populations in the microenvironment of ovarian cancer argues for specific targeting of tumor microenvironment components when applying tlr agonist therapies for cancer .
for example , tlr agonists can be prepared for their delivery to particular cells within the tumor microenvironment .
this type of strategy was successfully used to activate tumor - associated dcs in ovarian cancer , promoting antitumor immune response in vivo [ 44 , 45 ] .
specifically for ovarian cancer , zhou and colleagues have shown show that tlr2 , tlr3 , tlr4 , and tlr5 are highly expressed on the normal ovarian epithelium , as well as on neoplastic ovarian epithelial cells .
further , it has been recently shown that tlr4 is also expressed in granulosa tumor cells in the ovary .
in addition , it has also been demonstrated that tlr9 expression is associated with poor differentiation in ovarian cancer specimens , and that its overexpression and stimulation enhances the migratory capacity of ovarian cancer cells .
importantly , tlr4 expression in ovarian cancer cells has been shown to exert protumor activities and to hamper the efficacy of paclitaxel therapy [ 49 , 50 ] .
this is caused by the ability of paclitaxel to interact with this tlr , activating the myd88 signaling pathway and inducing the generation of tumor cell survival and proliferation [ 4951 ] .
this pathway points to a mechanism by which infections can promote tumor progression by stimulating cancer cells to generate inflammatory cytokines . tlr4
interacts with products of bacterial infection , but signaling through other tlrs in cancer cells can generate similar responses . to investigate this , we decided to use the myd88 negative a2780 human ovarian cancer cell line [ 50 , 51 ] to stimulate it via tlr3 .
this tlr is a largely endosomal prr that recognizes dsrna , such as viral rna , and may also be able to recognize intrinsic dsrna , such as that which can arise from normal events like cell lysis , for example [ 17 , 52 ] . while tlr3 has no known endogenous ligands , it is suspected that various dsrna duplexes can activate it , and synthetic dsrna analogs which successfully bind to and stimulate tlr3 signaling have been developed .
one of these synthetic ligands is polyinosinic : polycytidylic acid ( poly [ i : c ] ) which has been used for many years to stimulate tlr3 . as poly(i : c ) has been the gold standard
ligand for tlr3 stimulation , in vitro studies have been carried out using this ligand to investigate the tlr3 signaling pathways in immune cells .
it has been demonstrated that poly ( i : c ) can directly cause apoptosis in tumor cells in a caspase - dependent manner .
interestingly , it was found that certain nf-b proteins must be active for this tlr3-mediated apoptosis .
poly ( i : c ) has indeed been tried as an adjuvant to chemotherapy , but it was found that it was too toxic to be used for therapy [ 16 , 52 ] . as shown in figure 3 , tlr3 signaling can activate nf-b in a myd88-independent way . upon stimulation with poly(i : c ) , tlr3 signals through adapter trif ( ticam-1 ) to initiate a signaling cascade that can activate transcription factors nf-b and interferon regulatory factor 3 ( irf3 ) , as well as the jnk and p38 pathways .
trif is the largest adaptor known of the tlr adapters and can initiate several distinct signaling cascades .
trif has an n - terminal region called the effector - driving site , which is able to recruit the tnf receptor - associated factor ( traf ) family proteins to result in the eventual activation of irf3 and nf-b .
irf3 activation can result in the upregulation of - and -type interferons ( ifns ) and activation of cytotoxic lymphocytes and nk cells , while nf-b can either upregulate certain cytokines and potentiate chronic inflammation and angiogenesis , or act to promote apoptosis , depending on the particular nf-b members that are activated .
trif also has a c - terminal binding site , which can recruit factors such as receptor interacting protein ( rip-1 ) and fas - associated death domain ( fadd ) , the signaling pathways of which result in apoptosis and autophagy through the activation of the jnk and p38 pathways .
thus tlr3 signaling can contribute to tumor eradication via upregulation of ifn- and ifn- , ctl , and nk cell activation , and by signaling through the rip-1/fadd pathway , whereas it can also indirectly contribute to tumor progression via activation of nf-b , which can result in proangiogenic factors and subsequent tumor progression .
as shown in figure 4 , 24 h treatment of human ovarian cancer a2780 cells with 10 g / ml of poly(i : c ) admixed with liposomes is able to induce upregulation of several cytokines and chemokines , il-6 among others , which has been shown to promote tumor cell growth and survival .
altogether these data indicate that the non - targeted use of tlr agonists for ovarian cancer therapy can generate adverse effects .
thus , targeted therapies to activate only particular components of the tumor microenvironment will be more suitable . in this context , pioneering research from dr .
conejo - garcia 's lab has shown in a murine model of ovarian carcinoma that specifically targeting tlrs in tumor - associated antigen - presenting cells can induce a robust antitumor immune response and tumor regression [ 44 , 45 , 54 , 55 ] .
in closing , it is necessary to further elucidate all the distinct signaling pathways of tlr in both tumor and immune cells in order to develop effective and safe immunotherapies using this target . on the one hand
, tlr stimulation can result in apoptosis , but on the other , it can lead in proangiogenic factors , which can stimulate tumor growth . effectively being able to select for and specifically turn on and off these pathways is the ultimate goal . as details concerning tlr signaling in tumor cells and immune cells
are clarified , it may be possible to design therapeutic agents , which target specific pathways in specific cells and create a proapoptotic , antiangiogenic tumor microenvironment , which is conducive to tumor eradication . | ovarian cancer ( oc ) is an aggressive disease that affects approximately 1 in 70 women and has a poor prognosis ( < 50% , 5-year survival rate ) , in part because it is often diagnosed at a late stage .
there are three main types of oc : neoplasms of surface epithelial , germ cell , or stromal origin , with surface epithelial tumors comprising about 80% of all ocs .
in addition to improving diagnostics , it is necessary to develop more effective treatments for epithelial - origin oc . here , we describe the paradoxical roles of toll - like receptor ( tlr ) signaling in the progression of cancer and discuss how its modulation may result in decreased tumor growth and metastasis via the attenuation of proangiogenic cytokines and potentiation of proapoptotic factors .
in particular , it has been found that tlr activity can behave like a double - edged sword
, as its signaling pathways have been implicated as having both tumor - suppressive and tumor - promoting effects . with particular emphasis on oc , we discuss the need to consider the signaling details of tlrs and associated proteins in the multiple cell types present in the tumor milieu to achieve safe and effective design of tlr - based cancer therapies . |
patients taking lithium usually require to take the drug on a long - term basis , which makes the issue of long - term toxicity an important one . among the various side effects recognized , one of the major side effects is the development of various types of thyroid abnormalities .
both the mechanism of its therapeutic action and the mechanisms of its various adverse effects are still incompletely understood .
lithium is concentrated by the thyroid at levels 3 - 4 times that in plasma and might exert a direct toxic effect on the thyroid gland . the most important clinically relevant action purposed is the inhibition of thyroid hormone release .
independent effects on the hypothalamic - pituitary - thyroid axis and the receptor mediated mechanism of thyroid hormone action may contribute .
the immunological influence of lithium on thyroid antibody concentrations leading to a more rapid onset of thyroid autoimmunity resulting in goiter and hypothyroidism and possibly also a state of hyperthyroidism in some cases has been hypothesized .
lithium - induced direct damage of thyroid cells with consequent release of thyroglobulin and thyroid hormones into the circulation has been purposed as possible mechanism of hyperthyroidism associated with long - term lithium use .
additional risk factors such as iodine deficiency , smoking , or goitrogens may leads to thyroid dysfunction . although clinical inspection and palpation of the thyroid gland are fundamental to any physical examination of lithium - treated patients , the assessment of findings varies widely depending on the investigator .
biochemical assessment includes measurement of serum concentrations of thyroid - stimulating hormone ( tsh ) , free triiodothyronine ( ft3 ) , free thyroxine ( ft4 ) , and thyroid peroxidase antibody and ultrasonographic scanning of thyroid has therefore been performed in lithium - treated populations and found to be a sensitive tool . unlike other imaging techniques , ultrasonography does not involve radiation exposure and is low in cost , fast , and relatively accurate . only few studies up to
now have used both ultrasonography measurement and biochemical assessment along with clinical palpation in age and gender matched lithium - treated patients and controls .
decades of clinical use of lithium and availability of new diagnostic tools for thyroid abnormalities have extended the interest to other aspects , including autoimmunity , hyperthyroidism , and morphological changes .
most of the earlier study have used clinical inspection , palpitation and measurement of thyroid hormone to assess the change in thyroid status in lithium - treated patients of mood disorders . only few studies up to now have used both ultrasonography measurement and biochemical assessment along with clinical palpation in age and gender matched lithium - treated patients and controls . unlike most of the previous studies where normal was taken as control , we include patients with mood disorder as a control because impairment of the hypothalamic pituitary thyroid axis stemming from the mood disorder may leads to changes observed in the thyroid state .
although cross - sectional in design , the main advantage of our study is that this is the first study using ultrasonography to determine thyroid volume along with clinical assessment and thyroid hormone measurement in age and gender matched indian population .
the primary aim of this study was to see the ultrasonographically measured changes in thyroid gland volume , biochemical parameters ( tsh , t3 and t4 ) and to compare the above findings with gender and age - matched lithium nave patients of mood disorder receiving other mood stabilizers such as sodium valproate , divalproex sodium , carbamazepine , oxcarbazepine , and lamotrigine .
this was a cross - sectional study of lithium - treated patients with mood disorders and of gender - and age matched lithium naive patients on other mood stabilizers .
subjects attending outpatient or in - patient departments of central institute of psychiatry ( cip ) were included in the study .
cip is an academic psychiatry center with tertiary care facility , has a total bed capacity for 673 patients with more than 55,500 patients attending the outpatient clinic every year , which includes males , females and children .
it kept detailed documentation of patient history , course of illness , and pharmacological treatment .
lithium - treated subjects of both sexes who fulfilled the following criteria were eligible for the study : age 18 - 60 years , a diagnosis of mood disorder according to diagnostic criteria of international classification of diseases-10-diagnostic criteria for research ( icd-10-dcr ) ( who , 1992 ) , continuous and documented maintenance treatment with lithium at adequate blood levels ( 0.6 - 1.2 mmol / l ) for one or more years .
control subjects were gender - and age - matched , who fulfilled the following criteria : age 18 - 60 years , age and gender matched patients with mood disorder according to icd-10 dcr , ( who , 1992 ) , who have been on mood stabilizer other than lithium including sodium valproate , carbamazepine , divalproex sodium , oxcarbazepine , and lamotrigine for 1 year or more , no prior use of lithium .
the study 's exclusion criteria for case group : were use of mood stabilizers other than lithium , the study 's exclusion criteria for control group , a history of use of lithium and the study 's exclusion criteria applied to both groups , any past or family history of thyroid disorder , a history of complete / subtotal thyroidectomy , a history of exposure to thyroid supplements or antithyroid drugs , iodide , sulfonamide , amiodarone interferons interleukins-2 , -blockers , or any special diets that might interfere with iodine metabolism .
all subjects also underwent a clinical examination and palpation of the neck and thyroid gland .
a semi - structured pro - forma was used for recording demographic details like age , sex , marital status , religion , education , occupation , socioeconomic status , habitat , and family history of thyroid disorder , as well as clinical data such as duration of illness total duration of treatment , continuous duration of treatment , age of onset of mood disorder , history of medical or psychiatric illness , family history of medical or psychiatric illness and premorbid personality .
it also included details of physical examination of all organ systems and mental status examination . finally , diagnosis of the patient according to icd-10 dcr , ( who , 1992 ) and the drugs taken were also recorded .
the ultrasound images were acquired with a conventional ultrasound scanner , envisor hd ( phillips ) ultrasound scanner .
ultrasonographic examination of the thyroid was performed using a linear 3 - 12 mhz transducer with the patient in a supine position and his or her neck hyper extended .
the volume of the thyroid lobes was determined using the ellipsoid model by measuring the maximum transversal ( t ) , horizontal ( h ) , and longitudinal ( l ) diameters .
the volume ( ml ) was calculated with the formula for ellipsoids as described by brunn et al .
: t ( cm ) h ( cm ) l ( cm ) 0.479 .
venous blood was drawn on the day of sonographic evaluation and analyzed for tsh , total thyroxine ( t4 ) , and total triiodothyronine ( t3 ) at the clinical laboratory of the cip , ranchi , india , with the commercially available syntron elisa test kit , bioresearch , inc . ,
normal reference value was taken as t3 = 72 - 184 g / dl , t4 = 5 - 13 g / dl , tsh = 0.5 - 4 -iu / l .
venous blood was drawn after 24 h of last lithium dose and serum lithium estimation was done at the clinical laboratory of the cip , ranchi , india .
ion selective electrode method was used , with the commercially available esylyte tranasia biomedicals , mumbai , india .
all analyses were performed with the computer software program , statistical package for social sciences , version 16.0 ( spss-16.0 ) for windows .
demographic and clinical differences between the groups were tested with mcnemar 's chi - square test to compare categorical variables .
the paired t - test was employed for continuous variables such as age , thyroid volume , and thyroid hormone levels .
descriptive statistics was used to calculate mean , percentage , and standard deviation of the sample .
the level of significance was taken as p < 0.05 ( two - tailed ) .
this was a cross - sectional study of lithium - treated patients with mood disorders and of gender - and age matched lithium naive patients on other mood stabilizers .
subjects attending outpatient or in - patient departments of central institute of psychiatry ( cip ) were included in the study .
cip is an academic psychiatry center with tertiary care facility , has a total bed capacity for 673 patients with more than 55,500 patients attending the outpatient clinic every year , which includes males , females and children .
it kept detailed documentation of patient history , course of illness , and pharmacological treatment .
lithium - treated subjects of both sexes who fulfilled the following criteria were eligible for the study : age 18 - 60 years , a diagnosis of mood disorder according to diagnostic criteria of international classification of diseases-10-diagnostic criteria for research ( icd-10-dcr ) ( who , 1992 ) , continuous and documented maintenance treatment with lithium at adequate blood levels ( 0.6 - 1.2 mmol / l ) for one or more years .
control subjects were gender - and age - matched , who fulfilled the following criteria : age 18 - 60 years , age and gender matched patients with mood disorder according to icd-10 dcr , ( who , 1992 ) , who have been on mood stabilizer other than lithium including sodium valproate , carbamazepine , divalproex sodium , oxcarbazepine , and lamotrigine for 1 year or more , no prior use of lithium .
the study 's exclusion criteria for case group : were use of mood stabilizers other than lithium , the study 's exclusion criteria for control group , a history of use of lithium and the study 's exclusion criteria applied to both groups , any past or family history of thyroid disorder , a history of complete / subtotal thyroidectomy , a history of exposure to thyroid supplements or antithyroid drugs , iodide , sulfonamide , amiodarone interferons interleukins-2 , -blockers , or any special diets that might interfere with iodine metabolism .
all subjects also underwent a clinical examination and palpation of the neck and thyroid gland .
a semi - structured pro - forma was used for recording demographic details like age , sex , marital status , religion , education , occupation , socioeconomic status , habitat , and family history of thyroid disorder , as well as clinical data such as duration of illness total duration of treatment , continuous duration of treatment , age of onset of mood disorder , history of medical or psychiatric illness , family history of medical or psychiatric illness and premorbid personality .
it also included details of physical examination of all organ systems and mental status examination . finally , diagnosis of the patient according to icd-10 dcr , ( who , 1992 ) and the drugs taken were also recorded .
the ultrasound images were acquired with a conventional ultrasound scanner , envisor hd ( phillips ) ultrasound scanner .
ultrasonographic examination of the thyroid was performed using a linear 3 - 12 mhz transducer with the patient in a supine position and his or her neck hyper extended .
the volume of the thyroid lobes was determined using the ellipsoid model by measuring the maximum transversal ( t ) , horizontal ( h ) , and longitudinal ( l ) diameters .
the volume ( ml ) was calculated with the formula for ellipsoids as described by brunn et al .
: t ( cm ) h ( cm ) l ( cm ) 0.479 .
venous blood was drawn on the day of sonographic evaluation and analyzed for tsh , total thyroxine ( t4 ) , and total triiodothyronine ( t3 ) at the clinical laboratory of the cip , ranchi , india , with the commercially available syntron elisa test kit , bioresearch , inc . ,
normal reference value was taken as t3 = 72 - 184 g / dl , t4 = 5 - 13 g / dl , tsh = 0.5 - 4 -iu / l .
venous blood was drawn after 24 h of last lithium dose and serum lithium estimation was done at the clinical laboratory of the cip , ranchi , india .
ion selective electrode method was used , with the commercially available esylyte tranasia biomedicals , mumbai , india .
all analyses were performed with the computer software program , statistical package for social sciences , version 16.0 ( spss-16.0 ) for windows .
demographic and clinical differences between the groups were tested with mcnemar 's chi - square test to compare categorical variables .
the paired t - test was employed for continuous variables such as age , thyroid volume , and thyroid hormone levels .
descriptive statistics was used to calculate mean , percentage , and standard deviation of the sample .
the level of significance was taken as p < 0.05 ( two - tailed ) .
in our study , 60 patients ( 30 cases , 30 controls ) with the diagnosis of mood disorder fulfilling the inclusion and exclusion criteria were selected .
the patients who were receiving lithium treatment were similar to the control group patients who were receiving other mood stabilizers with respect to age , education , marital status , religion , occupation , and habitat .
the mean age of patients was 29.40 6.81 years in the lithium group and 28.56 7.40 years in the other mood stabilizers group .
both the groups had 25 ( 83.3% ) males and 5 ( 16.7% ) females [ table 1 ] .
more than 50% of the patients were from lower socioeconomic status in both the group because the catchment area of our institute is economically underdeveloped .
thyroid was palpable only in 5 ( 16.6% ) patients in lithium group and not palpable in any of the patient of other mood stabilizer group .
both the groups had similar duration of illness , age of onset of mood disorder , continuous duration of treatment , and education in years .
the mean age of onset was 23.58 5.82 years for the lithium group and 21.01 5.27 years for the other mood stabilizer group .
the total duration of treatment was 4.33 2.94 years for the lithium group and 3.53 2.38 years for the other mood stabilizers group [ table 2 ] .
no statistically significant difference could be observed in any of the sociodemographic or clinical parameters between two groups .
there was no significant group difference in terms of past and family history of any psychiatric or any major medical illness , treatment history , and premorbid personality .
comparison of sociodemographic profiles between patients on lithium and on other mood stabilizers comparison of clinical profiles between patients on lithium and on other mood stabilizers significantly higher right , left , and total thyroid volume were observed in lithium group subjects as compared to subjects taking other mood stabilizers ( all p < 0.001 ) [ table 3 ] .
significantly increased mean t3 ( microgram / dl ) value was observed in the lithium group ( 151.12 4.40 ) as compared to the other mood stabilizers group ( 123.33 5.18 ) ( p = 0.029 ) [ table 3 ] .
similar trend toward higher mean value of t4 ( microgram / dl ) ( 10.87 4.30 ) was noted in the lithium group as compared to the other mood stabilizers group ( 9.10 2.74 ) ; although , it was not statistically significant . a slight lower mean value of tsh ( micro - iu / ml ) was observed in the lithium group ( 3.22 2.06 ) as compared to other mood stabilizers group [ table 3 ] . however , the difference between groups was not statistically significant .
our finding suggests trends toward hyperthyroidism in lithium - treated patients as compared to lithium nave patients receiving other mood stabilizers .
comparison of thyroid profile between mood disorder subjects on lithium and on other mood stabilizers in the lithium group , the age of patients in years was found to positively correlate with left lobe volume ( r = 0.391 ) and total volume ( r = 0.376 ) .
similarly , the age of onset of mood disorder was found to correlate positively with right lobe volume ( r = 0.392 ) , left lobe volume ( r = 0.394 ) , and total thyroid volume ( r = 0.407 ) [ table 4 ] .
correlation of sociodemographic profile with ultrasonographically measured thyroid volumes and biochemical thyroid parameters ( t3 , t4 , tsh ) in lithium group patients ( n = 30 ) significantly negative correlation was found between age of patients in years and t4 ( r = 0.379 ) duration of illness and t3 ( r = 0.376 ) , t4 ( r = 0.380 ) [ table 4 ] .
there was no significant correlation between gender , duration of lithium treatment , age of onset of mood disorder , serum lithium level , and serum level of t3 , t4 , and tsh [ table 4 ] .
no significant correlation was found between ages , gender , duration of illness , age of onset , duration of treatment , and with any of the thyroid parameters in control group [ table 5 ] .
correlation of sociodemographic profile with ultrasonographically measured thyroid volumes and biochemical thyroid parameters ( t3 , t4 , tsh ) in other mood stabilizers group patients ( n = 30 )
although cross - sectional in design , this study has the advantages as it included gender and age matched control belonging to same geographical area , so to eliminate the chances of normal variation in the prevalence of thyroid disorder primarily due to regional differences in iodine availability . to best of our knowledge , in most of the previous studies healthy
volunteer has been used as a control , this study includes patients with mood disorder as a control , to eliminate the chances of variation in thyroid status due to impairment of the hypothalamic pituitary thyroid axis stemming from the mood disorder itself .
a single ratter using same laboratory and same laboratory technique , ensured uniformity in biochemical assessment of thyroid status . for assessment of thyroid status both ultrasonography and laboratory measurement along with clinical inspection and palpation of thyroid gland was done .
to best of our knowledge , only four studies up to now have used ultrasonography both in lithium patients and controls . although clinical inspection and palpation of the thyroid gland are fundamental to any physical examination of lithium - treated patients , the assessment of findings varies widely depending on the investigator .
ultrasonographic measurement of thyroid size has therefore been performed in lithium - treated populations and found to be a sensitive tool . unlike other imaging techniques
, ultrasonography does not involve radiation exposure and is low in cost , fast , and relatively accurate .
ultrasonographically measured thyroid volume was found significantly increased in lithium - treated patients , as reported in previous studies , increased volume was found more common in patients treated with lithium for 1 - 5 years ( 44% ) or more than 10 years ( 50% ) than in patients who never received lithium ( 16% ) .
the volumes of the right and the left thyroid lobes were significantly higher in the lithium - treated than among normal controls .
several mechanisms have been hypothesized to explain the development of increase thyroid volume , like lithium is concentrated by the thyroid and inhibits thyroidal iodine uptake .
various studies have used the laboratory assessment of serum t3 , t4 , and tsh to study the effects of lithium on thyroid gland . in our study , significantly increased mean t3 value was observed in the lithium group as compared to the other mood stabilizers group .
similar trend toward higher mean value of t4 was noted in the lithium group as compared to the other mood stabilizers group ; although , it was not statistically significant . a slight lower mean value of tsh was also observed in the lithium group as compared to other mood stabilizers group .
our finding suggests trends toward hyperthyroidism in lithium - treated patients as compared to lithium nave patients receiving other mood stabilizers .
whereas , hypothyroidism is described as commonly known effect of lithium use hyperthyroidism associated with lithium use has also been described .
a large retrospective review demonstrated that lithium - associated silent thyroiditis and lithium - associated hyperthyroidism had a much higher incidence ( 1.3 and 2.7 cases per 1000 person - years , respectively ) than that seen in the general population ( 0.03 - 0.28 and 0.8 - 1.2 , respectively ) . increased t3 and t4 along with decreased tsh found in our study could be possibly due to increased concentration of lithium in thyroid gland and direct damage of thyroid cells with compensatory hypertrophy leading to increase thyroid volume and release of thyroid hormones into the circulation
as in our study starting lithium at a later age has been reported as the main risk factors for increased thyroid volume in lithium - treated patients .
however , we have found no significant correlation between gender , duration of illness , duration of lithium treatment , habitat , serum lithium level , palpable thyroid , and thyroid volume in both groups .
however perrild et al . reported no gender difference with regard to thyroid volume , in our study we could not find any correlation between female gender and total thyroid volume ( p = 0.280 ) [ table 4 ] , this could be due to small sample size of female patients . unlike our study
significant positive correlation was found between duration of lithium treatment , serum lithium level and thyroid volume .
duration of lithium treatment and thyroid volume was not found to be significantly correlated also . in our study , significantly negative correlation was found between age of patients in years and t4 ( r = 0.379 ) duration of illness and t3 ( r = 0.376 ) , t4 ( r = 0.380 ) [ table 4 ] .
there was no significant correlation between gender , duration of lithium treatment , age of onset of mood disorder , serum lithium level , and serum level of t3 , t4 , and tsh [ table 4 ] .
significantly positive correlation has been found between female gender , duration of treatment , starting lithium at later age , serum lithium level , and hypothyroidism .
no correlation was found between serum lithium level and t3 , t4 , and tsh values .
the design of our study has few limitations like this was a cross - sectional study conducted at a random point in time , concomitant antipsychotics used in the patients could be a confounding factor , small sample size and measurement of thyroid volume by single radiologist .
our study has produced several significant findings like ultrasonographically measured thyroid volume was significantly increased in patients receiving lithium therapy as compared to the patients receiving other mood stabilizers , measurements of total triiodothyronine ( t3 ) , total thyroxine ( t4 ) and tsh , finding suggests trends toward hyperthyroidism in lithium - treated patients as compared to lithium nave patients receiving other mood stabilizers and enlargement of thyroid can be diagnosed reliably only by ultrasonography and not by clinical inspection or palpation .
starting lithium at a later age was found as the risk factor for increased thyroid volume in lithium - treated patients but duration of lithium treatment and serum lithium level was not associated with any change thyroid status .
ultrasonographic measurement of thyroid volume should be included as part of the standard thyroid work - up before initiating lithium prophylaxis and during follow - up of long - term lithium - treated patients .
hyperthyroidism can cause or exacerbate mania , so it is important to be aware of this association during lithium treatment .
| background : lithium , which is frequently used in the treatment of mood disorder , can lead to various types of thyroid dysfunctions .
although clinical examination and biochemical assessment are fundamental to any thyroid work - up of lithium - treated patients , assessment findings vary widely depending on the investigator .
ultrasonographic measurement of thyroid volume has , therefore , been performed in lithium treatment populations and found to be a sensitive tool.aim:we aimed to determine and compare thyroid gland volume using ultrasonography and laboratory parameters , ( thyroid - stimulating hormone [ tsh ] , t3 , and t4 ) in long - term lithium and other mood stabilizers treated patients with mood disorder.materials and methods : in this cross - sectional study , we performed ultrasonography examinations and thyroid function test of 30 patients on lithium treatment and 30 patients on other mood stabilizers.results:the ultrasonographically measured thyroid volume was significantly increased in patients receiving lithium therapy as compared to the patients receiving other mood stabilizers .
the total triiodothyronine ( t3 ) was significantly increased with trends toward increased total thyroxine ( t4 ) and decreased tsh in patients receiving lithium therapy as compared to the patients receiving other mood stabilizers.conclusion:these results highlight the need of including ultrasonographic measurement of thyroid volume as a part of standard thyroid work - up before initiating lithium prophylaxis and during follow - up .
additional studies on the incidence and mechanism of lithium associated hyperthyroidism are needed . |
medical professionalism is not a new concept and has always been present throughout the history of medicine in the form of a hippocratic oath taken by physicians .
patient relationship and a successful diagnosis , treatment , and prevention of diseases is the patient 's trust on the physician .
this trust is that the main goal of medical professionals is to ensure public health and that this goal is prioritized by them over all their own interests .
the accreditation council for graduate medical education ( acgme ) in the united states has emphasized 6 core competencies in all residency training and assessment programs .
professional competence is the constant and conscious use of communication , knowledge , technical skills , clinical reasoning , emotions , and values and is reflected in daily practices through serving the interests of individuals and the society .
international north american and european experts have helped define professionalism . in one of its publications ,
the general medical council describes the duties of a physician with good medical practice as providing good clinical care , maintaining good medical practice , learning and teaching , communication with patients , interaction with colleagues , probity , and health .
professionalism is an ancient and respectable concept in medicine exemplified by the hippocratic oath or its versions taken by medical students in their graduation ceremonies . in the modern age
, professionalism holds the same significance and relevance for the world that it held 2500 years ago for hippocrates .
the acgme defines professionalism as a commitment to perform professional responsibilities , adhere to ethical principles and be sensitive to the diversity of patients population .
this broad and universal definition of professionalism has both advantages and disadvantages . in recent years
although it is a good change , there is still a lack of common understanding about the meaning of the concept .
discussions on the subject have thus been unsystematic , as the term professionalism has multiple meanings , complexities , and subtle differences . despite this lack of common understanding , medical professionalism
is largely taken to consist of a set of behaviors , includingphysicians preferring others interests over their own.physicians adhering to high ethical standards.physicians responding to social needs and their behaviors reflecting a social contract to serve the community.physicians representing basic human values , including trustworthiness and honesty , compassion and sympathy , altruism and empathy , and respect for others and trust.physicians being accountable for their own and their colleagues actions.physicians being committed to excellence.physicians being committed to conducting research and updating their knowledge and using them in their professional field.physicians dealing with high levels of complexity and uncertainty.physicians reflecting on their actions and decisions .
physicians responding to social needs and their behaviors reflecting a social contract to serve the community .
physicians representing basic human values , including trustworthiness and honesty , compassion and sympathy , altruism and empathy , and respect for others and trust . physicians being accountable for their own and their colleagues actions .
physicians being committed to conducting research and updating their knowledge and using them in their professional field .
physicians reflecting on their actions and decisions . clinical competence , communication skills , and a correct understanding of the ethical and legal aspects of medicine
constitute the framework of medical professionalism and include features such as accountability , altruism , excellence , and humanism .
the capstone of this framework is the realization of medical professionalism or the making of a perfect physician .
the reasons for providing medical professionalism training and assessment to medical students and practicing physicians include the patients expectations ; the relationship between professionalism and improved clinical outcomes ( i.e. , the relationship between unprofessionalism and its consequences ) ; the accreditation of organizational needs ; and the observation that professionalism can be taught , learned , and assessed .
methods of teaching professionalism including lectures , group discussion , simulations and rolemodeling should be assessed to ensure that medical students and practicing physicians are competant in medical professionalism .
approaches to the assessment of professionalism include the use of various tools and the presence of observers .
the assessment of professionalism can be used for formative and summative feedback , the assessment of programs on professionalism , and the development of a hypothesis for research on professionalism .
arnold classified the assessment tools used for medical professionalism in 3 categories that consider professionalism as part of clinical competence and then argued that the development of good qualitative methods is necessary for strengthening quantitative methods and that further research is required into the environment in which assessments should be carried out .
methods of assessing medical professionalism include peer assessment , direct observation , patient assessment , objective structured clinical examination , standardized patient assessment , student evaluation forms , self - assessment , educational portfolios , teamwork , professionalism mini - evaluation exercise , supervisors reports , videotape analysis , and single best answer multiple choice situational judgment tests .
this competence is required to be incorporated into general medicine training and residency education in all fields of medicine so that all medical students are competent in medical professionalism .
in addition to teaching the knowledge and skills required for acquiring this competence , medical students and practicing physicians should be assessed on their professionalism in order to determine the attitude and performance of different medical professions with regard to this subject ; the results obtained through these short- and long - term evaluations can then be used for the development and promotion of this essential competence .
although apparently an important subject to iranian medical professionals , only a limited number of noncomprehensive studies have been conducted on medical professionalism in iran .
evidence suggests that the subject of professionalism is not taught or assessed as part of medical students curricula , especially in the case of general medicine and residency programs .
assessing the knowledge of medical professionalism in medical students and physicians helps identify the weaknesses of professionalism training and devise plans for future training on the subject .
the present study was conducted to explore the knowledge of students in both general medicine and residency programs as well as physicians practicing in teaching hospitals affiliated with shahid beheshti university of medical sciences about the subject of professionalism .
the results of this study can help the medical community in its efforts to develop competence in professionalism .
this cross - sectional , quantitative , observational , and prevalence study was conducted in 2014 to 2015 in tehran , iran . in this study
, the authors developed a questionnaire in 2 parts ; the first part examines participants familiarity with the term and the definitions of the term ( professionalism ) , and the second part examines their knowledge in areas related to professionalism based on the resources available ( books ; guidelines ; research papers by using search engines like medline , eric , hapi , psychinfo ; and search terms such as professionalism , duty , ethics in combination with evaluation , assessment , and measurement ) . after the collaborators of the project revised the questionnaire and prepared its final version , 15 professors and colleagues with an expertise in the field assessed its validity .
a total of 11 validity assessment forms were returned to the researchers for the first and second versions of the questionnaire .
the researchers modified the questionnaires according to the comments the reviewers had made on the items for ensuring their clarification .
the mean score of validity for the questionnaire ( including its relevance , clarity , and simplicity ) was 0.899 .
the final version of the questionnaire was distributed among 25 study participants for measuring its reliability .
internal consistency ( reliability ) of the questionnaire was assessed using cronbach alpha , which was obtained as 0.755 for the 21 items of the questionnaire .
first , the first part of the questionnaire was distributed among the participants . after collecting the completed first part ,
the second part of the questionnaire was distributed among the same group with a unique code .
the first part of the questionnaire examined familiarity with the term and the definitions of the term , and the second part of the questionnaire examined areas related to professionalism . in this study ,
the main outcome was evaluating knowledge of professionalism in medical interns , clinical residents , physicians , and professors .
the study population consisted of 150 medical interns , clinical residents , physicians , and professors working in hospitals affiliated with the shahid beheshti university of medical sciences .
the sample size required for the study was calculated using the following equation : where z is the standard normal score with 95% confidence interval ( ci ) ( = 0.05 ) , s is the standard deviation of the variable , and d is maximum acceptable error .
the variance was estimated at 0.06709 in the pilot study . the sample size required for the study
was estimated at 103 with a confidence level of 95% and a margin of error of 0.05 . to take account of potential errors and sample loss , which is common in cross - sectional studies ,
participants were selected from some hospitals in tehran ( capital city of iran ) by stratified random sampling method .
stratified sampling is a random sampling technique wherein the investigator divides the whole population into different strata or groups , then randomly selects the final subjects proportionally from the strata . in this study , the population was divided into 3 strata including medical interns ( students ) , general practitioner ( gp)s and residents , and specialists . then the needed sample size was randomly selected from each stratum proportionate to the population size of the stratum when viewed against the entire population .
baseline characteristics of the subjects were presented as mean standard deviation ( sd ) for continuous variables and frequency ( percentage ) for categorical variables with 95% ci .
the data were analyzed by independent samples student t test , analysis of variance ( anova ) , and linear regression model . in all analyses ,
stata software version 13 ( stata corp , college station , tx ) was used for data analyses .
ethical considerations will also be taken into account in the dissemination of the data and the publication of the results .
a total of 149 questionnaires were completed for the study , and their analysis then yielded a set of data .
one of the questionnaires was returned blank on the second part and was excluded from the analysis . as shown in table 1 , 3 out of 149 participants did not disclose their gender and of the remaining 146 , 90 were male ( 61.64% ) and 56 were female ( 38.36% ) .
a total of 146 participants disclosed their age , whose mean age was 30.81 years ( 95% ci : 29.2732.35 , with range 2364 years ) with the standard deviation of 9.39 .
regarding age distribution , 40.41% were younger than 25 years , 26.71% were 26 to 30 years old , 16.44% were 31 to 40 years old , and 21.48% were older than 41 years . of the participants , 44.30% were medical students ;
34.23% were medical residents and general practitioners ; and 21.48% were fellows , specialists , or had higher degrees of education . of the whole study population , 66 ( 44.29% )
( 95% ci : 36.44%52.44% ) had heard and 83 ( 55.70% ) ( 95% ci : 47.55%63.55% ) had not heard the term medical professionalism before .
the frequency distribution of demographic characteristics of participants . as shown in table 2 , mean of the percentage of correct answers in male and female was 47.67 ( sd : 17.5 ) and 51.87 ( sd : 14.54 ) , respectively , and t test revealed no significant difference between mean of percentage of correct answers in male and female ( p = 0.135 ) .
the mean percentage of correct answers in students , gp and residents , and specialist was 48.26 , 47.52 , and 55.50 , respectively .
based on anova test , there was no significant difference between the mean percentage of correct answers by degree levels ( p = 0.070 ) .
age was analyzed in continuous and categorized scale in the relation with knowledge of professionalism , and there was no significant relationship in both scale of age and knowledge of professionalism ( p > 0.05 ) .
the relationship of independent variables with participants knowledge of professionalism . after adjusting for potential confounders in the linear regression model , age and degree did not have a significant difference but the mean difference of male and female ( mean difference : 5.88 ) after adjusting for age and degree was statistically significant ( p = 0.045 ) , and the mean of the female was significantly higher than that of the male participants ( table 3 ) .
table 4 presents the results obtained on participants knowledge of professionalism separately for each question and in percentage .
as shown in fig . 1 , 3 participants ( 2.0% ) responded correctly to only 3 of the items on the knowledge of professionalism ( 14.29% of the items ) and 2 participants ( 1.3% ) responded correctly to 18 of the 21 items ( 85.71% of the items ) , and the maximum number of correct answers were given to 9 , 10 , 12 , and 13 items ( each of which received 16 correct responses or 10.7% ) .
the relative frequency of participants correct responses to the items on the knowledge of professionalism .
professionalism is a set of values and behaviors that reinforce the social contraction between patient and physicians .
weak professionalism in physicians is a fundamental cause of medical malpractice and mortality and morbidity in patients .
a study showed that cultural similarities and differences have a strong impact on the concept of professionalism in a manner that a universally acceptable concept about medical professionalism is difficult .
however , medical students learn these values during their education , many studies show that certain aspects of professionalism seem to be underdeveloped in medical students and need to be targeted for teaching and assessment in order to develop professionally responsible practitioners .
one of these studies that assessed knowledge in professionalism in 2 to 4 , sixth - year medical students showed that medical curricula are not sufficiently sophisticated for reaching this aim .
another study in 937 students in china revealed that medical curricula for undergraduate students are neither sophisticated nor comprehensive in teaching medical professionalism .
a similar study in assessing knowledge and attitudes toward medical professionalism among students and junior doctors showed that there is limited knowledge but good attitude toward medical professionalism and formal training , and educational programs must be revised in this regard .
byszewski et al suggested that the main values in patient care are compassion , empathy , altruism , and integrity ( honesty ) , which are taken to define medical professionalism . in their study , they investigated medical students perception of professionalism after having added it to their curricula .
although a large number of tools have been described for assessing professionalism , the majority of studies show the lack of a single tool specifically used for measuring all the aspects of medical professionalism .
many authors have noted the importance of a multifaceted approach through a range of tools used in school curricula for assessing formative and summative objectives .
the present study , therefore , examined the level of knowledge in physicians and medical students . for this purpose , the researchers interviewed 150 medical students , general practitioners , medical residents , and specialists working at shahid beheshti university of medical sciences for their knowledge on professionalism .
the results of this study can be used for determining the effectiveness of the changes made in the knowledge of medical professionals and students and for developing a proper curriculum .
blue et al and jiang et al have also noted this application in their papers . a study in canada also showed that medical curriculum for family physicians is not sophisticated for learning medical ethics and professionalism .
the results of the present study indicated that , despite the significance of the issue , only 44.7% of participants had heard the term medical professionalism .
an average of 49.57% of participants responded correctly to the items on the knowledge of professionalism .
after adjusting for age and degree , a statistically significant difference was observed between the genders in their mean level of knowledge , and females had a significantly higher mean than males ( p value 0.045 ) .
the review of the literature yielded no relevant studies that could be compared to the present one .
the leading countries in the discussion of medical professionalism appear to have focused more on the adherence to professional standards in medical and healthcare professionals and to have assessed their knowledge of medical professionalism through educational programs .
the researchers were unable to find relevant studies assessing medical professionals knowledge on the subject under examination . in terms of the level of education , the mean rate of correct answers was 48.26% in the medical students , 47.52% in the medical residents and general practitioners , and 55.50% in the specialists and those with higher degrees of education ; however , the difference was not statistically significant ( p 0.05 ) .
the results show that it is necessary to arrange teaching programs in the field of professionalism for both general practitioners and medical residents . despite the importance of the issue
, the medical professionals working in the national healthcare system appear to have an unfavorable theoretical knowledge about medical professionalism ; although this unfavorable status does not indicate that their practices are unethical , it should be noted that one of the prerequisites for possessing a high level of medical professionalism and for establishing a proper relationship between the medical community and the patients is to have a proper knowledge of this essential concept .
improving behaviors and performances in medical professions requires adequate training on the concepts of medical professionalism and consequently the assessment of the levels of professionalism achieved in medical professionals .
the study evaluated just the knowledge of physicians and medical students on medical professionalism and other aspects of medicine , including the attitudes and behavior , were not studied .
although the validity of the questionnaire is checked by researchers , there is no certainty about whether this questionnaire really reflects the true awareness of professionalism among responders .
the study was conducted in teaching hospitals , and the results can not be generalized to all hospitals and medical community .
it must be considered that medical professionalism is under the category of medical ethics and can be different in different countries according to their culture and values .
according to the results obtained from this study , a course on medical professionalism is recommended to be added to the curriculum of students in all fields of medicine covering any relevant concept and to then be followed by a pregraduation assessment of the professional performance of the students .
the medical council and related organizations are also recommended to pursue education on medical professionalism for medical professionals more actively .
more comprehensive studies are also recommended to be proposed , approved , and carried out in areas related to medical professionalism , with adequate sample sizes and with the support of medical universities and the medical council for achieving more comprehensive results .
the researchers would like to express their gratitude to the school of traditional medicine at shahid beheshti university of medical sciences for funding the study ; the faculty 's deputy of education and research for supporting the project ; and all the professors , physicians , and students who participated in the study and completed the questionnaires .
there is no funding source for this study and shahid beheshti university of medical sciences and school of traditional medicine supported in performing the study such as gathering data and approving in ethics in research committee . | abstractalthough medical professionalism is a fundamental aspect of competence in medicine and a distinct facet of physicians competence , evidence suggests that the subject of professionalism is not taught or assessed as part of medical students curricula in iran and many other countries .
assessing the knowledge of medical students and physicians about medical professionalism seems to be helpful in identifying the weaknesses of training in the field of professionalism and devise plans for future training on the subject.the present cross - sectional , quantitative , observational , and prevalence study recruited 149 medical interns , clinical residents , physicians , and professors working in hospitals selected through stratified random sampling using a questionnaire designed by the researchers and confirmed for its validity and reliability .
the results were analyzed by stata at a significance level of 0.05.out of 149 cases , 61.64% were male with the mean age of 30.81 years .
a total of 66 participants ( 44.29% ) ( 95% confidence interval [ ci ] : 36.44%52.44% ) had heard and 83 ( 55.70% ) ( 95% ci : 47.55%63.55% ) had not heard the term medical professionalism before the study . after adjusting for potential confounders , age and degree did not have statistically significant difference in assessed knowledge of medical professionalism ,
but sex had ( mean difference : 5.88 , p = 0.045 ) , and the mean of the female was significantly higher than that of the male participants .
the mean percentage of correct answers was 47.67.the present study demonstrated that the medical professionals working in the national healthcare system have an unfavorable theoretical knowledge about medical professionalism in iran ; although this does not indicate that their practices are unethical , it should be noted that one of the prerequisites of possessing a high level of medical professionalism and for establishing a proper relationship between the medical community and the patients is to have a proper knowledge of this concept .
improving behaviors and performances in medical professions requires adequate training on the concepts of medical professionalism and consequently the assessment of the levels of professionalism achieved in medical professionals . |
peptides are small ( low molecular weight , lmw ) proteins , built up of amino acids connected by peptide bonds .
the shortest peptide is two amino acids long , and with increasing length of the amino acid chain , the name changes from peptide over polypeptide to protein , with a fuzzy border between them .
also the international union of pure and applied chemistry ( iupac ) has no clear weight or amino acid chain length limit .
a somewhat arbitrary but quite generally acknowledged definition puts the boundary between a peptide and a protein at a chain length of 50 amino acids ( wikipedia ) . in literature ,
often pragmatic definitions are used , such as the small proteins typically running off a typical 2d polyacrylamide gel or the small proteins with zero or maximally one tryptic cleavage site , and , therefore , different upper molecular weight limits for peptides can be found from 10 kda and even beyond [ 16 ] . as proteomics
is for proteins , peptidomics is the comprehensive study of all ( native ) peptides in a biological sample . in the last decade , proteomics has gained increasing recognition as a reliable and reproducible approach to study molecular processes in high throughput at a global level .
hot topic as it is recognized that peptides play complex regulatory roles in many if not all biological processes , for example , intercellular signaling [ 7 , 8 ] . as such , peptide signals secreted into the extracellular medium , reflect the state of a cell in a certain condition , and , by definition , are potential biomarkers indicative for specific physiological / pathological processes [ 911 ] .
currently , with a general proteomic approach , it is possible to detect and identify several hundreds to a few thousands of proteins in a single experiment [ 1214 ] . with modern mass spectrometers excelling in sensitivity and dynamic range , also the cell 's peptidomes become much more comprehensively accessible for analysis , and the discovery of novel biomarkers becomes possible , such as in innovative cancer research . for example , ueda et al . used size
- exclusion - based enrichment of the peptidome in combination with label - free quantitation by nano - lc - ms / ms for peptidome profiling in lung carcinomas .
body fluids , especially blood serum or plasma , and , in particular cases , ( primary ) cell culture media , serve as typical and readily available sources for a
however , the detection of peptide biomarkers typically present at low concentrations is hampered by the masking effect caused by a number of highly abundant proteins [ 11 , 1619 ] .
the large dynamic concentration range , in which peptides and proteins are present in a biological system presents a major bottleneck for peptidomic discovery of new biomarkers .
figure 1 reflecting the large dynamic range of proteins and peptides in human blood plasma is very illustrative in this respect .
especially - the presence of albumin in a sample has been shown to prevent the successful identification of low - abundance biomarkers in many peptidomic studies [ 17 , 20 , 21 ] .
hence , different methods for capturing , partitioning , fractionating , depleting , or enriching a sample have been developed [ 22 , 23 ] .
liquid chromatography ( lc ) coupled with tandem mass spectrometry ( ms / ms ) is the analytical method of choice in today 's proteomics and peptidomics research .
its major benefits include enhanced specificity ( particularly over the gc - ms technologies of 25 years ago , which had very limited applicability for peptide separations ) , its potential for high - throughput analyses , no requirement for expensive analyte - specific reagents , high speed of assay development , and a relatively low cost per assay ( the instrument itself , however , not being that cheap ) . in an ideal world
, no sample preparation would be required for the analysis of a sample as every manipulation can lead to problems such as loss of sample and , even worse , loss of quantifiability .
in particular , for the proteomic / peptidomic analysis of native peptides , which typically do not require protease ( trypsin ) digestion prior to lc - ms / ms analysis , a so - called top - down
however , as the complexity of samples still far exceeds the capacity of currently available analytical systems , specific sample preparation remains a crucial part of the analysis in a whole .
peptidomics sample preparation is often time consuming and laborious , involving multiple steps [ 25 , 26 ] . in this paper , we present an overview of different techniques ( see figure 2 ) used for the simplification of complex biological samples and review advantages and possible problems related to them ( see table 1 ) .
our focus will be on biofluids such as blood , urine , or cerebrospinal fluid ( csf ) , but some issues related to the extracellular peptidome ( part of the so - called secretome )
conditioned media are cell culture media whose cells have grown in for a certain period of time . the cells
condition the media by releasing / secreting proteins , cytokines , and other biomolecules . as such , culture supernatants or conditioned media ( cm ) can be considered yet another ( body ) fluid that can serve as a source for the identification of novel biomarkers , for example , in cancer research .
it is important to note that in order to promote a healthy growth of cells in culture , the culture medium has to be supplemented with a standard cocktail of nutrients and growth factors .
in mammalian cell cultures , this is typically achieved by the addition of a substantial volume of fetal calf serum ( fcs , up to 10% solution ) . when studying the intracellular proteome ( or peptidome ) ,
cells are washed several times with fcs - free cell culture medium and/or phosphate - buffered saline ( pbs ) prior to lysis to reduce contamination of the sample with ( bovine ) serum proteins . however ,
this is not possible when the extracellular peptidome is under investigation . as a compromise , in most studies
, the medium containing fcs is replaced by fcs - free medium or medium containing reduced amounts of fcs just before starting the secretome experiment ( e.g. , giving a biological / physiological stimulus ) . for adherent cells
however , when studying cells in suspension , several centrifugation steps are required before the medium can be replaced , which arguably is a source of unnatural stress to the cells , possibly even causing cell lysis .
a main disadvantage of working with reduced amounts of fcs ( or no fcs at all ) is that also this is known to cause metabolic stress to the cells , not seldom inducing cell death and consequently altering the cell culture 's secretome [ 11 , 2834 ] .
a traditional well - established technique in proteomics is one- or two - dimensional ( 1d or 2d ) polyacrylamide gel electrophoresis ( page ) .
although more a protein than a typical peptide separation technique , 1d and 2d page are used in
the latter can then be analyzed by ms after extraction from the gel as proteolytic fragments , after in - gel digestion .
denaturing agents , such as sds , are used to unfold the macromolecules and disrupt noncovalent intra- and intermolecular protein / protein interactions .
its poor resolving power , however , often poses a problem in the analysis of complex mixtures .
1d and particularly 2d page are employed to increase the depth of proteome / peptidome analysis , that is , through fractionation of the sample components and removal of lmw impurities , particularly salts , which interfere with subsequent ms analyses [ 3541 ] .
2d page is very sensitive predominantly to molecular charges of a protein ( by the isoelectric focusing step ) , making it a very effective method to reveal / separate certain posttranslational modifications like phosphorylations , sulfations , or glycosilations .
limitations are that proteins / peptides with extreme pi values can not be separated and that the smaller peptides are typically not retained in the second ( mw separation ) dimension .
the dynamic range for detection in 2d page is 1010 , which is less than the protein expression range observed in biological systems . in order to achieve the detection of low abundant proteins ,
such higher loads , however , often further compromise the technique 's resolution due to spot fusion and comigration [ 19 , 21 , 39 , 4246 ] .
important in page is the visualization of the separated proteins , although selected areas of a gel can be processed for ms from unstained preparative gels after comparison / alligning with an analytical reference or master gel .
the most commonly used visible stains are coomassie brilliant blue ( cbb ) and silver nitrate staining .
cbb staining is easy , ms compatible , and linear over , at least , one order of magnitude , so it is usable for quantification to a limited extent .
silver nitrate is a more sensitive staining method0.5 ng versus 50 ng for cbb but the staining procedure is more labor intensive and has a more limited linear range ( due to its polychrome results ) . although widely regarded as the standard of rigor by which all other ultrasensitive staining methods are judged , silver staining remains quite a complex and variable protein - gel - staining methodology , with many dozens of published protocols , all of them requiring several steps .
silver staining quantitation is never simple , due to the complex polychromatic nature of the color development and to considerable differences in response factors between different proteins .
fluorescent dyes are also popular to visualize proteins in gels ; however , they are not cheap reagents and require expensive scanners / image analyzers .
they exhibit detection sensitivity rivaling that of silver staining , with workflow advantages similar to cbb staining .
fluorescence offers linear quantitation ranges 10100-fold greater than other colorimetric methods [ 4750 ] . because of the limitations associated with gel - based techniques , recently with respect to detection of the smaller proteins and peptides ( see above ) , attention has gone to off - gel methods for peptide / protein separations , in particular in solution - pi - based peptide separations without the need for carrier ampholytes .
it focuses proteins and peptides on an immobilized ph gradient ( ipg ) gel , which is sealed against a multichamber frame that contains both sample and focusing solutions . the sample is separated by migration through the gel , followed by diffusion into the well adjacent to the section of the ipg strip .
it allows for multiple samples to run simultaneously and requires only small sample volume and no prior sample cleanup .
disadvantages are that it has a rather long separation time and requires an insulated cooling system [ 5153 ] .
recently described a variation of the mostly used offgel system ( agilent technologies , usa ) .
off - gel separation has not only shown applicability for proteins but also for peptides .
hubner et al . compared the technique to in - gel digestion and found that using peptide off - gel separation led to a third more protein / peptide identifications comparing off - gel separation to reversed phase liquid chromatography ( rplc ) at high ph , manadas et al .
conclude that rplc leads to the identification of more peptides and also more unique peptides .
in general , off - gel separation has clear advantages over a gel - based approach with respect to focusing and concentrating peptides , but it still requires further optimization to reach the same level of identifications as an rplc - based separation .
many different approaches exist to separate proteins based on their biochemical and biophysical properties such as molecular weight , mass , and hydrophobicity .
another way to reduce a sample 's complexity is to specifically remove the most abundant protein(s ) , by doing ( immuno- ) affinity capturing [ 17 , 20 , 32 , 39 , 5661 ] .
an example is the removal of albumin from serum , plasma , or cell culture samples .
the most used dye for removal of albumin is cibacron blue ( often in combination with protein g for the removal of igg ) .
this dye however does not only show affinity for albumin but also for nad , fad , and atp binding sites of proteins , which often results in the unwanted removal of proteins of interest [ 6264 ] .
several comparisons have been made on cibacron - blue - based depletion of highly abundant proteins from a complex sample versus immunodepletion of the same sample .
the overall conclusion is that the dye method is less performant than the immuno - based affinity removal : it does not only incompletely bind all albumin from the sample ( lower efficacy ) , but it also appears to remove a substantial portion of proteins other than the targeted albumin ( lower specificity ) [ 62 , 65 , 66 ] .
immunodepletion based on monoclonal antibodies ( mabs ) is generally not preferred as , besides being very expensive , these antibodies typically remove only proteins or protein fragments with the specific targeted epitope , whereas other fragments of the protein remain untouched . therefore , immunodepletion systems are generally based on polyclonal igg and/or igy antibodies , targeting multiple epitopes on the same proteins .
moreover , a mixture of polyclonal antibodies to distinct proteins are nowadays commonly used for removing multiple highly abundant proteins at once .
antibody - based depletion of a sample can be performed in ( low - pressure ) spin cartridges or ( high - pressure ) liquid chromatography ( lc ) columns . when using antibodies , one has to consider the number of proteins that has to be depleted from the sample .
depending on the system used , it is possible to remove between 1 and 20 abundant proteins .
they observed that increasing the number of depleted proteins from 12 to 20 had only little beneficial effect and could in fact even increase the removal of peptides and proteins of interest which are associated with the abundant proteins .
also tu et al . did a comparison of 2 types of multiple affinity removal system ( mars ; agilent technologies , inc . ) depleting , respectively , 7 ( mars-7 ) or 14 ( mars-14 ) abundant proteins .
the mars-14 column removed 7 extra proteins but showed no substantial advantage over the mars-7 in improving peptide analysis / global protein identifications from plasma . recently ,
a creative way of depleting a sample was developed , the so - called hexapeptide library of combinatorial peptide ligands .
high abundant proteins are expected to quickly saturate their specific affinity ligands leaving nonbound high abundant proteins to be washed away .
in contrast , low and medium abundant proteins and peptides do not saturate their ligands and hence are concentrated on the beads .
this technique has the advantage that peptides and proteins are adsorbed under native conditions and thus allow monitoring of their biological activity [ 68 , 69 ] , although its efficacy is still debated .
limited comparative studies are published on the different depletion and enrichment methods . in these few comparisons ,
typically , the methods compared all lead to identification of a number of peptides and proteins , a part of which is generally identified by all methods under investigation and another part which has been identified uniquely in a sample that was treated with one of the methods [ 58 , 68 ] .
the only exception to this is the comparison of dye - based depletion to immunodepletion of albumin ( see above ) . in this case ,
immunodepletion invariably resulted in the most efficient enrichment of low abundant proteins and peptides [ 62 , 65 , 66 ] .
they , however , did not compare based on identification of proteins but on number of spots found by 2d page and also took into account the costs of all methods .
when only looking at protein spots , they concluded that one column ( seppro mixed12-lc20 , genway biotech ) had the best overall performance leading to the largest number of new proteins spots . a second column ( multiple affinity removal column human 6 , agilent technologies ) in the same study led to almost the same quality results , while being cheaper and thence representing a more economical option which could become the preferred method when budgets are limited .
in those cases where larger members of the peptidome need to be addressed , another crucial part of sample pretreatment is alkylation and digestion of the peptides / small proteins .
this can be performed prior to or after the previously described techniques , that is , just before lc - ms / ms analysis . to break the tertiary structure of peptides
, disulfide bridges have to be disrupted ( reduced ) and blocked to prevent reoxidation .
breaking of disulfide bonds is traditionally achieved using reducing agents such as dithiothreitol ( dtt ) or tris(2-carboxyethyl)phophine hydrochloride ( tcep ) [ 72 , 73 ] . after reduction , the thiol groups are blocked , typically by alkylation , with iodoacetamide ( iam ) being the mostly used alkylating agent .
another alkylating agent is 2-bromoethylamine ( brea ) , which transforms cysteine into s - aminoethyl cysteine , a pseudo - lysine , hereby creating an extra cleavage site for proteolytic enzymes such as trypsin [ 74 , 75 ] , which can be an advantage in certain cases .
when larger ( poly)peptide members of the peptidome are envisaged , a bottom - up approach , requiring proteolytic digestion prior to mass spectrometric analysis , is sometimes to be considered .
trypsin by far is the most used proteolytic enzyme for the degradation of proteins or peptides .
this protease has the advantages of having a high cleavage specificity and being stable in a wide range of conditions .
it cleaves c - terminal to arginine or lysine residues ( except where the subsequent amino acid in the parent sequence is a proline ) .
thanks to the biological distribution of these amino acids among all proteins , the resulting peptide masses typically fall within the range required for analysis by mass spectrometry .
some larger peptides or even proteins have been described in literature that contain only 1 tryptic cleavage site , producing a peptide still too large to be readily detected by mass spectrometry . in those cases , a combination of 2 or more proteases and/or alkylating with brea i.s.o .
trypsin is very similar to chymotrypsin in primary structure , however chymotrypsin prefers cleaving c - terminal to amino acids with bulky aromatic residues such as phenylalanine , tyrosine , and tryptophan [ 48 , 7679 ] .
also other sequence - specific proteases have their advantages in view of subsequent ms analysis of the resulting peptides .
a good example is metalloendopeptidase lys - n , which guarantees a prominent positive charge at the aminoterminus of the cleaved product .
ultrafiltration is a rather easy and widely used technique to fractionate a proteomics sample into almw fraction ( the peptidome ) and ahmw fraction ( the rest of the proteome ) by centrifugation .
for example , in a recent study on csf , zougman et al . demonstrated the power of ultrafiltration to separate neuropeptides from the set of larger proteins present in csf .
the ultrafiltrate low molecular weight fraction of the csf was directly separated by nano - lc - ms / ms . ultimately , this led to the identification of 563 peptides derived from 91 protein precursors .
for example , they exhibited evidence of being produced by proconvertase cleavage , having a high cysteine content , bearing an aminoterminal pyroglutamate and/or carboxyterminal amidation .
interestingly , in the same study , a parallel proteome profiling by 1d page was performed , and this yielded 798 proteins . for many of the identified neuropeptides in the ultrafiltrate
, the corresponding precursor protein was not identified in the proteome screen , which evidently demonstrates the necessity of separating peptides from larger protein species .
many different devices are available , each with specific molecular weight cut - off ( mwco ) and type of membrane .
ultrafiltration is not seldom reported as a poorly reproducible technique , with possible removal of proteins and peptides below and above the stated mwco [ 8284 ] . to disrupt potential protein - protein / peptide interactions , acetonitrile ( acn )
. acn this way improves the recovery of lmw peptides [ 20 , 85 ] .
harper et al . investigated several factors which might influence the ultrafiltration such as centrifugation time and speed . in their study , slower speed and longer time than advised by the manufacturer lead to improved recovery of lmw proteins and peptides .
however , at the same time , also more hmw proteins appeared to pass through the filters .
they concluded that devices with vertical or angular membrane configuration reduce membrane fouling , allowing continuous flow rates , even with high protein concentrations .
this approach is known as filter - aided sample preparation ( fasp ) and it can be used to combine the advantages of in - gel and in - solution digestion [ 88 , 89 ] .
the addition of organic solvents to serum causes high molecular weight ( hmw ) proteins to precipitate , leaving the lmw protein fraction including the peptides in solution . by also adding ion - pairing reagents such as trifluoroacetic acid , peptides , and smaller proteins can be dissociated from high abundant proteins , thereby facilitating their extraction [ 9092 ] . in general , proteins are precipitated by adding ice - cold acetone or acn .
tucholska et al . compared several solvents for organic precipitation and concluded that acetone precipitated the serum proteins most completely .
they found that methanol was actually the least favorable solvent for precipitation as it still contained the largest amount of albumin in solution .
report that the use of methanol also leads to a much larger and less dense precipitate which was more difficult to separate from the supernatant .
polson et al . likewise compared several precipitation methods , concluding that acn is the most efficient precipitant for protein removal . as an alternative to organic solvents ,
although as is a very efficient precipitant , it can cause interface contamination when combining with lc - ms .
this method consists of 2 steps , the first of which denatures proteins and peptides and by dropping the denatured sample in ice - cold acetone causes all proteins and peptides to precipitate . in the second step ,
the lmw proteins and peptides which dissolve in 70% acn containing 12 mm hcl are separated from most of the other proteins . when compared to normal organic precipitation ( with acn ) and ultrafiltration
, they conclude this method gives a much better yield for low molecular weight peptide extraction from human serum ( see figure 3 ) .
another analytical tool to separate lmw compounds from hmw compounds is size exclusion chromatography ( sec ) .
this is a widely used technique for the purification and analysis of synthetic and biological polymers based on their size , which is not by definition the same as their molecular weight .
the material used for sec consists of porous beads , which either exclude the peptide / protein analytes from the internal space or allow them to enter based on their size . peptides and
smaller proteins , which can enter the beads , will move at a slower rate through the column than bigger proteins which can not penetrate the beads , thus migrating faster .
a disadvantage of this technique is its low resolving power , which can be improved by using it in combination with other separations , such as in multidimensional separation approaches ( see below ) .
other drawbacks are the high elution volumes which cause dilution of the sample , increased costs when having to use multiple columns , and the need for high sample loads . in literature
, some reports have appeared on the use of a one - step peptidome enrichment method based on sec [ 15 , 95 , 96 ] .
mention the separation of over 12,000 molecules by using sec followed by c18 nano - lc - ms .
they show that their procedure allows a precise separation of serum proteins and peptides based on their molecular weights .
tucholska et al . showed that sec can be used to remove substantial amounts of albumin from serum ( although a small amount could still be detected in the depleted fraction ) .
performed a comprehensive peptidome analysis of mouse liver by combining sec prefractionation with nano - lc - msms . from the low molecular weight fractions ( <3 kda ) that eluted from the sec column ,
reversed phase liquid chromatography ( rplc ) separates molecules based on differences in their hydrophobicity .
the mobile phase is a water and nonpolar organic solvent mixture , whereas the stationary phase is hydrophobic .
factors influencing the selectivity and resolution of separation include the hydrophobic ligand , the particle size , sample volume , column length , and ph . the most commonly used hydrophobic ligand for peptide analysis is c18 , but c4 and c8 are preferred for the larger peptides and proteins . in terms of peptides extraction
, the solid phase material can be packed in syringe - shaped cartridges , or even in 96-well plate formats , which allows for high throughput extractions .
another advantage of this technique is its capability to desalt samples , which is desirable prior to mass spectrometric analysis [ 43 , 44 , 98 , 99 ] . in proteomics / peptidomics analysis , nowadays ,
capillary reversed phase columns with an internal diameter of 75 to 100 m are commonly used , mainly because of the increased sensitivity this offers .
because of these small columns , low flow rates have to be used , without decreasing the sensitivity , and ( often environmentally unfriendly ) solvents are saved . reducing the particle size to 2 m or less
allows the use of a wider variety of linear velocities while maintaining acceptable chromatographic resolution in substantially reduced analysis times .
this , however , requires new hardware since typical hplc systems generally are not able to handle the high pressures required to pump the mobile phase through a column packed with such minute particles .
ultrahigh - pressure lc ( uplc ) systems have been developed for this purpose , allowing pressures of 400 bar and higher .
thanks to the greater analytical power of uplc , downscaling of sample volumes and increasing the throughput of sample handling have become possible [ 98 , 100103 ] .
ion exchange chromatography ( iex ) separates peptides ( and proteins ) based on their charge in a specific salt environment and ph of the mobile phase .
two types of iex exist , namely , cation or anion exchange ( respectively cx and ax ) .
iex has a disadvantage , being the use of salts which makes the eluate incompatible with ms .
the use of salts can also lead to irreversible peptide or protein absorption to the resin , resulting in sample loss .
the principles of iex are well understood , and other advantages include the high resolution that can be achieved , high capacity , selectivity , and robust operation . usually , simple salt buffers are sufficient and concentrations are used in a defined range , in which the so - called salting - in effect on proteins is observed .
cation exchangers are negatively charged , and anion exchangers ; are positively charged . above its pi
, a protein is negatively charged and binds to an anion exchanger ; below its pi , it is positively charged and binds to cation exchangers .
the ion exchanger itself behaves like an acid or base , and the disproportionation of the charges depends on the ph .
strong ion exchangers behave like a strong acid or base and do not change the charge within a wide range of ph , whereas weak ion exchangers do .
this property can also be exploited to gain selectivity or by applying ph gradients for elution [ 44 , 105 , 106 ] .
restricted access material ( ram ) has been described for use in the separation of large biomolecules and the extraction of lmw analytes .
ram could be considered to be an upgrade of normal size exclusion material .
the outer surface of the ram particles is coated with a protective , nonadsorptive hydrophilic packing , while the surface of the pores can be coated with a variety of different affinity matrices . in principle , it prevents access of macromolecules ( e.g. , proteins ) to the bonded phase in the pores , which can be compared to the size exclusion process .
simultaneously , lmw target compounds ( such as peptides ) are selectively retained on the affinity matrix in the pores [ 108 , 109 ] .
the most used type of internal selection material is strong cation exchange ( scx ) as this type of material is highly suitable for the online extraction of target peptides from complex biological samples such as plasma [ 110 , 111 ] .
the method allows repetitive , direct injections of untreated sample matrices and can also handle higher injection volumes in comparison to sec [ 95 , 98 ] .
a few setups have been described , in which ram is even used in an online lc - ms / ms setup [ 108 , 110113 ] .
even described the use of a macroscopic ram cartridge online with nano - lc - ms for the quantification of a peptide analyte in samples containing high amounts of bovine serum albumin ( bsa ) . although the separation process can be automated , important parameters in ram chromatography , such as loading flow rate , ph , and amount of dilution and injected volume should be optimized as they have a large effect on the amount of adsorbed peptides and proteins as well as on their recovery from the column .
the use of ram has been demonstrated for the analysis of peptides in complex biological matrices .
isolated and detected more than 1500 peptides and protein fragments from only 3 ml of urine using an online ram - scx system . because of the high complexity of biological samples , often a single fractionation or separation step is insufficient
therefore , several techniques can be combined to what is referred to as a multidimensional separation .
combining 2 ( different ) separation techniques leads to an increased number of peptides measurable , an enlarged overall dynamic range and thus an improved peptidome coverage .
a multidimensional approach can be achieved both offline and online . for the offline separation ,
fractions are collected after the first dimension , which are later reinjected into the second dimension separation . in between both steps
a disadvantage of this method is the potential sample loss when transferring between both dimensions .
when doing online multidimensional separation , the samples are automatically transferred from the first to the second dimension .
also the solvents should not cause salt precipitation or immediate elution of the compounds in the second dimension .
most often , rplc is the second dimension for its high speed , desalting capability , and compatibility with mass spectrometry [ 19 , 43 , 115118 ] .
it is clear that , besides selecting the best methodology to comprehensively separate peptides from a biological mixture , an essential part of peptidomics sample preparation is the preservation of the integrity of the in vivo peptidome .
various protease inhibitor cocktails can be introduced in the sample as early as viable , to try and inhibit ubiquitous peptide degrading enzymes [ 118 , 119 ] .
in addition , peptidomics samples are traditionally snap - frozen , at a very low temperature as soon as possible , and subsequently freeze dried . yet
, such strategies donot always appear adequate ( rapid enough ) to prevent bioactive peptide degradation ( several peptidases survive repeated freeze - thaw cycles ) .
this has clearly been demonstrated for the ( neuro)peptidome , which appears particularly sensitive to rapid ex vivo ( postsampling ) degradation ( figure 4 ) .
microwaves were found to be effective to rapidly and permanently inactivate enzymes [ 104 , 120 ] , although poorly controllable .
an elegant automated instant thermal denaturation system , based on conductive heating , at controlled pressure under vacuum , was developed for successful and reproducible blocking of ex vivo sample degradation , thereby stabilizing the peptidome ( denator , gothenburg , sweden ) .
depending on the sample - origin - specific indicator peptides can be used to monitor / assess the sample quality , prior to analysis .
one example is the post mortem appearance in mammalian neuronal tissue of the peptide stathmin 220 , which indicates proteolytic degradation .
in general , it can be concluded that sample preparation for the purpose of capturing the peptidome from biofluids still has room for improvement , and a single generic methodology is not ( yet ) available .
many methods exist for the analysis of proteins which can , sometimes with minor adjustments , be used for peptides as well .
ideally , sample preparation / handling should be minimal , but current mass spectrometers are not quite able to handle complex biological matrices .
for example , over the last decades , the sensitivity of mass spectrometers has tremendously increased , where at the moment they have reached the low attomole level . also , improvements in mass accuracy and advances in peptide fragmentation techniques permit us now to obtain highly confident identification and even fully de novo sequence novel peptides . despite these ongoing improvements
, it should be noted that at the moment , a peptidomics analysis ms system that can operate without sample preparation is still far way , which is mainly due to the high complexity of the biological matrices .
the biggest bottleneck in biofluid research is the high dynamic range at which the proteins / peptides occur .
for example , albumin concentration is ~40 mg / ml in serum , whereas the concentration of biologically active compounds such as cytokines is ~1 pg / ml .
the removal of abundant plasma / serum proteins by dye- or immuno - based depletion is at moment the most popular strategy to mine
however , one should also realize that albumin and other major proteins in blood plasma carry some number of other
adsorbed peptides and even small proteins on their surface , and , therefore , these will risk to get lost during this depletion step .
the precipitation of the abundant protein fraction in human biofluids by organic solvents has probably the longest history among all the methods used for abundant protein removal .
this procedure is still one of the most effective methods as it permits fast and , although not always highly reproducible , cheap obtainment of the peptide fraction for their analysis by mass spectrometry .
several other analytical strategies are at hand that aid in the reduction of the complexity of samples , and some specifically focus on capturing the lmw fraction , that is , the smaller proteins and peptides from the bulk of larger proteins , for example , ultrafiltration as method to split a sample into a proteome and a peptidome fraction .
however , one should be aware that several studies have shown dramatic differences in the performance ( both in terms of reproducibility , recovery , and separation ) of commercially available ultrafiltration devices .
a more preferred analytical tool to separate biomolecules on basis of size is liquid chromatography .
size exclusion chromatography and more specifically restricted access material have high potential in peptidome research , due to their high resolution and selectivity .
these last two chromatography techniques also can be miniaturized , making them even more favorable in term of sensitivity . in general , as sample preparation is often time consuming and laborious , high - throughput and automated approaches are highly desirable . whereas up to now , when doing multidimensional lc , the first dimension led to the collection of fractions in an offline setup , online multidimensional lc setups gain popularity
. also the column material used in liquid chromatography is continually being improved , and the possibility of combining separation based on different physicochemical properties , such as ram , is a big step forward .
even though it usually takes some effort to completely set up and optimize an lc - based method , once done , it usually results in a very robust and reliable technique , which can be fully automated .
also miniaturization is an important aspect of the evolution , as a miniature system ( e.g. , lab - on - a - chip ) will allow for lower amounts of solvents and less complicated equipment to be used , decreasing the cost of an analysis . with the development of new sample pretreatment methods and lower detection limits on mass spectrometers , future peptidome analysis will aid substantially in biological and medical research , for example , by discovering new biomarkers and uncovering novel signaling pathways . | although big progress has been made in sample pretreatment over the last years , there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices .
being the little brother of proteomics , peptidomics is a relatively new field of research aiming at the direct analysis of the small proteins , called peptides , many of which are not amenable for typical trypsin - based analytics . in this paper , we present an overview of different techniques and methods currently used for reducing a sample 's complexity and for concentrating low abundant compounds to enable successful peptidome analysis .
we focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification and indicate their advantages as well as their shortcomings when it comes to the untargeted analysis of native peptides from complex biological matrices . |
multiplex pcr ( esensor respiratory viral panel ; genmark dx , carlsbad , ca , usa ) for patients seen at a major teaching hospital in alachua county indicated that > 95% of illnesses were caused by h3n2 strains .
influenza a(h3n2 ) virus ha , neuraminidase ( na ) , and matrix ( m ) gene sequences from 5 specimens collected from patients in november 2014 were directly sequenced from the specimens , and corresponding first - passage viruses were isolated in mdck cells by using previously described primers ( 6,7 ) .
these viruses were designated as a / gainesville ( h3n2 ) isolates 59 from 2014 ( e.g. , a / gnvl/05/2014 ) ( table 1 ) .
for all , the consensus viral genomic sequences determined directly from clinical specimens and their matched virus isolates were identical . *
the deduced ha major antigenic epitopes a and b1 of the 5 viruses isolated had some amino acids not found in the ha protein of vaccine strain a / texas/50/2012 ( h3n2 ) ( table 1 ) .
no changes were observed for major antigenic epitopes b2 , c1 , c2 , d , and e. changes at the major antigenic epitopes included n144s , n145s , f159y , k160 t , n225d , and at other epitopes l3i and q311h ; those changes are characteristic of ha gene clade 3c.2a viruses ( 4 )
. substitutions at residues 144 and 160 have been associated with the loss and the gain , respectively , of possible n - linked glycosylation sites of the ha protein ( 8,9 ) .
an h3n2 virus isolated in february 2014 in gainesville , designated a / gnvl/01/2014 ( h3n2 ) , was from a different ha gene clade ( table 1 ) , suggesting that the clade 3c.2a viruses were introduced locally later .
the na proteins of the 5 viruses from november 2014 also differed from those of the northern hemisphere h3n2 vaccine strain at 3 amino acids , whereas the m proteins were identical .
data on frequency of medically attended influenza - like illness ( ili ) for the 201415 influenza season were obtained from florida s electronic surveillance system for the early notification of community - based epidemics ( essence ) for alachua county ; the 13-county florida department of health region 3 ( 2010 population : 2.2 million ) , which includes alachua county ; and the state of florida ( 10,11 ) .
ili - associated emergency department ( ed ) visits were defined as visits for which a chief complaint contained the word influenza or
region 3 and florida are comparison areas , so they exclude alachua county data . for the 201415 influenza season
( september 28 , 2014may 23 , 2015 ) , the rate of reported ili for alachua county residents was 499 cases per 100,000 population .
sixteen percent of cases were among children < 17 years of age , a significant increase over 201314 , when 11% of cases were among children in this age group ( p = 0.001 , pearson test , 2-tailed ) .
the pattern for laboratory - confirmed influenza cases in our hospital ed was similar ; although the rate of confirmed influenza cases among adult ed patients did not increase , the rate of confirmed cases among pediatric ed patients increased significantly from 6/1,000 unique ed patients in 201314 to 43/1,000 in 201415 ( p<0.0001 , ) .
alachua county has a nationally recognized school - based influenza vaccination program that administers live attenuated influenza vaccine ( flumist ; astrazeneca , wilmington , de , usa ) ; parenteral influenza vaccine is administered by private physicians ( 12 ) . since 2010 , the average overall influenza vaccination rate for children 017 years of age in alachua county has been approximately 45% . as previously reported ( 11 ) , this rate has correlated with a reduction in rates of ed visits for ili ( as reported through the florida essence surveillance system ) among children in alachua county , compared with those in florida region 3 and the state of florida .
corresponding estimates of vaccine effectiveness ( relative reductions in risk ) in alachua county , by year , are shown in table 2 .
accompanying the increase in cases among children for 201415 was a small but significant decrease in estimated effectiveness of vaccination for children in florida region 3 ( table 2 ) .
* estimates are based on reports of influenza - like illness from emergency departments reported through florida s electronic surveillance system for the early notification of community - based epidemics .
region 3 and florida are comparison areas , so they exclude alachua county data . as a marker of disease severity
, we identified the number of patients at our hospital admitted to the medical intensive care unit ( micu ) because of influenza by querying the university of florida health integrated data repository for patients with international classification of diseases , ninth revision , diagnostic codes for influenza ( 487 ) or novel influenza ( 488 ) listed in the first 10 diagnoses . in the 201415 influenza season
, 1.8% of micu patients carried the diagnosis of influenza , a significant reduction from the 4.8% of micu admissions in 201314 , when an h1n1 dominated ( p<0.0001 , fisher exact test , 2-tailed ) .
the mean age for persons admitted to micu with influenza in 201415 was 60 years , compared with 50 years for 201314 ( p = 0.006 , mann - whitney u test , 2-tailed ) .
seven of these micu patients died ; all were > 50 years of age ( mean 70 years ) .
patient ages were significantly higher than for persons with fatal cases in 201314 ( mean 48 years ) ( p = 0.04 , mann - whitney u test , 2-tailed ) , with a significantly higher charleston co - morbidity index ( 4.9 vs. 2.4 , p = 0.04 , mann - whitney u test , 2-tailed ) .
the predominant influenza strain in this community in 201314 was an h1n1 variant bearing the d225 g polymorphism , which was associated with more severe disease outside of elderly age groups ( 10 ) .
rates of illness severe enough to warrant micu admission with the h3n2 clade 3c.2a strain circulating in 201415 were significantly lower than those with h1n1 in 201314 and occurred in a significantly older age group with a higher rate of co - morbidities , more closely matched observations from in the 201213 h3n2 influenza season .
these findings highlight the substantial strain - related differences in virulence that occur from year to year and the importance of ongoing genetic monitoring of strains circulating within communities .
we noted an increase in influenza incidence in children < 17 years of age in alachua county during 201415 .
this increase was probably due , at least in part , to a decrease in vaccine effectiveness with the clade 3c.2a strain in this highly vaccinated population of children .
our hospital - based ed and micu data use laboratory - confirmed cases , whereas the regional and state data and vaccine effectiveness calculations are based on ili data from the florida essence system , which are potentially less reliable . although we found data trends in the 2 systems to be consistent ,
our findings underscore the need for expanded surveillance of laboratory - confirmed cases statewide , particularly with the increasingly widespread distribution of clade 3c.2a strains in the americas and europe ( 13 ) .
these issues might be particularly relevant in light of the recent world health organization recommendation to substitute the ha gene clade 3c.3a virus a / switzerland/9715293/2013 ( h3n2 ) ( rather than a clade 3c.2a virus ) for a / texas/50/2012 ( h3n2 ) in the 2015 vaccine for the northern hemisphere . | influenza a(h3n2 ) strains isolated during 201415 in alachua county , florida , usa , belonged to hemagglutinin gene clade 3c.2a .
high rates of influenza - like illness and confirmed influenza cases in children were associated with a decrease in estimated vaccine effectiveness .
illnesses were milder than in 201314 ; severe cases were concentrated in elderly patients with underlying diseases . |
hepatocellular carcinoma ( hcc ) is one of the most common malignancies worldwide , especially in asian countries .
although the mainstay of therapy is surgical resection , the majority of patients are not treated by surgery because of the tumor extent or underlying liver dysfunction . instead of surgical resection , several treatments
have been developed , including radiofrequency ablation ( rfa ) , transcatheter arterial chemoembolization ( tace ) , and radiation therapy ( rt ) .
tace has become the main treatment modality , especially in cases of intermediate - stage hcc . however , repeated tace or combination therapy with rt is often needed to control tumor progression [ 2 - 4 ] . as tace has become more widely used ,
serious complications are acute liver failure ( 7.5% ) , acute renal failure ( 1.8% ) , encephalopathy ( 1.8% ) , ascites ( 8.3% ) , upper gastrointestinal bleeding ( 3.0% ) , and hepatic or splenic abscess formation ( 1.3% ) . among these complications ,
we describe herein a case of hepatoduodenal fistula caused by an abscess after repeated tace combined with rt , which was successfully treated with endoscopic histoacryl injection .
a 71-year - old male patient was readmitted to our hospital 1 month after discharge because of relapse of abdominal pain .
one year prior , he had been diagnosed with hcc associated with chronic hepatitis b. he could not undergo surgical resection because of invasion of the hepatic and main portal veins .
although he underwent five sessions of tace over 10 months , newly viable hcc was found in the left lobe of the liver with thromboses of the portal vein , hepatic vein , and inferior vena cava .
palliative rt was administered to the hcc in the left lobe of the liver and area of left portal vein thrombosis using a dose of 33 gy comprising daily doses of 3 gy .
one month after the seventh tace session , the patient presented to our institution with a complaint of persistent abdominal pain .
abdominal computed tomography ( ct ) showed a gas - containing cavity suggestive of an abscess within a necrotic mass in the left lateral segment of the liver ( fig .
he was hospitalized , received intravenous empirical antibiotics , and underwent ultrasound - guided abscess aspiration , which produced 0.5 ml of turbid yellowish fluid with a foul odor .
percutaneous drainage catheter insertion was difficult because of the small size of the abscess pocket .
the patient 's symptoms , including abdominal pain , fever , and chills , resolved completely after 2 weeks of intravenous antibiotic therapy , and he was discharged with oral antibiotics . although he continued to take oral antibiotics , his abdominal pain recurred only 2 weeks after discharge , and the frequency of abdominal pain gradually increased .
he complained of persistent abdominal pain ; however , other generalized symptoms , including fever and chills , did not reappear . on physical examination , right upper quadrant tenderness was observed , and his serum c - reactive protein level was higher than that during the previous hospitalization .
although the abscess had decreased in size , we detected a new communication between the remaining abscess and the duodenal bulb ( fig .
gastroduodenoscopy was performed to confirm the fistula through a 0.3-cm hole in the proximal portion of the duodenal bulb ( fig .
follow - up endoscopy was performed 2 days after clipping ; however , there was still a fistula opening beside the clips ( fig .
a ~1-cm fistulous tract was confirmed by guidewire insertion from the fistula hole of the duodenal lumen . a 1:3 mixture of histoacryl and lipiodol solution ( 0.5 ml )
follow - up endoscopy performed 2 days after this intervention revealed that the previous fistula orifice was filled with histoacryl ( fig .
the patient was then discharged from the hospital because he had no abdominal pain after starting oral intake .
one month after the intervention , a follow - up abdominal ct scan showed obliteration of the fistulous tract and a decrease in the extent of the abscess ( fig .
eight months later , he underwent four sessions of additional tace without abscess recurrence ( fig .
the most recent ct scan , taken 7 months after histoacryl injection , showed advanced hcc without abscess or fistula formation .
only a few cases have been reported in the literature [ 3,6 - 9 ] .
three mechanisms of hepatoenteric fistula formation have been suggested . in the f irst mechanism ,
a f istula is formed by the spread of inflammation from an inadequately treated liver abscess in cases of fistulas that form secondary to a liver abscess .
this chemoembolization causes ischemic damage to the surrounding liver tissue and induces tumor necrosis , subsequently resulting in abscess formation by secondary bacterial infection in about 0.2% to 1.1% of all patients . in the second mechanism , in cases of a direct perforation of the adjacent bowel by sharp electrodes during rfa or a delayed perforation by thermal injury during rfa ,
infection is caused by the leakage of bowel contents , resulting in a fistula or abscess .
finally , in the third mechanism , a fistula is formed after treatment of direct invasion of the gastrointestinal tract by advanced hcc . in our patient
, the f istula was detected 1 month after abscess treatment ; therefore , the mechanism of the fistula appears to be liver abscess formation . in particular , the combination of tace and rt caused more severe ischemic damage to the tumor and surrounding liver tissue .
the risk of abscess formation is higher in combination therapies than in tace alone . in our patient ,
if a hepatoenteric fistula that formed during hcc treatment is not completely treated , it will become a cause of recurrent ascending infection and intractable liver abscess formation , which deteriorate liver function .
furthermore , it can shorten the life expectancy of the patient because of a delay in appropriate treatment of hcc .
therefore , early diagnosis and treatment of hepatoenteric fistulas is vital . in the literature ,
most patients with fistulas have complained of abdominal pain after an initial symptom - free period , and persistent fever and chills have been seen in some patients ( table 1 ) .
however , the symptoms usually disappear in patients treated for the abscess without repairing the fistula .
thus , the abscess , rather than the fistula itself , is a more probable cause of the abdominal symptoms . for early diagnosis of liver abscess or fistula formation , clinicians should be alert for patients complaining of such symptoms and should immediately perform contrast - enhanced ct .
when only an abscess is treated , the fistula remains , and in some cases , it causes recurrent infection or secondary complications such as massive gastrointestinal bleeding .
therefore , aggressive treatment such as surgical excision of the fistula is needed ; however , excision is usually difficult because patients have decreased liver function and poor performance .
in addition , the benefit from surgery is likely to be limited due to the short life expectancy of patients with advanced hcc . in our patient ,
a nonsurgical approach was attempted first because of the inoperability of the fistula due to the advanced hcc state and residual poor liver function .
initially , endoscopic clipping was performed to close the fistula opening , but this failed .
histoacryl was then injected into the fistula endoscopically and was successful in treating the fistula .
treatment of an abscesso - colonic fistula caused by rfa with histoacryl injection through a percutaneous catheter during fistulography has been reported . in our case
, a percutaneous catheter could not be inserted because of the small size of the abscess . instead of fistulography
, ercp was performed to visualize the fistula and inject the histoacryl . in conclusion , although hepatoenteral fistula formation after treatment of hcc is a rare complication , it can be intractable and fatal .
for early diagnosis of fistulas , clinicians should be alert for symptoms such as persistent fever and abdominal pain after an initial symptom - free period .
if a fistula is identified , histoacryl injection should be considered as an alternative to major surgical intervention for treatment .
endoscopic histoacryl injection can be a useful treatment choice , especially if pcd is difficult to insert because of the small size or location of the abscess . | a 71-year - old male patient was readmitted to our hospital 1 month after discharge because of relapse of abdominal pain .
he had been diagnosed with hepatocellular carcinoma ( hcc ) 1 year prior and had undergone repeated transcatheter arterial chemoembolization and radiotherapy . during the last hospitalization , he was diagnosed with a liver abscess complicated by previous treatments for hcc and was treated with intravenous antibiotics and abscess aspiration .
follow - up abdominal computed tomography revealed a liver abscess with a duodenal fistula , which was successfully treated with endoscopic histoacryl injection into the fistula .
liver abscesses with duodenal fistulas rarely occur , but they are intractable and possibly fatal in patients with hcc . in the literature ,
they have frequently been managed only with abscess treatment without fistula management .
we herein report the first case of a patient with a liver abscess complicated by a fistula between the duodenum and the abscess , which was treated with endoscopic histoacryl injection . |
our diet is composed of a variety of dietary macronutrients carbohydrates , proteins , fats and fibres .
changes in those nutritional components can act as priming triggers for autoimmunity , whereas the overconsumption of others can lead to cell damage and inflammation .
for instance , the amount of fibre and fat in the diet shapes large - bowel microbial ecology that has been associated with many inflammatory diseases .
this is in line with a study showing that consumption of dietary fibre has globally declined below the recommended daily intake , particularly in westernised societies .
meanwhile , in mediterranean societies where high intake of fibre from vegetables , fruits and nuts is preferred to intake of highly processed meats and industrialised goods , diet - associated complications , such as cardiovascular diseases , have considerably low prevalence .
foods high in fibre provide many health benefits , as it becomes the source of energy for both our own gut cells and the microbial communities that reside there in symbiosis .
industrialised diets might deeply alter the gut microbiota and affect beneficial microbes and their effects on gut , immune and metabolic homeostasis a topic that we are going to discuss later throughout this review .
the importance of diet and its effects on the gut microbiota are reflected in a recent study showing changes in microbiota diversity through evolution in people following ancestral lifestyles relative to westernized societies , indicating that changes in the gut microbiota critically shape human biology .
recent studies have shown that resistant starches mediate many of the effects ascribed to fibre , and their supply is critical for optimal gut function .
resistant starches that can be obtained from vegetable , fruits , wheat , corn and nuts are one such form of dietary fibre .
they are aptly named because of their strong ability to resist degradation by the body 's digestive processes , which continues through to the caecum and large intestine , where they are fermented by the gut microbiota .
this property of resistant starches is often utilised in commercial foods to reduce energy density because of the inability of the human body to digest them . in the mammalian gut ,
primarily the colon , resistant starches are degraded and fermented by gut microbiota that subsequently produce metabolites , the most prominent being scfa : acetate ( two carbons ) , propionate ( three carbons ) and butyrate ( four carbons ) .
these metabolites are produced at varying ratios , with acetate being the most abundant in the colon ( ~60% ) , followed by propionate ( ~25% ) and then , to a much lesser degree , by butyrate ( ~15% ) .
in addition , acetate may itself fuel the production of fellow scfa such as butyrate via alternate biochemical pathways .
more than 95% of scfas are absorbed by the colon , with butyrate being the preferential energy source for colonocytes , as well as having a profound effect on maintaining gut epithelial homeostasis and function .
a ' leaky gut ' in humans and mice , referring to increased gut permeability , disturbed microbial balance and impaired mucosal immunity , has been linked as the preceding step to the initiation of inflammatory diseases and autoimmunity . this is possibly because alteration in microbial ecology and decreased production of scfas altered mechanisms of mucosal barrier function .
for instance , the gut epithelial layer acts as a barrier , preventing the translocation of gut bacteria that can become pathogenic once they reach other organs .
this study inferred that acetate production could be one of the principal features of probiotic bacteria that are thought to provide immune benefits and protection against certain pathogens .
a ' leaky ' intestinal mucosal barrier underpins the breakdown of immune tolerance and leads to intestinal inflammation and diseases , including coeliac disease , colorectal cancer , allergies , asthma , chronic kidney disease , as well as autoimmune t1d . in murine models ,
colonic epithelial cells can suffer dna damage from harmful dietary by - products , such as those generated from protein fermentation , which alarmingly can lead to colon cancer .
clarke et al . observed that rats with azoxymethane - induced colorectal cancer that were fed diets high in resistant starches have a significantly reduced number of tumour formations compared with rats fed control diets with highly digestible starches .
interestingly , increasing butyrate concentrations in the caecum , as well as in the proximal and distal colon , were negatively associated with tumour formation in the large bowel .
in addition , conlon et al . identified an inverse relationship between increased caecal butyrate concentrations and the amount of dna single - strand breakage in colonocytes .
consequently , epithelial cells treated with butyrate regain gut motility and have reduced intestinal permeability . the diet - derived microbial metabolites accumulating in the gut environment
interact with epithelial and immune cells via specific receptors to modulate their respective molecular pathways .
targets of these metabolites include specific gpcrs , which bind free fatty acids such as scfas .
over the years , a vast number of gpcrs have been identified , some currently with unknown ligands or function ; however , only a select few have been characterised as molecular sensors of diet - related microbial products .
of particular interest to this review are the receptors of scfas , namely gpr43 ( ffa2 ) , gpr41 ( ffa3 ) and gpr109a .
whereas under normal physiological conditions niacin levels are not high enough to activate the receptor , levels of butyrate , obtained from the gut environment , and its oxidised form -hydroxybutyrate , are sufficient to stimulate a response .
expression of gpr109a has been found on a variety of immune cells , as well as on adipocytes , hepatocytes , gut and retinal epithelium , vascular endothelium and neuronal tissue .
owing to its connection to the nf-b pathway , gpr109a activation can lead to suppression of pro - inflammatory mediators such as inos , cox2 , tumour necrosis factor ( tnf)- , interleukin ( il)-1 and il-6 .
thus , focus on gpr109a as a therapeutic target to treat inflammatory diseases has been growing . in patients suffering from colitis ,
a form of inflammatory bowel disease , experimental treatment with butyrate enemas has reduced clinical signs of inflammation and even led to remission in some cases . as butyrate
potently activates gpr109a , many studies have focused on the effects of gpr109a activation in treating inflammatory conditions of the bowel .
colonic epithelial cells from neonatal mice cultured with butyrate in vitro generate an enhanced production of mrna for anti - inflammatory il-18 ; yet colonic epithelial cells from mice lacking gpr109a failed to have this upregulating response . adding to this , our group demonstrated recently that nlrp3 inflammasome activation in colonic epithelial cells required two signals .
first , the priming , induced by gut microbial products and second activation mediated by membrane hyperpolarization .
showed that dietary fibre had beneficial effects on epithelial integrity by promoting epithelial nlrp3 activation through effects on both signals one and two . by reshaping the gut microbial composition ,
the scfas , released by anaerobic fermentation by colonic bacteria , activated the inflammasome through their binding to gpr43 and gpr109a .
this beneficial role on epithelial integrity was confirmed in a model of dextran sulphate sodium ( dss)-induced colitis in vivo in which the protective role of dietary fibre was mediated through nlrp3 activation in the epithelial compartment following gpcr activation .
interactions with the external environment in vertebrates occur at various sites including the mucosal surfaces of airways , skin and the gastrointestinal tract .
the gut , being the largest immunological organ in our body and in constant contact with food antigens , commensal microbiota and foreign pathogens , has to be extremely adept at innate and adaptive immune regulations . in order to effectively manage these interfaces ,
the gut has evolved with a highly dynamic anatomy that interacts with the resident microbiota and the mucosal immune system .
the gut mucosal immune system consists of three distinct mucosal lymphoid structures : the mucosa - associated lymphoid tissue found in the gastrointestinal tract either in clusters ( peyer 's patches ) or in isolated lymphoid tissue , the lamina propria where cytokines and immunoglobulins are secreted by effector cells and the epithelium layer in which intraepithelial lymphocytes reside .
another distinct population of immune cells named the innate lymphoid cells ( ilcs ) is essential for the maintenance of intestinal homeostasis .
the ilcs are responsive to microbial composition , and their development and function depends on the specific expression of transcription factors : t - bet for group 1 ilcs , gata-3 for group 2 ilcs or rort for group 3 ilcs .
numerous studies have established a role for ilcs in maintaining a healthy intestinal barrier through the production and secretion of cytokines such as il-22 and il-17 , or activity of the transcription factor aryl hydrocarbon receptor . whereas more research is needed to fully uncover the role of ilcs in regulating host microbe interactions ,
it is clear that ilcs confer another level of protection to the epithelial cells from pathogenic exposure by repairing tissue damage , promoting gut barrier function and preventing systemic inflammation .
recent studies have shown that scfas produced from bacterial fermentation of fibre have anti - inflammatory and immunomodulatory effects through the impact of regulatory t ( treg ) cells as an important factor in immune tolerance .
the scfa butyrate promotes inducible treg ( itreg ) number and function in the colon of mice .
il-10-producing itregs develop after tgf- cytokine exposure in the periphery from naive cd4 t cells .
in addition , adoptive transfer of cd4 cd45rb naive t cells into rag1 mice showed their conversion into treg cells when mice were fed a butyrylated diet .
indeed , it is likely that commensal bacterial species that promote itregs in the gut do so through production of high amounts of acetate or butyrate . in parallel , smith et al . further expanded on this finding by demonstrating a direct effect of scfa on colonic tregs through the increased expression of gpr43 mrna ; however , this effect was absent in mice deficient in gpr43 .
a portion of diet - derived microbial metabolites passes across the mucosa into the lamina propria , where it enters the systemic circulation via the portal vein . whereas butyrate enacts strong effects in the gut , its levels in circulation throughout the body are often negligible or undetectable .
acetate , the most abundantly produced scfa , is , however , readily detectible in the peripheral circulation at ~50150 m .
therefore , the scfa acetate is one means by which the microbiota may regulate the immune system beyond the gut .
the western diet underlies obesity , t2d , as well as asthma and cancer .
all these conditions are elements of the metabolic disorders , where diet contributes to the chronic inflammation of visceral adipose tissue , insulin resistance and increased intestinal permeability , allowing dissemination of gut bacteria or bacterial products ( endotoxaemia ) .
genetically obese mice had increased intestinal permeability and lipopolysaccharide ( lps ) levels in the portal blood , which promote inflammatory liver damage .
this is evidenced by the increased levels of tnf- and reduced zona occludens 1 mrna in the proximal colon of obese c57bl/6j mice , which correlated with increased macrophage infiltration and levels of inflammatory cytokines tnf- and il-6 in the mesenteric fat .
in contrast , the gut anti - inflammatory agent 5-aminosalicyclic acid was shown to improve metabolic parameters in diet - induced obesity ( dio ) mice , with associated regulation of gut adaptive immunity and reduced gut permeability , thus implicating the role of gut leakiness and inflammation in dio mice . a similar link between obesity - induced abnormalities in lipid homeostasis ,
gut permeability and non - alcoholic steatohepatitis was also found in human subjects , similar to increased circulating zonulin and il-6 in obesity - associated insulin resistance .
two groups have demonstrated that induction of il-22 produced by group 3 ilcs is impaired in obese mice , and il-22-deficient mice fed a high - fat diet are more susceptible to developing metabolic disorders .
lymphoid tissue - inducer cells secrete large amounts of il-22 that maintains gut mucosal barrier integrity and keeps the host microbial balance .
in addition , il-22 is involved in the recruitment of b cells and other lymphocytes to the germinal centres of isolated lymphoid tissues important for pathogen clearance , in line with the theory of endotoxaemia induced by inflammation and increased intestinal permeability .
the effects of il-22 extend beyond the gut as il-22 is a natural regulator of beta - cell insulin biosynthesis and secretion , protecting beta - cells from stress and preventing insulin hypersecretion , ultimately suppressing islet inflammation in obesity .
administration of exogenous il-22 to db / db or dio mice improves obesity - driven insulin sensitivity and gut barrier dysfunction , and reduces chronic inflammation in the liver and adipose tissues . the potential link between gut microbiota and the obese phenotype
was established a decade ago . since then obese mice treated with prebiotics selectively increased bifidobacterium and showed a decrease in concentrations of lps and inflammatory cytokines in blood , and this associated with improvements in gut barrier function .
faecal microbiota transferred to germ - free mice from mothers with gestational diabetes induced increased adiposity and insulin sensitivity , thus demonstrating the association between human metabolic disorders and altered microbiota composition .
more recently , a study assessing the role of drug effect on gut microbiota of t2d subjects showed that the t2d subjects lacking butyrate - producing gut bacteria could be restored following treatment with metformin , an antidiabetic therapy , suggesting a role for scfa - producing microbes in disease and health .
in addition , scfas stimulate the release of the gut hormone glucagon - like - peptide-1 and 2 ( glp-1 and glp-2 ) , which is responsible for modulating gut barrier function and reducing uptake of inflammatory compounds that may trigger the chronic low - grade inflammation often linked with obesity and cardiovascular disease . indeed ,
prebiotic - treated mice show an increased glp-2 production associated with lower plasma lps levels and oxidative stress markers . the scfa acetate has also been demonstrated to regulate production of leptin , an adipose - based hormone crucial for regulating energy homeostasis .
some studies have elucidated the roles of scfas and gpcrs and production of leptin in vitro and in vivo . , although concentrations of propionate in serum are quite low or undetectable , treatment of adipose tissue explants with propionate significantly downregulated the production of tnf- and ccl5 by macrophages , and increased the expression of lipoprotein lipase and glut4 ( associated with lipogenesis and glucose uptake ) .
meanwhile , gpr109a promotes lipolysis , as niacin treatment in mice deficient in gpr109a fails to increase the secretion of adiponectin .
in contrast , tang et al . showed that mice with dio and t2d displayed increased plasma acetate in correlation with higher expression of gpr43 and gpr41 in the islets , and this contributed to compromised capacity of beta - cells to respond to hyperglyceamia .
this is in line with increased local glucose - dependent acetate formation by pancreatic islets , also seen in people with diabetes independent of fibre intake
. however , several studies show inconsistent results using gpr41- or gpr43-deficient mice . given scfas
modulate immune responses , the extent to which diet and the gut microbiota account for progression of metabolic syndrome through immune regulation is still poorly understood .
mathis and co - workers showed that low - grade of inflammation in the adipose tissue correlates with reduced treg cell numbers with downregulated expression of gut - homing markers cd103 and gpr83 .
in addition to metabolite - sensing gpcrs , scfas also exert activities through epigenetic effects , particularly the hdacs .
hdacs regulate chromatin remodelling and gene expression , as well as the function of numerous transcription factors .
hdacs are a group of enzymes that remove acetyl groups from the histones that bind dna .
removing acetyl groups alters the binding of histones to dna , which changes the expression patterns of different genes . through this activity
in adipose tissue , a high - fat diet impairs adipogenic differentiation of c / ebp , ppar , fabp4 and adiponectin associated with elevated expression of hdac 9 .
the pro - inflammatory obese state can also lead to the development of chronic kidney disease due to a ' leaky ' intestinal mucosal barrier , possibly because compromised epithelial integrity allows the dissemination of gut bacteria or bacterial products ( endotoxaemia ) resulting in kidney damage .
feeding mice with high - fibre diets prompted a reduction in markers of kidney damage including serum concentration of creatinine and urea .
similarly , inhibition of hdac activity by acetate led to reduced dna methylation in kidney tissue .
epigenetic modifications are essential for development and proper functioning of the kidney , as they modulate tgf signalling , inflammation , profibrotic genes and the epithelial - to - mesenchymal transition , promoting renal fibrosis and progression of chronic kidney disease . as such ,
hdacs have been shown to have integral roles in the regulation and activity of different immune cells .
in leukocyte cells , such as macrophages , neutrophils and eosinophils , hdacs have been linked to controlling cell survival and proliferation , as well as the regulation of cytotoxicity . in b cells
, hdacs have been shown to be important for inducing the apoptosis of proliferating cells .
hdacs are also important for promoting cd8 t cells , particularly in regards to increased function and differentiation . besides influencing immune cell survival , hdacs have also been linked to the suppression of cytokine production , having a role in controlling the inflammatory response .
hdacs are a very important part of immune regulation , both in promoting and regulating the immune system , and are a potential target for microbial metabolites in influencing the immune system .
studies into such diseases , including t1d and certain variants of inflammatory bowel disease , emphasise not only genetic factors but also environmental and dietary factors
. twelve - week - old non - obese diabetic ( nod ) mice that are pre - diabetic exhibit increased intestinal permeability and , when infected with a bacterial pathogen citrobacter rodentium , show increased activation and proliferation of diabetogenic cd8 t cells , which accelerate the onset of insulitis . increased intestinal permeability
is associated with clinical diagnoses of t1d , with a link between serum zonulin levels and development of t1d in patients and their relatives .
moreover , diabetes - prone biobreeding rats fed with hydrolysed casein diet reduced disease incidence by 50% , correlating with decreased lactulose : mannitol ratio and serum zonulin levels , indicative of a tighter intestinal barrier .
variances in gut microbiota in children diagnosed with t1d , although conflicting , have been widely examined .
children who develop t1d have a less diverse gut microbiota with a decreased presence of firmicutes phylum correlated with decreased fecal butyrate than children with no t1d that presented an increase in bacteroidetes phylum . in line with these findings , nod mice deficient in the adaptor protein myeloid differentiation factor 88 ( myd88 ) , important for the detection of microbial antigens , fail to develop t1d under spf conditions ; yet germ - free conditions lead to an exacerbated development of t1d . in addition , a following study by markle et al
. demonstrated the role of the gut microbiota in the marked gender differences that characterise t1d in nod mice .
similar to humans , male nod mice display a considerably delayed onset and a reduced incidence of t1d . remarkably , the female cohort gavaged with male gut microbiota were protected from t1d development , in comparison with female cohorts gavaged with a female gut microbiota or left untreated , which displayed typical disease incidence .
treatment of nod mice with probiotics coincides with maintenance of beta - cell function and prevention of t1d , and probiotic treatment in genetically susceptible children for the prevention of t1d is currently the focus of the ongoing prodia study in finland .
whereas these studies provide compelling evidence for the role of gut microbiota in modulating t1d development , the specific metabolites responsible for preventing or ameliorating the diabetic immune response remain to be identified .
as alluded to throughout this review , scfas may have a major role in prevention of autoimmune diseases , and may underlie at least some microbiota - related associations with human disease .
we have shown that scfas from the mother cross the placenta and protect against inflammatory asthma in offspring through epigenetic imprinting , mediating changes in gene transcription such as foxp3 target genes important for tolerance / autoimmunity .
scfas produced from bacterial fermentation of fibre not only promote itreg number and function in the colon but also induce the promotion of extrathymic generation of tregs via epigenetic effects .
this , in turn , allows tregs to better control autoreactive lymphocytes and prevent the development of autoimmune disease .
for instance , epigenetic alterations such as histone modifications of the foxp3 locus are important for proper foxp3 expression and the functional activity of tregs .
foxp3 also epigenetically modulates transcriptional activity of target gene loci by altering dna methylation , transcription factor associations and histone modifications .
tregs , driven by the foxp3 transcription factor , are particularly important for limiting autoimmunity and chronic inflammation .
b cells have been implicated in the pathogenesis of certain inflammatory diseases , including t1d and lupus , because of their ability to produce autoantibodies , as well as cross present self - antigens to autoreactive t lymphocytes . in a mouse model of lupus ,
treatment with butyrate and synthetic hdac inhibitors led to the suppression of mechanisms that promote hypermutated antibody responses and class - switching , which culminate in the generation of high - affinity autoantibodies .
inhibition of hdacs to limit autoreactive b cells will likely be relevant to other inflammatory diseases such as t1d ( our unpublished findings ) .
a potential use for hdac inhibitors to modify autoreactive b cells relates to individuals diagnosed with t1d , who also develop celiac disease .
celiac disease is an autoimmune condition involving the inflammation of the small intestine , specifically in response to the presence of gluten food antigen .
b cells and gluten - specific cd4 t cells from the intraepithelial lymphocyte compartment and lamina propria lead the inflammatory response . apart from priming an immune response , studies in balb / c and nod mice have shown a 15% decrease in treg cell number in response to dietary gluten .
this effect is because of the overexpression of il-15 in celiac disease , which suppresses treg activation .
owing to the highly regenerative ability of the small intestine , however , function can be recovered when individuals diagnosed with celiac disease adhere to a strict gluten - free diet .
targeting the gut microbiota is becoming a revolutionary therapy to correct metabolic dysfunction and inflammatory responses to treat diseases .
it is now evident that the western diets , possibly because of the lack of fibre , contribute not only to the loss of microbiota diversity but also promote an unbalance towards pathogenic gut bacteria associated with many inflammatory diseases . in a recent study , sonnenburg et al
. demonstrated that humanised gnotobiotic mice on a westernised diet ( lacking fermentable carbohydrates ) related with decreased gut microbiota diversity , particularly clostridiales and bacteroidales predominant producers of scfas .
worryingly , feeding with this diet over subsequent generations led to the extinction of those bacterial phyla by the fourth generation , and the missing phyla could only be recovered via faecal microbiota transplant in combination with a diet high in fermentable carbohydrates .
thus , we believe that dietary scfas could be an excellent alternative approach to preventing or correcting the deterioration of western gut microbiota , given it is the safest and most cost - effective way to have an impact on the large global patient population . targeting the gut microbiota using therapies such as probiotics ( treating the individual with healthy bacteria ) , prebiotics ( treating the individual with nutrients to promote good bacteria ) and the relatively crude fecal transplant has been largely studied .
so far , the outcomes from those methods are controversial or not so efficacious , given targeting specific components of the diets or a specific type of bacteria could be beneficial for some but detrimental for others .
the advantages of using dietary fibre to target individual microbial metabolites relate to its properties of being highly resistant to human digestion and , therefore , directly modulating the whole microbiota community rather than individual bacteria species .
furthermore , this dietary manipulation of microbial metabolites can be used to naturally tailor a beneficial microbial ecology for each individual based on their personal gut microbial diversity .
figure 1 illustrates the mechanisms of action for the microbial scfas , that is , through metabolite - sensing gpcrs and/or hdac epigenetic remodelling on epithelial cells and/or immune cells such as tregs .
one simple model is that reduced production of scfas through western style diet , or antibiotic use and so on , contributes to altered microbial ecology and altered mucosal barrier function , resulting in exposure of the mucosal immune system to bacteria or their products , which then in turn could affect immune tolerance .
therefore , targeting microbiota through dietary scfas could be a promising therapeutic approach to prevent or treat autoimmunity and inflammatory diseases associated with metabolic syndrome , where it has been observed that gut dysbiosis predates the development of the disease .
in developing our understanding of how dietary components shape the overall panorama of the gut microbiome , and the subsequent metabolite profile , we can identify the likelihood of events leading to inflammation and autoimmunity .
emerging dietary treatments are not only economical , but also offer a non - invasive approach alternate to the risks of surgical procedures for chronic states of inflammation .
although it is in its early days , the implementation of diet and/or microbial metabolites or engineering the gut microbiota as a tool to prevent or treat inflammatory diseases is an exciting prospect that may have a great impact on human health . | it is now convincingly clear that diet is one of the most influential lifestyle factors contributing to the rise of inflammatory diseases and autoimmunity in both developed and developing countries .
in addition , the modern ' western diet ' has changed in recent years with increased caloric intake , and changes in the relative amounts of dietary components , including lower fibre and higher levels of fat and poor quality of carbohydrates .
diet shapes large - bowel microbial ecology , and this may be highly relevant to human diseases , as changes in the gut microbiota composition are associated with many inflammatory diseases .
recent studies have demonstrated a remarkable role for diet , the gut microbiota and their metabolites the short - chain fatty acids ( scfas)in the pathogenesis of several inflammatory diseases , such as asthma , arthritis , inflammatory bowel disease , colon cancer and wound - healing .
this review summarizes how diet , microbiota and gut microbial metabolites ( particularly scfas ) can modulate the progression of inflammatory diseases and autoimmunity , and reveal the molecular mechanisms ( metabolite - sensing g protein - coupled receptor ( gpcrs ) and inhibition of histone deacetylases ( hdacs ) ) .
therefore , considerable benefit could be achieved simply through the use of diet , probiotics and metabolites for the prevention and treatment of inflammatory diseases and autoimmunity . |
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